Browsing by Subject "Metabolomics"
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Item 2-hydrazinoquinoline as a novel derivatization agent for LC-MS-based metabolomic investigation of ketoacidosis in streptozotocin-elicited diabetes(2013-07) Lu, YuweiShort-chain carboxylic acids, aldehydes, and ketones are important intermediates and end products of many metabolic processes. Their levels in biofluids and tissues can reflect the status of specific metabolic reactions, the homeostasis of whole metabolic system, and the well-being of a biological entity. Traditionally, GC-MS has been widely used for analyzing carboxylic acids, aldehydes, and ketones in biological samples after chemical derivatization. However, due to poor compatibility of common GC columns with water, the derivatization process in GC-MS is often complicated and time consuming, especially for the metabolites in biofluids and tissues. Recently, new chemical derivatization techniques have been developed to enhance the sensitivity and performance of LC-MS for analyzing these metabolites. In this study, the use of 2-hydrazinoquinoline (HQ) as a novel derivatization agent for LC-MS analysis of carboxylic acids, aldehydes, and ketones in biological samples was explored, and the conditions for the derivatization reaction were optimized. The metabolites in urine, serum, and tissue extracts can be conveniently derivatized in a 60-min process. The formation of carboxylic acid derivatives is attributed to the esterification reaction between HQ and carboxyl group, while the production of aldehyde and ketone derivatives is through the formation of Schiff bases between HQ and carbonyl group. Compared to other known hydrazine derivatization agents, including 2-hydrazinopyridine, 2-picolylamine and dansyl hydrazine, HQ can react with a broader spectrum of intermediary metabolites in biological samples, and can achieve better chromatographic performance in reversed phase LC system and higher ionization efficiency in electrospray source. Using this HQ-based approach, the metabolic disorder induced by streptozotocin-elicited diabetes was examined by the LC-MS-based metabolomics. The results showed the time-dependent separation of mouse urine samples from STZ treatment in a multivariate model of urinary metabolites. Both known and novel small-molecule biomarkers associated with STZ-induced ketoacidosis were conveniently identified and subsequently elucidated, reflecting the dramatic changes in nutrient (glucose, amino acid, and lipid) and energy metabolism after STZ treatment. Overall, HQ derivatization of carboxylic acids, aldehydes, and ketones can be an effective platform for the LC-MS-based metabolomic investigation of endogenous metabolism.Item Application of Systems Biology Analysis to Hepatic Injury Following Hemorrhagic Shock(2014-05) Determan, Charles Edward JrIntroduction: This dissertation is focused on the metabolomic and transcriptomic changes that occur as a result of carbohydrate prefeeding during hemorrhagic shock and trauma within the liver of a porcine model. The risk of trauma and hemorrhagic shock continues to be an important issue in both military and civilian sectors. As such, we explored the impact of a prior fed state upon the overall response to hemorrhagic shock and resuscitation. The primary hypotheses were that changes in metabolism at the metabolomic and transcriptomic levels would be dependent upon the fed state. In addition, this thesis explores a more comprehensive analysis of metabolomics datasets to standardize analysis and improve overall consistency.Materials and Methods: Algorithm comparison was accomplished using six commonly applied methods to three synthetic datasets, of different sample sizes, and three openly accessible published datasets. This comparison also incorporated metrics to measure consistency of identified features (i.e. stability) to provide further confidence in results. Metabolomics analysis was accomplished with nuclear magnetic resonance spectroscopy (NMR) and Chenomx software to profile and quantify metabolites in liver extracts. The metabolome was subsequently analyzed with partial least squares discriminant analysis (PLS-DA). Transcriptomics analysis was conducted using next-generation sequencing (NGS) technology to employ RNA-sequencing (RNA-seq) on mRNA extracts from liver biopsies. The RNA-seq data was analyzed using typical processing techniques to generate a count matrix and subsequently analyzed with the Bioconductor package EdgeR. Results: The comparison of algorithms showed that the best algorithm is associated with differently structured datasets (e.g. number of features, number of groups, sample size, etc.). Analysis of the liver metabolome revealed changes in carbon energy sources, amino