Maize (Zea mays L.) is a global food staple and is at risk from infection by the pathogenic fungus Aspergillus flavus L. The ubiquitous, soil-borne fungus causes ear rot of maize and produces the carcinogenic secondary metabolite known as aflatoxin. Aflatoxin B1 is the most potent carcinogenic mycotoxin known, causing hepatocellular carcinoma, along with many other serious health problems such as immunosuppression. Previous studies have shown that maize cob tissue plays an essential role in both facilitating and limiting the spread of the fungal pathogen A. flavus, however, little attention in the literature has been given to the cob. To date, there have not been any studies published describing the metabolome of maize cob tissue. This study assessed three different methods for disruption of maize cob tissue and investigated the global metabolome of maize cob of two resistant (Mp313E and Mp420) and two susceptible (B73 and SC212m) genotypes via liquid chromatography-mass spectrometry. Three treatments (control, water-inoculated, and fungus-inoculated) and three-time points (3, 9, 15 days after inoculation) were included in the experimental design. For the first time in maize cob, 69 metabolites were identified via the mzCloud online database. Out of them, 28 metabolites showed statistically significant differences in abundance across the treatments. Twenty-two metabolites were identified via Fragment Ion Search, of which 14 were statistically significant differences in abundance across the treatments. The majority of the metabolites identified where from the phenylpropanoid, linoleic acid, and terpenoid biosynthesis pathways. Thousands of unknown ions were detected, and for 521 compounds the formula could be derived, based on accurate monoisotopic masses. In the targeted metabolomics analysis, the MS3 spectral tree was obtained for zealexin B1 for the first time via a highly induced sample and was subsequently used to identify zealexin B1 in maize cobs (Va35). To date, this work is the sole metabolomic profiling study of maize cob tissue, and it provides insight into constitutive and induced molecular antifungal defenses of resistant and susceptible genotypes. A list of significant fungal-induced metabolites related to the maize-A. flavus defense response was compiled for further targeted metabolomic identification.

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Metabolomics is the systematic analysis of low-molecular weight compounds (metabolites) within biological systems that represent molecular endpoints of gene expression and environmental exposures. A major goal of metabolomics is achieving better understanding of the pathophysiology of complex disease processes while elucidating mechanisms of action of nutrients, toxins, and/or drugs. Multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) is a high-throughput microseparation platform that is ideal for the analysis of polar/ionic metabolites from volume-restricted biological samples. This thesis includes two major metabolomics projects using MSI-CE-MS that are aimed at contributing new advances in public health and chronic disease prevention. Chapter II presents an analysis of the metabolome from patients with phenylketonuria (PKU) — a genetic disease affecting phenylalanine (Phe) metabolism that requires lifelong dietary restriction to prevent irreversible intellectual disabilities. A targeted and nontargeted metabolomics approach using matching urine and plasma samples was conducted to confirm known markers of PKU and identify new markers associated with dietary adherence and disease progression. Along with increased excretion of Phe catabolites in urine, high plasma Phe was associated with decreased excretion of acylcarnitines and greater excretion of histidine catabolites, suggesting impaired fatty acid oxidation and micronutrient deficiencies, respectively. Overall, this may provide a strategy to objectively monitor dietary adherence beyond standard dietary records or patient recall. Chapter III investigates the impact of electronic cigarette smoke exposure on the placental metabolome as a model cell line of fetal development. Evidence of altered amino acid metabolism, in addition to changes in acylcarnitines and metabolites associated with cellular proliferation, were observed in more susceptible first trimester placental cells and were attributed to flavouring agents irrespective of nicotine dosage. This work supports the hypothesis that flavoured e-cigarette formulations pose a significant health risk in comparison to unflavoured formulations and supports the need for further risk assessment and careful regulation of these products to prevent deleterious birth outcomes in pregnant mothers. / Thesis / Master of Science (MSc)

In the post-genomic era, one of the most important goals for the community of plant biologists is to take full advantage of the knowledge generated by the Arabidopsis thaliana genome project, and to employ state-of-the-art functional genomics techniques to assign function to each gene. This will be achieved through a complete understanding of what all cellular components do, and how they interact with one another to produce a phenotype. Among the proteins encoded by the Arabidopsis genome are 24 related carboxyl methyltransferases that belong to the SABATH family. Several of the SABATH methyltransferases convert plant hormones, like jasmonic acid, indole-3-acetic acid, salicylic acid, gibberellins, and other plant constituents into methyl esters, thereby regulating the biological activity of these molecules and, consequently, myriad important physiological processes. Our research aims to decipher the function of proteins belonging to the SABATH family by applying a combination of genomics tools, including genome-wide expression analysis and gas-chromatography coupled with mass spectrometry-based metabolite profiling. Our results, combined with available biochemical information, provide a better understanding of the physiological role of SABATH methyltransferases, further insights into secondary plant metabolism and deeper knowledge of the consequences of modulating the expression of SABATH methyltransferases, both at the genome-wide expression and metabolite levels. / Ph. D.

