Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated sludge wastewater treatment plant, overproduces exopolysaccharide (EPS) leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole genome phenotypic sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants were subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type genome. We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The FTIR spectra of EPS isolated from the wild-type showed reduced C=O peak of the N-acetyl group at 1665 cm-1 as compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated the deacetylase gene transcript increased before flocculation occurred. The results suggest that the deacetylation of MZ1T EPS is crucial for flocculation. The information obtained from this study will be useful for preventing viscous bulking and wastewater treatment system failure, and may have potential applications in the biotechnology sector for …

Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.

With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the production costs and wastes of high-value products such as medicines, biofuels, and chemical feedstocks that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. To achieve these complex goals, information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed. A genetic Boolean AND logic gate is a device that computes the presence or absence of two inputs (signals, stimuli) and produces an output (response) only if both inputs are present. Here, we optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate two hormonal inputs in whole plants. These AND gates produce an output only in the presence of both abscisic acid and auxin, but not when either or neither hormone is present. Furthermore, we demonstrate the AND gate can also integrate two plant stresses, cold temperature and bacterial infection, to produce a specific response. The design principles used here are generalizable, and therefore multiple orthogonal AND gates could be assembled and rationally …

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 …

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 …

The lipoxygenase (LOX) pathway plays an important role in the oxidative metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). The LOX pathway functions in conjugation with hydrolysis by fatty acid amide hydrolase (FAAH) and to produce oxidized NAEs during seed germination and early seedling development. When Arabidopsis seedlings were grown in low micromolar concentrations of lauroylethanolamide (NAE 12:0), growth retardation and elevated endogenous PU-NAE levels were observed due to the competitive inhibition of LOX by NAE 12:0. The elevated levels of endogenous PU-NAEs were more pronounced in genotypes with reduced NAE hydrolase capacity (faah knockouts), and less evident with overexpression of FAAH. Alterations in PU-NAE metabolism were studied in seedlings of various lox and FAAH mutants. The partitioning of PU-NAEs into oxylipin metabolites was exaggerated in the presence of exogenous linolenoylethanolamide (NAE18:3) and resulted in bleaching of cotyledons. The bleaching phenotype was restricted to a narrow developmental window (3-to-5 days after sowing), and was attributed to a reversible disruption of thylakoid membranes in chloroplasts. Biochemical and genetic evidence suggested that 9-hydro(pero)xy and 13-hydro(pero)xy octadecatrienoylethanolamides (9- and 13-NAE-H(P)OT), but not their corresponding hydro(pero)xy free fatty acids, induced cotyledon bleaching. The LOX-mediated metabolites of NAE18:3 shared some overlapping effects on seedling development with those of …

The ovarian hormone 17β-estradiol (E2) is one of the central regulators of the female reproductive system. E2 is also a pleiotropic regulator since it can exert its non-reproductive role on other organ systems. E2 is neuroprotective, it maintains body's energy homeostasis, participates in various repair mechanism and is required for neural development. However, there is a substantial evidence suggesting that there might be a molecular reprogramming of E2's action when it is supplied exogenously after E2 deprivation. Though the length of E2 deprivation and age has been linked to this phenomenon, the molecular components and how they activate this reprogramming is still elusive. Our main goal was to perform global proteomics and metabolomics study to identify the molecular components and their interaction networks that are being altered in the brain and serum after a short-term E2 treatment following ovariectomy (OVX) in Sprague Dawley rats. One of the strength of our global study is that it gave us extensive information on the brain proteome itself by identification of a wide number of proteins in different brain sections. By analyzing the differentially expressed proteins, our proteomics study revealed 49 different networks to be altered in 7 sections of the brain. Most of …

Fiber-yielding plants is an area of increased interest due to the potential use in a variety of green-based materials. These biocomposites can be incorporated into multiple uses; for example, to replace building materials and interior vehicular paneling. The research here aims to focus in on the crop Hibiscus cannabinus for utilization into these functions. H. cannabinus is economically attractive due to the entire process being able to be accomplished here in the United States. The plant can be grown in a relatively short growth period (120-180 days), and then processed and incorporated in a biocomposite. The plant fiber must first be broken down into a useable medium. This is accomplished by the retting process, which occurs when microbial constituents breakdown the heteropolysaccharides releasing the fiber. The research aims to bridge the gap between the primitive process of retting and current techniques in molecular and microbiology. Utilizing a classical microbiological approach, which entailed enrichment and isolation of pectinase-producing bacteria for downstream use in augmented microbial retting experiments. The tracking of the bacteria was accomplished by using the 16S rRNA which acts as “barcodes” for bacteria. Next-generation sequencing can then provide data from each environment telling the composition and microbial diversity of …