acid metabolism, oxidative stress, and membrane maintenance. Transcriptomic analysis revealed changes in carbohydrate metabolism, cytokine inflammation, cholesterol synthesis and apoptosis. In addition, there is evidence of increased cytoskeleton reorganization which may correspond to a shrunken, catabolic state which provides and anti-inflammatory condition to mitigate cellular damage.Conclusion: The response to hemorrhagic shock and resuscitation is altered with respect to a fasted or carbohydrate prefed state. Metabolomics and transcriptomic analyses suggest altered metabolic pathways as a result of fed state. Altered carbohydrate metabolism was readily identified thereby confirming both methods were successful. Additionally, indications of membrane maintenance that follow cytoskeletal remodeling and cellular shrinkage are potentially reflected by 3-Hydroxyisovalerate and sn-Glycero-3-phosphocholine. These results provide further evidence for pre-conditioning (e.g. altered diet) and hypertonic resuscitation methods to possibly improve patient outcome. Further research is required in alternative prefeeding substrates (e.g. protein, lipid, etc.) as well as improving the integration of different systems level datasets to understand more thoroughly the systemic effects of hemorrhagic shock and resuscitation.Item Diagnostic and Prognostic Test for Periodontitis Using Nuclear Magnetic Resonance and Mass Spectrometry(2021-03) KORMAS, IOANNISAbstract Background: The technological advances in the field of metabolomics have shown promise for the development of a site-specific diagnostic test for periodontitis using the gingival crevicular fluid (GCF). We hypothesize that metabolites, identified through Mass Spectrometry (MS) will distinguish between health and disease. Methods: Ten stage III/IV periodontitis patients were recruited and ten healthy controls and GCF samples from the 6 Ramfjord teeth and 2 randomly selected sites were harvested from subjects with a porous silver disc. The samples were analyzed with Hydrophilic interaction liquid chromatography (HILIC) and reverse-phase liquid chromatography (RPLC) coupled with tandem MS. Statistical analysis was performed using mixed linear models and the p-value was adjusted to control false discovery rate (FDR) using the method of Benjamini and Hochberg. Metaboanalyst 5.0 (www.metaboanalyst.ca) was utilized for pathway analysis. Results: 3,491 metabolites were identified from the sample analysis and 108 could be matched to existing libraries. Those metabolites can be statistically assigned (p<0.05) to 12 human metabolic pathways. Butyric acid was associated healthy sites of healthy subjects, while Thymine, L-Theanine, Cytokinin B, L-Aspartic acid, D-Galactose, Citruline and Stachyose were associated with diseased sites of diseased subjects with an FDR <5%. The remaining identified compounds presented with a higher FDR. More compounds were detected with FDR <5%, but could not be identified due to warranting a targeted approach. Conclusion(s): GCF metabolites identified through MS could be a potential future means to diagnose periodontitis. Butyric acid was associated with periodontal health and thymine, L-theanine, Cytokinin B, L-aspartic acid, D-galactose, citrulline and stachyose were associated with periodontitis. Longitudinal comparisons and a larger sample could show value in the development of a predictive test/model as well.Item Evaluating the chemical diversity and biological activity of plant extracts for commercialization(2014-11) Martin, Amanda C.Perennial plants are a manageable natural resource with the potential to provide both highly valuable biologically active chemicals and ecosystem services. Ecosystem services include various benefits that are provided by an ecosystem such as food, fuel, recreation, as well as water, air, and land quality for society. Biologically active chemicals from plants have a long history of use by humans in botanical medicines and pharmaceuticals, food and dietary supplements, agricultural inputs, and home and personal care products. There are different strategies that can be used to incorporate plants into an economic and ecosystem service role. Method development and application studies were used to facilitate use of plant derived bioactive compounds for commercial use. Methodological studies, using the technique of metabolic fingerprinting, resulted in the determination of extraction conditions that maximize chemical diversity and yield. Maximum chemical diversity in a plant extract was most efficiently approached if solvent partitioning was performed on an extract made with 70 percent ethanol. Additionally, strategies to integrate extract chemical analysis with information regarding extract quality, such as cytotoxicity