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. …

Myosin subfragment-2 (S2) is an intrinsically unstable coiled coil. This dissertation tests if the mechanical stability of myosin S2 would influence the availability of myosin S1 heads to actin thin filaments. The elevated instability in myosin S2 coiled coil could be one of the causes for hypercontractility in Familial Hypertrophic Cardiomyopathy (FHC). As hypothesized FHC mutations, namely E924K and E930del, in myosin S2 displayed an unstable myosin S2 coiled coil compared to wild type as measured by Fluorescence Resonant Energy Transfer (FRET) and gravitational force spectroscopy (GFS). To remedy this, anti-S2 peptides; the stabilizer and the destabilizer peptides by namesake were designed in our lab to increase and decrease the stability of myosin S2 coiled coil to influence the actomyosin interaction. Firstly, the effectiveness of anti-S2 peptides were tested on muscle myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by the stabilizer and decreased by the destabilizer across the cardiac and skeletal myosin S2 isoform but not for the smooth muscle isoform. The destabilizer peptide had dissociation binding constants of 9.97 × 10-1 μM to MYH7 isoform, 1.00 μM to MYH2 isoform, and no …

To better understand the nature and function of N-acylethanolamines (NAEs) in Camelina sativa, we used mass spectrometry analysis to identify and quantify NAE types in developing seeds, desiccated seeds and seedlings. Developing seeds showed a differential increase in individual NAE species and an overall increase in NAE content with seed development and maturation. The NAE composition in mature, desiccated seeds mostly reflected the total fatty acid composition in the seed tissues, except for a noted absence of 11-eicosenoic (20C monounsaturated) fatty acid in the NAE pool. During seed stratification and seedling growth, individual NAE species were depleted at similar rates. Simulated drought treatments during seedling development resulted in a significant rise in NAE levels for the major 18C NAE types compared with untreated seedlings. Arabidopsis and Camelina mutants with reported altered fatty acid profiles were analyzed for their NAE compositions; both Arabidopsis and Camelina had relatively similar changes between compositions of total seed fatty acids and NAEs. Furthermore, seeds were analyzed from transgenic Arabidopsis and Camelina with engineered, non-native, long-chain polyunsaturated fatty acids (18C, 20C and 22C), and the results showed the production of novel N-acylphosphatidylethanolamines (presumed precursors of NAEs) and NAEs with the same long acyl chains. These results …

Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. Previously, it has been shown that the RNA released from damaged blood cells activates clotting. However, the nature of RNA released from hemolysis is still elusive. We found that after hemolysis, the red blood cells from both zebrafish and humans release 5.8S rRNA. This RNA activated coagulation in zebrafish and human plasmas. Using both natural and synthetic 5.8S rRNA and its synthetic truncated fragments, we found that the 3'-end 26 nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor blocked 3'-26 RNA-mediated coagulation activation of both zebrafish and human plasma. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activates normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via FXII-like protein. Since zebrafish has no FXII and hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from …

In this study, we have developed a novel way of generating and exposing biological organisms (both prokaryotic and eukaryotic) to silver nanoparticles (AgNPs) and studying the biochemical changes induced by these particles. We analyzed the various organs of Wistar rats for localization and quantification of these particles using mass spectrometric and molecular biological techniques. Highest levels of AgNP was found in the lung tissue in addition to being present in the liver and kidneys. Analysis of the of the blood plasma from AgNP exposed rats revealed elevated levels of glutathione-disulfide, which is indicative of reactive oxygen species (ROS) generation, which was further validated using ROS specific immunofluorescence staining of liver tissue. Quantification of blood lactate levels of the AgNP exposed rats showed increased lactate levels, which is indicative of anaerobic respiration and may result from AgNP-induced oxidative stress. Further analysis of bone marrow cells from AgNP exposed rats showed a higher number of micronuclei formation in developing erythrocytes and bone marrow cytotoxicity. Finally, analysis of the genes involved in the renin-angiotensin system (RAS) and inflammatory response revealed upregulation in transcript levels of many of these important genes in the liver tissue. Taken together, our study provides an initial road map …