Forensics face a complicated problem when evaluating intoxications induced by opioids and non – intoxications of opioid abusers since the in vitro concentrations of the said opioid overlap. Researchers found that acylcarnitines role as biomarkers for a diversity of diseases may also be used as biomarkers postmortem, easing the complications that occurs of evaluating the cause of death.  A reversed phase ultra high performance liquid chromatography (UHPLC) method in combination with mass spectrometer detection was developed for a quantitative analysis of different acylcarnitines in authentic blood samples. The hypothesis investigated was the altercation of acylcarnitine concentration depending on the cause of death, specifically when induced by opioids. Separation was achieved using ACQUITY UPLC HSS T3 1.8 µm (2.1 x 100 mm) Waters column along with a gradient elution consisting of Mobile phase A: 0.05% HFo in 10 mM Ammoniumformate and Mobile phase B: 0.05% HFo in Methanol. Flowrate was 0.4 mL/min. The method was validated in respect to linearity and range, accuracy, precision, LOD and LOQ as well as stability and degradation of acylcarnitines. Linearity was acceptable with R2 – values   for all the substances. Results from the authentic sample analysis showed no statistically significant difference between the investigated groups based on Kruskal – Wallis non-parametric tests and median comparison, however a trend in the data was found correlating to the investigated hypothesis suggesting it may be true.

Intracellular sterol redistribution is an important step in the lipid homeostasis of organisms, a process directly linked to the organizational arrangement in the plasma membrane (PM) of cells. Previous studies in the budding yeast Saccharomyces cerevisiae have demonstrated that the ARV1 (ACAT-related enzyme-2 required for viability 1) protein is a major regulator of sterol transport from the endoplasmic reticulum to the plasma membrane, contributing to the structural organization of the PM, rendering it resistant to anti-fungal compounds as well as maintaining ER integrity. This study assessed the significance of ARV1 in the plant pathogenic fungus Cryphonectria parasitica (Cparv1) and investigated its role in the pathogenesis and virulence of the fungus. C. parasitica is the causative agent of Chestnut blight, which has wreaked havoc on the American chestnut species. Genomic analysis revealed that the Cparv1 gene is very closely linked to another gene that putatively encodes a cyanamide hydratase (Cpcah). An initial gene deletion event resulted in the elimination of both genes and a highly deformed phenotype in C. parasitica that was fully recoverable by complementation. PCR-based expression analysis determined that the lack of Cparv1 was responsible for the debilitated phenotype of the double mutant, with no transcript detectable from Cpcah. Subsequent complementation of the Cparv1 gene was also observed to restore the wildtype phenotype. Mass spectrometry-based (MS) results indicated a decrease in sterol content of the DCparv1 mutant strain compared to wildtype EP155 thus confirming a role for Cparv1 in sterol homeostasis. It has been shown that infection of C. parasitica with virulence-attenuating hypoviruses altered intracellular lipid content and protein secretion. Ultrastructure studies conducted on the Cparv1 strain showed disrupted organelle integrity and the presence of cytoplasmic double membrane stretches. Decreased sterol content in C. parasitica infected with CHV1-EP713 was observed similar to DCparv1 suggesting a connection between the hypovirus-infected phenotype and Cparv1. Furthermore, a non-targeted metabolomic study on all three strains identified 324 metabolites. Through the subsequent pathway analysis, we have investigated the pleiotropic effects in the C. parasitica strains and established a mechanistic linkage between this the activity of the ARV-1 protein and the hypovirus-infected phenotype.

Background: A significant portion of lactating women in less developed countries have vitamin A (VA) deficiency. Lactation has substantial effects on a mother’s metabolism and VA is known to be needed in multiple biological processes, including growth, vision, immunity, and reproduction. Objective: The objective of this pilot study was to utilize metabolomics profiling to conduct a broad, exploratory assessment of differences in plasma metabolites associated with low VA status versus adequacy in lactating women. Methods: Plasma samples from lactating women who participated in a survey in Samar, Philippines, were selected from a cross-sectional study based on plasma retinol concentrations indicating low (VA-; n=5) or adequate (VA+; n=5) VA status (plasma retinol <0.7 or >1.05 µmol/L). The plasma results collected from six metabolomics assays (oxylipins, endocannabinoids, bile acids, primary metabolomics, aminomics, and lipidomics) were compared by group, using liquid chromatography mass spectrometry. Results: Twenty-eight metabolites were significantly different in the VA- versus VA+ status, with 24 being lipid mediators (p<0.05). The lipid mediators demonstrated lower concentrations of the arachidonic acid- and eicosapentaenoic acid-derived oxylipins, as well as lysophospholipids and sphingolipids, in the VA- group (p<0.05). Chemical similarity enrichment analysis identified HETEs, HEPEs, and DiHETEs as significantly different oxylipin clusters (p<0.0001, false discovery rate (FDR) p<0.0001), as well as sphingomyelins, saturated lysophosphatidylcholines, phosphatidylcholines, and phosphatidylethanolamines (p<0.001, FDR p<0.01). Conclusions: The multi-assay nutritional metabolomics profiling of low VA status compared with adequacy in lactating women demonstrated reduced lipid mediator concentrations. Future studies with stronger study designs and a large and more diverse population are needed to validate these preliminary results.