measurements, were developed and used to evaluate commercial kava samples obtained from multiple sources. These approaches were then applied to two different perennial plant species (Comptonia peregrina, and (Glycyrrhiza lepidota,) with the aim of developing their commercial value based on their extractable chemical composition. These studies resulted in the isolation of two small molecules from (C. peregrina, with strong antimicrobial activity and the identification of two (G. lepidota, populations with the potential to be developed into a cultivar with optimal characteristics for the cultivation of biologically active compounds.Item A Fungal secretome tailored to enable a radical (oxidative) wood decay mechanism in brown rot fungi(2021-07) Castano Uruena, JesusWood is one of the most important carbon sinks on earth, and it is composed mainly of cellulose, hemicellulose, and lignin, which together form what is known as the lignocellulosic complex. The lignin component in wood is highly recalcitrant and prevents wood from being degraded by most organisms. Wood degrading fungi have evolved mechanisms to handle the lignin barrier effectively and harness the carbon present in wood, which makes them also plausible biological templates for industry-related applications such as the production of biofuels. However, there are still several knowledge gaps about how fungi orchestrate the complex mechanisms behind wood decay, which prevents further usage of these fungi in biotechnological fields. Wood degrading fungi were initially classified by the physical properties of the rotted wood as white, brown, and soft rot fungi – with brown and white rot fungi being the most efficient. These properties are correlated with important genetic and regulatory differences that distinguish the different modes of decay. Recently, it was shown that brown rot fungi evolved from white rot fungi losing an important number of carbohydrate-degrading enzymes (CAZy) and oxidoreductases. This loss was accompanied by regulatory changes that included the overexpression of retained CAZys, and the development of a two-step mechanism that segregated a Fenton-based oxidative phase from a hydrolytic phase in early and late wood decay, respectively. However, although some main differences between white and brown rot fungi have been identified, several details remain obscure. For example, it is unknown how brown rot fungi manage to produce highly oxidative and unspecific hydroxyl radicals while producing some glycosyl hydrolases necessary for the depolymerization of pectin and hemicellulose at early decay stages. It seems reasonable, that brown rot fungi could protect their enzymes by making them more naturally tolerant of reactive oxygen species (ROS) radicals than other wood degraders associated with a different rot type. Also, although transcriptomics and proteomics data suggest brown rot fungi use some Fenton chemistry at early decay, there is little information about how these fungi regulate the concentration of the chemical species that enable this chemistry. This dissertation contributes to widen the knowledge about fungal wood decay mechanisms, especially those used by brown rot fungi. For starters, by studying the wood decay progression with the brown rot fungus Rhodonia placenta, we found that this fungus displays different mechanisms to harness the use of ROS during wood decay without inflicting damage on itself. First, R. placenta controls the extracellular production of ROS by regulating the concentrations of H2O2 and Fe2+ in the media (avoidance mechanism). Second, this fungus presents a high antioxidant capacity as decay progresses, potentially to quench any possible leaks of ROS from earlier decay stages (suppression mechanism). Thirdly, several R. placenta secreted CAZys, important for early wood decay, displayed tolerance of high concentrations of ROS compared to the soft rot fungus Trichoderma reesei (an industrially relevant cellulase producer), which enables these enzymes to work under harsh operating conditions (tolerance mechanism). Collectively, this indicates that R. placenta uses avoidance, suppression and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood. After finding these results, we decided to incorporate a white rot fungus (Trametes versicolor) in the tolerance comparison and observed similar results, with tolerance of ROS only present in the side-chain hemicellulases of R. placenta. Proteomics analysis, meant to examine the presence of oxidative modifications induced after an in-vitro oxidative treatment, revealed that not only side-chain hemicellulases but also several other enzymes such as laccases, glutathione-S-transferases, and proteases were differentially tolerant of ROS compared to white and soft rot