Flavonoids are polyphenolics compounds that constitute a major group of plant specialized metabolites, biosynthesized via the phenylpropanoid/polymalonate pathways. The resulting specialized metabolites can be due to decoration of flavonoid compounds with sugars, usually glucose, by the action of regiospecific UDP-glycosyltransferase (UGT) enzymes. In some cases, glycosylation can involve enzymatic attachment of other sugar moieties, such as glucuronic acid, galactose, rhamnose or arabinose. These modifications facilitate or impact the bioactivity, stability, solubility, bioavailability and taste of the resulting flavonoid metabolites. The present work shows the limitations of utilizing mammalian UDP-glucuronosyltransferases (UGATs) for flavonoid glucuronidation, and then proceeds to investigate plant UG(A)T candidates from the model legume Medicago truncatula for glucuronidating brain-targeted flavonoid metabolites that have shown potential in neurological protection. We identified and characterized several UG(A)T candidates from M. truncatula which efficiently glycosylate various flavonoids compounds with different/multiple regiospecificities. Biochemical characterization identified one enzyme, UGT84F9, that efficiently glucuronidates a range of flavonoid compounds in vitro. In addition, examination of the ugt84f9 gene knock-out mutation in M. truncatula indicates that UGT84F9 is the major UG(A)T enzyme that is necessary and sufficient for attaching glucuronic acid to flavonoid aglycones, particularly flavones, in this species. Finally, the identified UG(A)T candidates were analyzed via homology …

Alteration in diet and knockdown of detoxification genes impacts the response of C. elegans to oxygen deprivation stress. I hypothesized that feeding worms a vitamin D3-supplementation diet would result in differential oxygen deprivation stress response. We used a combination of wet lab and transcriptomics approach to investigate the effect of a vitamin-D3 supplemented diet on the global gene expression changes and the anoxia response phenotype of C. elegans (Chapter 2). C. elegans genome consists of 143 detoxification genes (cyp and ugt). The presence of a significant number of genes in these detoxification families was a challenge with identifying and selecting specific cyp and ugt genes for detailed analysis. Our goal was to understand the evolution, phylogenetic, and expression of the detoxification enzymes CYPs and UGTs in C. elegans (Chapter 3). We undertook a phylogenetic and bioinformatics approach to analyze the C. elegans, detoxification family. Phylogenetic analysis provided insight into the association of the human and C. elegans xenobiotic/endobiotic detoxification system. Protein coding genes in C. elegans have been predicted to be human orthologs. The results of this work demonstrate the role of C. elegans in the identification and characterization of vitamin D3 induced alterations in gene expression profile and anoxia …

Plant yield is an agronomic trait dependent on the transport of photosynthate from mature source leaves to sink tissues. Manipulating phloem transport may lead to increased yield, however in a previous study, Arabidopsis thaliana overexpressing sucrose transporter AtSUC2 in the phloem resulted in stunted growth and an apparent P-deficiency. In the course of further characterizing the phenotype and identifying the causative mutation, this research included 1) reverse genetics to test genes hypothesized to modulate carbon-phosphate interactions; 2) whole genome sequencing to identify all T-DNA insertions in plants displaying the phenotype; 3) genetic crosses and segregation analysis to isolate the causative mutation; and 4) transcriptomics to capture gene-expression profiles in plants displaying the phenotype. These phenotypes were traced to a T-DNA insertion located on chromosome 4. Transcriptomics by RNA-Seq and data analysis through bioinformatics pipelines suggest disruptions in metabolic and transport pathways that include phosphate, but do not support a direct role of well-established phosphate acquisition mechanisms. Gene At1G78690 is immediately downstream of the T-DNA insertion site and shows modestly increased expression relative to wild type plants. At1G78690 encodes O-acyl transferase, which is involved in processing N-acylphosphotidyl ethanolamine (NAPE) to N-acyl ethanolamine (NAE). Exogenous NAE application causes stunted growth in specific …