The anti-S2 peptides, the stabilizer and destabilizer, were designed to target myosin sub-fragment 2 (S2) in muscle. When the peptides are coupled to a heart-targeting molecule, they can reach the cardiomyocytes and interfere with cardiac muscle contraction. Monoclonal antibodies, MF20 and MF30, are also known to interact with light meromyosin and S2 respectively. The MF30 antibody compared to anti-S2 peptides and the MF20 antibody is used as a control to test the central hypothesis that: Both the anti-S2 peptides and antibodies bind to myosin S2 with high affinity, compete with MyBPC, and possibly interact with titin, in which case the anti-S2 peptides have further impact on myosin helicity and reach the heart with the aid of tannic acid to modulate cardiomyocytes' contraction in live mice. In this research, the effects of anti-S2 peptides and antibodies on myosin S2 were studied at the molecular and tissue levels. The anti-myosin binding mechanism to whole myosin was determined based on total internal reflectance fluorescence spectroscopy (TIRFS), and a modified cuvette was utilized to accommodate this experiment. The binding graphs indicated the cooperative binding of the peptides and antibodies with high affinity to myosin. Anti-myosin peptides and antibodies competition with Myosin Binding Protein C …

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 …

Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By …

Leguminous plants are able to fix nitrogen by establishing a symbiotic relationship with soil dwelling bacteria, called rhizobia. The model plant Medicago truncatula forms a partnership with Sinorhizobium meliloti whereby the plant gains bioavailable nitrogen and in exchange the bacteria gains carbohydrates. This process occurs within nodules, which are structures produced on the roots of the plants within which nitrogen is fixed. M. truncatula incomplete root elongation (MtIRE) was localized to the infection zone, which is zone II of indeterminate nodules. It was shown to encode a signaling kinase so it was anticipated to play a role in nodulation. Mutants of MtIRE in the R108 background, mutagenized with the Tnt1 retrotransposon, were obtained from reverse screen, and were assessed to determine if a disrupted MtIRE gene was the cause of nitrogen fixation defective nodules. Mutant line NF1320, having a mutant phenotype, showed typical Mendelian segregation of 3:1 when backcrossed to R108. Experimental results show that MtIRE gene is not the cause of the mutant phenotype, but was linked to the causative locus. MtIRE co-segregated with the mutant phenotype 83%. Southern blot and the first version of the M. truncatula genome (version 3.5) reported a single MtIRE gene and this was …

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were …

Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin …

Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. So, in our study we have taken an metabolomics approach to identify the rapid response of plants to light stress. In the first part we have focused on the ultrafast (0-90 sec) metabolic response of local tissues to light stress and in the second part we analyzed the metabolic response associated with rapid systemic signaling (0-12 min). Analysis of the rapid response of Arabidopsis to light stress has revealed 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is dependent on an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for …

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 …

In this study, we have used genetic, cell biological and transcriptomic methods in the nematode C. elegans as a model to examine the impact of glucose supplementation during development. We show that a glucose-supplemented diet slows the rate of developmental progression (termed "glucose-induced developmental delay" or GIDD) and induces the mitochondrial unfolded protein response (UPRmt) in wild-type animals. Mutation in the insulin receptor daf-2 confers resistance to GIDD and UPRmt in a daf-16-dependent manner. We hypothesized that daf-2(e1370) animals alter their metabolism to manage excess glucose. To test this, we used RNA-sequencing which revealed that the transcriptomic profiles of glucose-supplemented wildtype and daf-2(e1370) animals are distinct. From this, we identified a set of 27 genes which are both exclusively upregulated in daf-2(e1370) animals fed a glucose-supplemented diet and regulated by daf-16, including a fatty acid desaturase (fat-5), and two insulin-like peptides (ins-16 and ins-35). Mutation of any of these genes suppresses the resistance of daf-2(e1370) to GIDD. Additionally, double mutation of ins-16 and ins-35 in a daf-2(e1370) background results in an increase in constitutive dauer formation which is suppressed by glucose supplementation. Further investigation of the insulin-like peptides revealed that ins-16 mutation in a wild-type background results in upregulation of …