fungi. This suggest that the fungal secretome in brown rot fungi has been tailored as a whole to better endure the presence of ROS. Also, we found that several of the oxidative modifications that occurred in the glycosyl hydrolases of T. reesei and T. versicolor happened in amino acid residues in the vicinity of the active site, which can be linked to the loss of enzyme activity after the oxidative treatment. In a follow-up study addressing the effects of these modifications, we used molecular dynamics to understand the effect of some of the oxidative modifications in an α-L-arabinofuranosidase of T. reesei. Even though the number of oxidative modifications that we could include in the modeled protein was limited due to the lack of force field parameters, the simulations still showed some of the negative potential outcomes when a number of amino acid residues become oxidized. For instance, there were significant alterations of the conformational stability of the protein when oxidized, as evidenced by changes in root mean square deviation (RMSD) and principal component analyses (PCA) trajectories. Likewise, enzyme-ligand interactions such as hydrogen bonds were greatly reduced in quantity and quality in the oxidized protein. In addition, free energy landscape plots showed that there was a more rugged energy surface in the oxidized protein, implying a less favorable reaction pathway. Collectively, these results revealed the basis for the loss of function in the α-L-arabinofuranosidase of the commercially-relevant fungus T. reesei. Finally, metabolomics experiments were carried to find out whether the different modes of decay translated into signature metabolite profiles that could be assigned to either brown or white rot fungi. For this purpose, we cultured two brown rot fungi (R. placenta and Gloephylum trabeum) and two white rot fungi (Pleurotus ostreatus and T. versicolor). The results showed that brown rot fungi have a distinct metabolite pattern at late decay stages that clearly distinguish them from white rot fungi. Different metabolites such as organic acids, sugars, pyranones, and furanones contributed to this result. The finding of several pyranones and furanones being differentially more abundant in brown rot fungi was interesting since it agrees with the expansion of polyketide synthase genes in brown rot fungi. In contrast to brown rot fungi, we could not find a lot of similarities in white rot fungi as deduced by the PCA plots and heatmaps. However, some commonalities were evident such as the presence of galactitol as a potential biomarker, and the higher efficiency of these fungi at removing phenolic compounds originally found in undecayed wood. When focusing on both types of decay, we found that wood degrading fungi tend to accumulate sugars and carboxylic acids at late decay stages. Also, as fungal decay progresses, we observed an accumulation of different furans such as furfural or 5-methylfurfural in all fungi.Item Improving Sustainability of Perennial Ryegrass (Lolium perenne) Seed Production: Integrated crop management practices and the development of a novel metabolomics-assisted technique to select for resistance to rust (Puccinia) pathogens(2014-05) Koeritz, EricThis project focused on three main areas of study which will have a significant impact on the perennial ryegrass seed industry and end users. They are: 1) the effects of alternative nitrogen and growth regulator management practices including their interaction on crop agronomic characteristics and stem rust , 2) the effect of seeding rate and row spacing and their interaction with N rate on crop agronomic characteristics, competition with weeds, and stem rust and 3) the development of a novel breeding strategy (metabolomics-assisted selection) to help with faster and more accurate selection for crown rust resistance in perennial ryegrass. To study the effects of alternative growth regulator and nitrogen management practices, experiments were conducted during 2009 and 2010. Three N application methods (single, two-split, and three-split), five growth regulators (split and single applications of prohexadione Ca or trinexapac ethyl) and two spring N rates (56 or 100 kg N ha-1) were evaluated in a split-split plot design. Seeding rates (SRs) and row spacing widths (RSWs) in perennial ryegrass seed production are variable and the impacts of these factors on perennial ryegrass seed yield and plant growth characteristics are not well characterized. Experiments were conducted during 2010 and 2011 where five seeding rates [1.3, 2.6, 5.2, 7.8, and 10.4 kg PLS (pure live seed) seed ha-1], three row spacing widths (10, 20, and 30 cm), and three N rates (67, 112, and 157 kg N ha-1) were evaluated. The goal of metabolomics-assisted selection research was to detect a set of metabolic biomarkers to be used for fast and accurate selection of crown rust resistant perennial ryegrass genotypes. These chemical biomarkers will serve as a "metabolic fingerprint" associated with crown rust resistance in perennial ryegrass. Termed metabolomics-assisted breeding, this technique will lead to faster cultivar development and ultimately reduce fungicide use, making perennial ryegrass seed production a more profitable, marketable and sustainable option for farmers in rural communities in northern Minnesota as well as the Pacific Northwest and Canada.Item Metabolic alterations associated with hemorrhagic shock and traumatic injury as measured in the urine.