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, …

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 …

In the past couple of decades, the zebrafish has been widely used to study hemostatic disorders. In this study, we generated a CRISPR/Cas9 mediated zebrafish mutant that contains a 55-nucleotide insertion in exon 29 of the von Willebrand factor (vwf) gene. The mutants had impaired ristocetin-mediated agglutination of whole blood, prolonged PTT and more bleeding in the lateral incision compared to wild-type fish. The bleeding phenotype observed here is similar to the phenotype observed in vwf knockout mice and patients with von Willebrand disease (VWD). The mutant model developed here can thus be used for exploring the role of Vwf in angiogenesis and for developing gene therapy. The deficiency of VWF causes VWD and the etiology remains unknown in 30% of Type 1 VWD cases. Previous studies have identified that the ABO blood group and ST3GAL4 (glycosyltransferases) are involved in the regulation of VWF levels. Since VWF is heavily glycosylated, we hypothesized that other glycosyltransferases may also be involved in regulating VWF. We performed a knockdown screen of 234 glycosyltransferase genes and identified 14 genes that altered Vwf levels. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for …

Tissue Factor Pathway Inhibitor (TFPI) is an anticoagulant protein containing three Kunitz domains, K1, K2 and K3. K1 inhibits Factor VIIa, K2 inhibits Factor Xa, and K3 enhances the Factor Xa inhibition by its interaction with Protein S. Since zebrafish is an excellent genetic model, we hypothesized that TFPI regulation could be studied using this model. As a first step, we confirmed the presence of tfpia in zebrafish. Subsequently, we performed knockdown of tfpia, and knockout of tfpia in K3 domain using CRISPR/Cas9. Both the tfpia knockdown and tfpia homozygous deletion mutants showed increased coagulation activities. Our data suggest that zebrafish tfpia is an orthologue for human TFPIα, and silencing it results in a thrombotic phenotype. We then optimized the piggyback knockdown method, where we could simultaneously piggyback 3 or 6 ASOs corresponding to 3 or 6 genes, respectively, using one VMO. These multiple gene knockdowns will increase the efficiency of genome-wide knockdowns. Since there are no studies on chromatin remodeling that control TFPI expression, we hypothesized that the genome-wide knockdowns of the Chromatin Binding and Regulatory Proteins (CBRPs) in zebrafish could help identify novel tfpia gene regulators. We chose 69 CBRPs and subjected them to simultaneous gene knockdowns. Our …

Symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. Within nodule cells, N-fixing rhizobia are surrounded by plant-derived symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. First, I investigated the impact of P deprivation on wild-type Medicago truncatula plants. My observations that plants had impaired SNF activity, reduced growth, and accumulated less phosphate in P-deficient tissues (leaves, roots and nodules) is consistent with those of similar previous studies. Galactolipids decreased with increase in phospholipids in all P-starved organs. Matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI-MSI) of phosphatidylcholine (PC) species in nodules showed that under low P environments distributions of some PC species changed, indicating that membrane lipid remodeling during P stress is not uniform across the nodule. Secondly, a metabolomics study was carried out to test the alterations in the metabolic profile of the nodules in P-stress. GC-MS based untargeted metabolomics showed increased levels of amino acids and sugars and decline in amounts of organic acids in P deprived nodules. Subsequently, …

The isoflavones in kudzu roots, especially the C-glycosylated isoflavone puerarin, have been linked to many health benefits. Puerarin contains a carbon-carbon glycosidic bond that can withstand hydrolysis. The C-glycosylation reaction in the biosynthesis of puerarin has not been thoroughly investigated, with conflicting reports suggesting that it could take place on daidzein, isoliquiritigenin, or 2,7,4ʹ-trihydroxyisoflavanone. Kudzu species were identified for use in comparative transcriptomics. A non-puerarin producing kudzu was identified as Pueraria phaseoloides and a puerarin producing kudzu was identified as Pueraria montana lobata. Through the use of the plant secondary product glycosyltransferase (PSPG) motif, glycosyltransferases (UGTs) were identified from the transcriptomes. The UGTs that had higher digital expression in P. m. lobata were examined further using additional tools to home in on the UGT that could be responsible for puerarin biosynthesis. One of the UGTs identified, UGT71T5, had previously been characterized from kudzu as a C-glycosyltransferase involved in puerarin biosynthesis through in vitro enzyme activity (with daidzein) and a gain of function approach in soybean hairy roots. Previous studies have not supported the end-product of a pathway such as daidzein as the target for C-glycosylation, and no genetic analysis of UGT function had been conducted in kudzu. The activity of …