Subsidiary mother cell (SMC) divisions during stomatal complex formation in Zea mays are asymmetric generating a small subsidiary cell (SC) and a larger epidermal cell. Mutants with a high number of abnormally shaped subsidiary cells include the brick1 (brk1) and discordia1 (dcd1) mutants. BRK1 is homologous to HSPC300, an ARP2/3 complex activator, and is involved in actin nucleation while DCD1 is a regulatory subunit of the PP2A phosphatase needed for microtubule generation (Frank and Smith, 2002; Wright et al. 2009). Possible causes of the abnormal SCs in brk1 mutants include a failure of the SMC nucleus to polarize in advance of mitosis, no actin patch, and transverse and/or no PPBs (Gallagher and Smith, 2000; Panteris et al 2006). The abnormal subsidiary mother cell division in dcd1 is due to correctly localized, but disorganized preprophase bands (PPBs; Wright et al. 2009). The observation that brk1 has defects in PPB formation and that the dcd1 phenotype is enhanced by the application of actin inhibitors led us to examine the dcd1; brk1 double mutant (Gallagher and Smith, 1999). We found that dcd1; brk1 double mutants demonstrate a higher percentage of aberrant SCs than the single mutants combined suggesting that these two mutations have …

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, …

Since phages use the host resources to replicate themselves after infection, the different sizes of the phage genome should influence the replication rate. We, therefore, hypothesized that the smaller genomes should burst the cell faster than the larger ones. As well, the shorter genomes would have greater burst sizes because they should replicate faster. Here, we obtained 16 phages of various genome length. All phages were isolated on Streptomyces griseus and available in our phage bank at the University of North Texas. We performed one-step growth studies for the 16 phages, as well as determined the host doubling time from its growth curve. The results show that S. griseus grown in nutrient broth has a doubling time of 5 hours and 22 minutes. This doubling time is used as a guideline for the phage growth studies. Because the filamentous nature of the host caused several difficulties during the experiment, we isolated single cells by sonication and centrifugation. After the cell number was determined by viable cell count, the cells were infected with each type of phage using a multiplicity of infection (MOI) of 0.5. The results show that phages' burst times range between 45 (±0, standard error) and 420 (±30) …

The process of symbiotic nitrogen fixation (SNF) in legume root nodules requires the channeling and exchange of nutrients within and between the host plant cells and between the plant cells and their resident rhizobia. Using a forward genetics approach in the Medicago truncatula Tnt1 mutant population followed by whole genome sequencing, two putative sulfate transporter genes, MtSULTR3;5 and MtSULTR3;4b, were identified. To support the hypothesis that the defective putative sulfate transporter genes were the causative mutation for the mutants' phenotypes, the M. truncatula Tnt1 population was successfully reverse screened to find other mutant alleles of the genes. The F2 progeny of mutants backcrossed with wildtype R108 demonstrated co-segregation of mutant phenotypes with the mutant alleles confirming that the mutated mtsultr3;5 and mtsultr3;4b genes were the cause of defective SNF in the mutant lines mutated in the respective genes. This finding was further established for mtsultr3;4b by successful functional complementation of a mutant line defective in the gene with the wildtype copy of MtSULTR3;4b. A MtSULTR3;4b promoter-GUS expression experiment indicated MtSULTR3;4b expression in the vasculature and infected and uninfected plant cells of root nodules. MtSULTR3;4b was found to localize to the autophagosome membrane when expressed in Nicotiana benthamiana. A transcriptomics study …

In this dissertation I am using M. truncatula as a model legume that forms indeterminate nodules with rhizobia under limited nitrogen conditions. I take advantage of an M. truncatula Tnt1 mutant population that provides a useful resource to uncover and characterize novel genes. Here, I focused on several objectives. First, I carried out forward and reverse genetic screening of M. truncatula Tnt1 mutant populations to uncover novel genes involved in symbiotic nitrogen fixation. Second, I focused on reverse genetic screening of two genes, identified as encoding blue copper proteins, and characterization of their mutants' potential phenotypes. Third, I further characterized a nodule essential gene, M. truncatula vacuolar iron transporter like 8 (MtVTL8), which encodes a nodule specific iron transporter. I characterized the expression pattern, expression localization and function of MtVTL8. Additionally, I characterized several residues predicted to be essential to function using a model based on the known crystal structure of Eucalyptus grandis vacuolar iron transporter 1 (EgVIT1), a homologous protein to MtVTL8. I identified several potential essential residues of the MtVTL8 protein, mutagenized them, and through complementation experiments in planta and in yeast assessed functionality of the resulting protein. This helped us to better understand the potential mechanism by …

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, …

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 …