(2011-12) Lusczek, Elizabeth RoseIntroduction: This dissertation focuses on profiling the changes in metabolism that occur as a result of hemorrhagic shock and traumatic injury as observed in urine obtained from a porcine model. Hemorrhagic shock and traumatic injury are responsible for the majority of deaths under the age of 44. A porcine model of hemorrhagic shock and traumatic injury was used to examine changes in metabolism due to hemorrhagic shock and traumatic injury. Changes in metabolism were hypothesized to be dependent upon the metabolic state the animal occupies upon injury (fed or fasted). It was also hypothesized that a metabolite or metabolites could serve as a marker of mortality. Materials and Methods: Nuclear magnetic resonance (NMR) spectroscopy and Chenomx software were used to generate individual profiles of metabolite concentrations (the metabolome) for each urine sample obtained. Because hemorrhagic shock produces drastic changes in urine concentration and large fluctuations in the concentrations of endogenous metabolites in the urine, urine must be normalized to urine output to allow for accurate comparisons between urine samples. The urinary metabolome was analyzed with partial least squares discriminant analysis (PLS-DA) and with a scale-free network. Results: PLS-DA models showed that metabolites associated with respiration, ischemia/reperfusion injury, and cell membrane damage were observed in the urine two hours after the initiation of resuscitation. Other metabolites which may be associated with injury sustained from surgical preparation were observed at 20 hours after the initiation of resuscitation. The metabolites 1,6-Anhydro β-D-glucose and mannose were associated with the metabolome of fed animals. The network analysis of the metabolome is reflective of the scale-invariant nature of metabolism. Respiratory metabolites were separated by whether they are involved in aerobic or anaerobic respiration. Anaerobic metabolites were also associated with markers of cell membrane rupture. Lactate, pyruvate, 2-oxoglutarate, succinate, taurine, glycine, dimethylamine, and creatinine were identified as well-connected hub metabolites in the network. No metabolites were associated with survival alone. Conclusions: The pathophysiology associated with hemorrhagic shock can be treated as a scale invariant process particularly with respect to metabolism. Results from a traditional metabolomics treatment of the data with PLS-DA models were congruent with results from a scale-invariant, weakly modular network of the urinary metabolome. Other samples obtained from these experiments (muscle, liver, and serum) could benefit from a similar treatment. Additional work in (a) integrating the metabolomes of all four biological samples and (b) creating a physics-based theory of hemorrhagic shock may provide important information about mortality and the propagation of injury.Item Origin, composition, and role of antimicrobial plant resins collected by honey bees, Apis mellifera(2014-05) Wilson, Michael BradleyThe use of plant resins in nest building by honey bees, Apis mellifera, is an oft ignored, but critical behavior to bee health. Termed `propolis' by beekeepers, deposited resins in the nest have many positive physiological effects on the colony. Honey bees are feral and abundantly managed in many regions of the United States, from the Sonoran Desert to icy Minnesota, yet a diversity of very different resinous plants exist in every environment they call home. We know very little about what resinous plants bees utilize in these different regions, or what benefits bees might derive from specific plants. It is thought that the antimicrobial properties inherent in resins, which are complex mixtures of phenolic and isoprenoid compounds, are important drivers of their derived benefits to bees. The research herein focuses on creating better methods to track resin forager behavior, and then using those methods to discover the botanical sources of bee-foraged resins, while also exploring how resins from different plants