Fibrinolysis pathway is an important mechanism for dissolution of fibrin clot by the action of plasmin which is formed from plasminogen, a zymogen via the action of plasminogen activators, i.e. tissue plasminogen activator and urinary plasminogen activator. The regulation of fibrinolysis system in vivo is maintained by plasminogen activators and natural inhibitors i.e. α2-antiplasmin, α2-macroglobulin, Thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor 1 and 2 (PAI-1and PAI-2). There are several fibrinolytic assays developed for human plasma but there are no reports describing fibrinolytic assay using zebrafish plasma. In this study, a fibrinolytic assay via using small amount of zebrafish plasma was developed. This assay was performed under different conditions; one by the addition of exogenous tissue plasminogen activator alone to the pooled zebrafish plasma along with calcium chloride and thromboplastin, second Dade ACTIN was used instead of tissue plasminogen activator and third Dade ACTIN along with thromboplastin was used. Epsilon amino caproic acid (EACA), a synthetic antifibrinolytic agent was used at different concentrations to inhibit fibrinolysis successfully. Similar experiments were performed on human plasma as well to check the applicability of the assay to humans and positive results were obtained. Furthermore, knockdown of tissue plasminogen activator and plasminogen genes …

In this study, I examined the use of mouse (Mus musculus) Fat Specific Protein 27 (FSP27) ectopically expressed in Arabidopsis thaliana and Nicotiana benthamiana as a means to increase lipid droplet (LD) presence in plant tissues. In mammalian cells, this protein induces cytoplasmic LD clustering and fusion and helps prevent breakdown of LDs contributing to the large, single LD that dominates adipocytes. When expressed in Arabidopsis thaliana and Nicotiana benthamiana, FSP27 retained its functionality and supported the accumulation of numerous and large cytoplasmic LDs, although it failed to produce the large, single LD that typifies adipose cells. FSP27 has no obvious homologs in plants, but a search for possible distant homologs in Arabidopsis returned a Tudor/PWWP/MBT protein coded for by the gene AT1G80810 which for the purposes of this study, we have called LIPID REGULATORY TUDOR DOMAIN CONTAINING GENE 1 (LRT1). As a possible homolog of FSP27, LRT1 was expected to have a positive regulatory effect on LDs in cells. Instead, a negative regulatory effect was observed in which disruption of the gene induced an accumulation of cytoplasmic LDs in non-seed tissue. A study of lrt1 mutants demonstrated that disruption this gene is the causal factor of the cytoplasmic LD …

Synthetic biology is a rapidly growing field that aims to treat cellular biological networks in an analogous way to electrical circuits. However, the field of plant synthetic biology has not grown at the same pace as bacterial and yeast synthetic biology, leaving a dearth of characterized tools for the community. Due to the need for tools for the synthetic plant biologist, I have endeavored to create a library of well-characterized TATA box variants in the cauliflower mosaic virus (CaMV) 35S promoter using the standardized assembly method Golden Braid 2.0. I introduced single nucleotide changes in the TATA box of the CaMV 35S promoter, a genetic part widely used in plant gene expression studies and agricultural biotechnology. Using a dual-luciferase reporter system, I quantified the transcriptional strength of the altered TATA box sequences and compared to the wild-type sequence, both in transient protoplast assays and stable transgenic Arabidopsis thaliana plants. The library of TATA-box modified CaMV 35S promoters with varying transcriptional strengths created here can provide the plant synthetic biology community with a series of modular Golden Braid-adapted genetic parts that can be used dependably and reproducibly by researchers to fine-tune gene expression levels in complex, yet predictable, synthetic genetic circuits.

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 …

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.

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.

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 …