directly affect the growth of two bee pathogens, the gram-positive bacteria Paenibacillus larvae and the fungus Ascophaera apis. I found that individual resin foragers can be chemically tracked to their resinous plant targets using metabolomic methods that hold great advantages over traditional chemical analyses, and that there is much diversity in the ability of resins from different Populus spp. to inhibit the in vitro growth of P. larvae and A. apis. I go on to further explore the benefits of different resins and find that propolis from Fallon, NV was particularly active against P. larvae and A. apis out of samples from 12 different regions in the U.S. Finally, I used bioassay-guided fractionation against P. larvae to isolated several flavanone-3-alkyl esters from NV propolis that displayed very high activity (IC50 = 17 µM to 68 µM) against P. larvae and A. apis. Re-examination of data from my previous studies indicated that these compounds were strong contributors to overall anti-P. larvae activity in regional propolis samples, and that Populus spp. are likely the botanical sources of these compounds.Item Peri-implantitis Prognosis Using Metabolomic Biomarkers in Peri-Implant Crevicular Fluid: A Longitudinal Study(2021-05) Alassy, HatemBackground: Peri-implant diseases, peri-implantitis (PI) and peri-implant mucositis (PIM), are highly prevalent in subjects with dental implants. Despite this prevalence, diagnosing peri-implant disease (PID) remains challenging because of lack of accuracy and precision of periodontal probing and dental radiographs. Furthermore, these diagnostic tools document history of disease rather than current disease activity. There is no current model to predict the progression of PID. Biomarkers are commonly used in medicine to objectively determine disease state, or responses to a therapeutic intervention. Biomarkers in peri-implant crevicular fluid (PICF) show promise in their diagnostic and prognostic value. Metabolomic analysis of PICF quantifies molecules associated with host and bacterial metabolism may reflect on pathophysiology of peri-implantitis. Un-targeted metabolomics allows the discovery of unknown biomarkers without bias to correlate them with peri-implantitis and its progression. Aim: We hypothesize that the simple metabolites in PICF are predictors of future peri-implantitis progression. We aim to define the unique set of metabolites in the PICF that establish a reliable method for early prediction of bone-loss progression in peri-implantitis. Methods: Clinical and radiographic examinations and PICF samples were collected from 130 implants in 71 subjects at baseline, 6, 12, 18 and 24 months. At baseline, 59 implants were healthy (bone loss < 2mm; PD 4mm), 33 implants had PI (bone loss ≥ 3mm; PD ≥ 6mm) and 38 other implants had bone loss ≥ 2mm and <3mm and PD 5mm. Radiographic bone level changes of 112 implants and relative metabolites in PICF samples were measured at each 6 months interval using proton nuclear magnetic resonance (H-NMR) spectroscopy. MetaboAnalyst 5.0 software correlated metabolite levels with radiographic bone changes of ≥ 1mm within a 6-month interval. Results: In the cross-sectional component at baseline, univariate ROC curve analysis demonstrated that the Cadaverine/Lysine signature was significantly correlated with peri-implantitis (AUC= 0.76; 95% CI 0.658-0.855, p< 0.000) versus healthy implants. While alpha ketoglutarate was significantly correlated with healthy implants (AUC= 0.706; 95% CI 0.593-0.819; p= 0.002). In the longitudinal component, the metabolite levels in PICF of untreated diseased implants that demonstrated progressive radiographic bone loss of ≥ 1mm within a 6-month interval (group A, n=6) were compared to the metabolites of healthy-non-progressing implants (group B, n=26) and to diseased-non-progressing implants (group C, n=8). Proline and 1-3-diaminopropane levels could predict future bone loss of ≥ 1mm (AUC= 0.917 and 0.854 respectively) whereas glucose and arginine levels could predict the absence of bone loss in group C and group B respectively (AUC= 0.896 and 0.801) although statistical significance was not reached for all 4 metabolites. Biotin and propionate levels were higher in group C compared group A and group B (ANOVA p< 0.001; AUC biotin= 0.889; AUC propionate= 0.87). Valine levels were higher in both group A and group C compared to group B (p= 0.002; AUC= 0.841). Conclusions: PICF metabolites identified using H-NMR spectroscopy mapped a specific metabolomic profile able to identify implants with peri-implantitis versus healthy implants with moderate accuracy. Furthermore, specific metabolites discriminated between progressive disease versus non-progressing disease status and health.Item Resolving the major dyslipidemia phenotypes and genetic risk factors for familial hyperlipidemia in Miniature Schnauzers(2022-09) Tate, NicoleHyperlipidemia is common in the Miniature Schnauzer breed, especially those over the age of 10. Hyperlipidemia is defined as an increased concentration of lipids (i.e., triglycerides and/or cholesterol) in the blood. Hyperlipidemia predisposes Miniature Schnauzers to severe consequences such as pancreatitis, gallbladder mucoceles, and glomerular proteinuria. However, the underlying molecular derangements and cause remains unresolved. It is suspected that hyperlipidemia in Miniature Schnauzers is due to an underlying genetic risk factor(s). Additionally, the varied responses to management strategies in the breed, suggests a potential for multiple subtypes. Thus, the goal of this thesis was to identify the spectrum of dyslipidemia subtypes and ascertain the metabolic and genetic risk factors underlying hyperlipidemia in Miniature Schnauzers. The possibility of multiple dyslipidemia subtypes within the breed was evaluated using lipoprotein profile data and unsupervised hierarchical cluster analysis. The results support the hypothesis that multiple dyslipidemia subtypes exist in Miniature Schnauzers and that the major distinguishing factor between the subtypes may be differences in low-density lipoproteins. Additional studies are warranted to confirm the range and number of distinct lipoprotein profiles within this breed. The lipidome and metabolome of Miniature Schnauzers with moderate-to-severe primary hyperlipidemia were compared to those from Miniature Schnauzers with normal serum triglyceride concentrations to elucidate the underlying pathophysiological processes of hyperlipidemia in the breed. Differences in the lipidome and metabolome were identified between the two groups. The differentiating lipid and metabolite species suggest involvement and/or disruption of the pathways and products of glycerolipid, glycerophospholipid, glycosphingolipid, and fatty acid metabolism. The results of this study provide insights into the underlying pathways. However, it is still unknown whether these pathways are causal of hyperlipidemia or if the disturbances are in response to elevated triglyceride (TG) concentrations. This thesis also used unsupervised hierarchical cluster analysis to compare the lipidome and metabolome of Miniature Schnauzers dogs with normal serum triglyceride concentrations, mild triglyceride elevations, moderate-to-severe triglyceride elevations, and triglyceride elevations due to endocrinopathies (i.e., secondary hyperlipidemia). The most notable finding being that Miniature Schnauzers with mild HTG cannot be definitively classified as having primary HTG, as their lipid disturbances do not reliably differ from dogs with NTG. Whole genome sequencing (WGS) of eight Miniature Schnauzers with primary hyperlipidemia was screened for risk variants in six HTG candidate genes: LPL, APOC2, APOA5, GPIHBP1, LMF1, and APOE. A monogenic cause for primary hyperlipidemia in the breed was not identified in the evaluated candidate genes. Two variants passed the filtering criteria, a deletion in the TATA box of APOE and a missense variant in GPIHBP1. While the two variants did not have sufficient evidence to support a strong impact, neither can be ruled out as contributors to the disease. These findings, and the growing data on dyslipidemia subtypes in Miniature Schnauzers, suggest that hyperlipidemia in the breed is likely a polygenic or complex trait. Finally, a key challenge in genetic studies is the prioritization of identified variants. Many in silico tools have been developed to use features of amino acids and proteins to determine if a variant is likely pathogenic. However, these methods are typically trained using human variants and have not been validated for use in non-human species. Thus, this thesis evaluates the performance of eight tools for pathogenicity prediction of missense variants (MutPred2, PANTHER, PhD-SNP, PolyPhen2-HumDiv, PolyPhen2-HumVar, Provean, SIFT, and SNPs&GO) for use in the dog and horse. The findings of this study suggest that these methods can be effectively used in veterinary species.Item Revisiting Some Useful Analytical Methods From the Past: New Applications of Existing Tools Under the Metabolomics Paradigm(2022-12) Brockman, StephenThanks to a confluence of several technological advancements over the past severaldecades metabolomics has grown into a prominent field of research. However, its advancement has suffered from a lack of tools. This has been especially problematic as metabolomics increasingly spreads to gas chromatography-mass spectrometry-based studies. In order to help overcome some of these challenges three studies were performed. First, an informative visualization technique originally developed by van Krevelen for petrochemical analysis was updated for high-resolution mass spectroscopy data from metabolomics experiments. The resulting script, OpenVanKrevelen, includes an empirically derived metabolite map and has been demonstrated using experimental metabolomics data. Second, a standard volatile organic compound sampling technique for gas chromatography called solid phase microextraction was adapted for cost effective high-throughput sampling that can be implemented in field settings for metabolomics analysis. Finally, stable isotope labeling and a traditional liquid-liquid extraction method were employed to investigate smoke taint in hybrid Vitis berries for the first time. Results were largely consistent with those from previous studies in V. vinifera demonstrating the susceptibility of hybrid cultivars to this economically damaging phenomenon. Together, all three studies show that while metabolomics has emerged from new technologies it can also benefit greatly from cleverly repurposed tools from the past.Item The Role of Hepatocyte D-Β-Hydroxybutyrate Dehydrogenase In Ketone Body Metabolism And Liver Health(2021-09) Stagg, DavidThroughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. This thesis seeks to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB). Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, dietary-induced mouse models of nonalcoholic fatty liver disease (NAFLD), and complementary physiological approaches in novel cell type-specific knockout mice, the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies, are quantified. Exogenously administered AcAc is reduced to D-βOHB, and increases hepatic NAD+/NADH ratio, reflecting hepatic BDH1 activity. Livers of hepatocyte-specific BDH1 deficient mice produced no D-βOHB, but due to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte specific BDH1 deficient mice, maintained on either a chow or NAFLD-inducing western-style diet, showed diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis but normal overall hepatic energy charge. Furthermore, the livers of knockout mice maintained on a 60% high fat diet were less fibrotic, with reduced markers of oxidative stress, than littermate controls. Collectively, this thesis illustrates how ketone bodies and BDH1 activity influence liver homeostasis and health. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production, with a beneficial effect on liver fibrosis. Therefore, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis, and augmentation of hepatic BDH1 activity could prove beneficial in the treatment of NAFLD.Item Serum metabolomics proton NMR data from a porcine model of burn injury(2018-08-22) Lusczek, Elizabeth R; Beilman, Gregory J; lusc0006@umn.edu; Lusczek, Elizabeth RBackground: Burn injury initiates a hypermetabolic response that leads to muscle wasting and organ dysfunction in burn patients. We examined the transition between “ebb” and “flow” in post-burn metabolism using proton nuclear magnetic resonance (1H-NMR) spectroscopy and serum from a porcine model of severe burn injury. We hypothesized serum metabolomes of porcine subjects would be distinguishable by time point, and changes in individual metabolite concentrations would characterize the shift from ebb to flow after burns. Methods: Fifteen pigs received 40% total body surface area (TBSA) burns with additional pine bark smoke inhalation. Arterial blood was drawn at baseline (pre-burn) and every 24 hours until 72 hours post-injury or death. The aqueous portion of each serum sample was analyzed using 1H-NMR spectroscopy and metabolite concentrations were used for principal component analysis (PCA). Results: Thirty-eight metabolites were detected in 39 samples. Thirty-one showed significant concentration changes between time points (p<0.05). PCA captured 51.6% of metabolite variability in the first two principal components and revealed clustering of samples by time point on a 2D scores plot. The first 48 hours post-burn, indicative of the shift from ebb to flow, were characterized by high concentrations of histamine, alanine, phenylalanine, and tyrosine. Later time points were characterized by rising concentrations of 2-hydroxybutyrate, 3-hydroxybutyrate, acetoacetate, and isovalerate. Conclusions: Post-burn metabolomes of porcine subjects were distinguishable by time point using 1H-NMR and principal component analysis. Our work identifies metabolites indicative of the transition from the ebb to flow phases of the response to burn injury, while also highlighting the accumulation of organic acids resulting from fatty acid catabolism and oxidative stress. Further studies will be required to elaborate on the post-burn metabolic pathways suggested by this analysis.