Thesis Ph. D. Michigan State University. Chemistry 2014.

The main bottleneck in today's plant metabolomics lies with the identification of new metabolites. A number of plant metabolite databases that report liquid chromatography - mass spectrometry data have been constructed. However, more than 95% of compounds reported in these databases remain unannotated. The extensive range of unknown metabolites presents a significant challenge in interpreting metabolome data, and therefore developing methods that accelerate annotation and identification of previously unknown metabolites has great importance when metabolome data are used for functional genomic research. The challenge of metabolite annotation was addressed by using relative mass defect (RMD) filtering of ion masses measured using liquid chromatography-mass spectrometry. Such calculated RMD values reflect the fractional hydrogen content of each detected ion, and reflect the biosynthetic precursors and transformations that generate metabolites in vivo. RMD filtering aids grouping of compounds of similar relative mass defect independent of absolute mass and chromatographic retention time. Therefore, metabolites and metabolite precursors are grouped together enabling potential associations among related metabolites to be developed. Furthermore, a systematic variation of RMD among the fragment/product ions observed in multiplexed collision-induced dissociation (CID) MS or liquid chromatography-tandem mass spectrometry (LC-MS/MS) data for compounds of interest allowed for the identification of terpene glycosides in complex matrices. However, once the metabolites are annotated in metabolomics data sets, establishing the structure of these compounds requires the purification of the compound followed by de novo structure elucidation that relies heavily upon 1D and 2D NMR.Chapter 2 of this dissertation discusses the application of RMD filtering based data analysis to both parent and fragment ions generated in LC-multiplexed CID MS metabolite profiles generated from wild tomato species Solanum habrochaites LA1777. This resulted in the discovery of over 24 novel sesquiterpene glycoside chemical formulas, with multiple isomers comprising a group of more than 200 sesquiterpenoid glycosides. Chapters 3 and 4 of this dissertation discuss the purification and de novo structure elucidation of seven example compounds from wild tomato glandular trichomes using NMR. The structures of the sesquiterpenoid cores established for these compounds are different from the structures of known volatile sesquiterpenoid compounds found in S. habrochaites LA1777 suggesting that the synthesis of these non-volatile terpenoids involves different biosynthetic enzymes from those involved in the synthesis of known volatile terpenoids. Similarly, Chapter 5 of this dissertation discusses the application of these techniques to the analysis of metabolite profiles of the medicinal plant Hoodia gordonii generated using LC-multiplexed CID MS. This research led to the identification of 24 novel diterpene…

Thesis Ph. D. Michigan State University. Chemistry 2013.

PROFILING AND DATA PROCESSING STRATEGIES FOR PEPTIDOMIC ANALYSISBySiobhan Shay Peptide functions have been underappreciated compared to proteins. With a few notable exceptions, peptides have been thought to function largely as degradation intermediates between proteins and individual amino acids, but there is growing recognition that peptides have their own significant biological functions. Peptides, from both endogenous and exogenous sources, including diet, play essential roles regulating beneficial and detrimental physiological functions. Some bioactive peptides are unusual in that to exhibit physiological functions, they show resistance to proteolytic enzymes. The elucidation of identities of bioactive peptides is essential for determination of their abundances, structures and functions. Peptidomics, the analysis and description of total peptide content within a biological sample, however, is often hindered both by the nature of the peptide and current analytical approaches. Peptides are exposed in vivo to multiple proteolytic enzymes, and the constituents of the resulting digestion-resistant and bioactive peptidome are not guaranteed to have a specific N- or C-termini. As a result, digestion-resistant peptides often lack terminal amino acids with basic side chains that yield mass spectrometric fragments sufficient for their identification. In addition, bioactive peptides are also hindered by the effects of chromatographic co-elution, as peptidomes are more complex than can be resolved in a single liquid chromatographic separation. To mitigate the challenges that inhibit peptidomic analyses from detecting, identifying and characterizing the maximum number of peptides within a complex biological sample, this dissertation describes an alternate method that applies metabolomic-like data processing strategies to non-targeted profiling of the peptidome using a simulated digestion procedure to generate a peptide mixture of great complexity. The application of a data-independent LC/TOF MS analysis followed by multivariate statistical analysis allows for the survey of the entire complement of a peptidome, providing abundances are above limits of detection. Multivariate analysis also allows for the recognition of peptides within a peptidome that differentiate physiological states, genotypes, treatments, or temporal changes. In this work, the extent of post-translational deamidation of glutamine was found to be a distinguishing characteristic of protein grains of wheat (Triticum aestivum) and its relative spelt (Triticum spelta). In the model digestion-resistant peptidome generated from proteolysis of wheat storage proteins using gastrointestinal enzymes, surviving peptides exhibit a narrow range of hydrophobicity, and chromatographic co-elution hinders the analysis. The use of non-traditional stationary phase/solvent system combinations for peptide separation can spread peptide elution over a wider range of retention times. This dissertation describes use of a…

Thesis M.S. Michigan State University. Forensic Science 2017

The identification of emerging designer drug analogs, such as synthetic cathinones and phenethylamines, is a necessary step in the control and regulation of these illicit compounds. The standard technique for controlled substance analysis, gas chromatography-mass spectrometry, provides reproducible results useful for comparison to a library. But, this technique is hindered by low-resolution mass data that limits accurate molecular formulae assignments and extensive compound fragmentation that makes determination of the intact molecule ambiguous. Collision-induced dissociation mass spectrometry (CID-MS) is an alternative analysis method that provides high-resolution mass data for accurate molecular formulae assignment and controlled fragmentation through using multiple collision energies, providing molecular formulae for both the intact compound and fragments that reveal its structural features.Using CID-MS, this work aims first to facilitate class assignment of cathinones and phenethylamines by combining commonly observed fragmentation pathways for both drug classes into a single flowchart. This scheme is built and tested using compounds that represent the diversity of possible structural features within the drug classes. Additionally, this work addresses the issue of identifying the structural arrangement within cathinone isomers by specifically examining fragmentation differences between isomers under multiple collision energies.

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Thesis Ph. D. Michigan State University. Chemistry 2015.

Influenza virus causes substantial public health risk worldwide. Influenza is an enveloped virus and hemagglutinin membrane protein present in the viral membrane plays an important role in the viral infection process. Hemagglutinin protein is composed of two subunits called HA1 and HA2. Binding of the virus to the host cell is governed by HA1 subunit, and HA2 subunit is responsible for the fusion of viral membrane and endosomal membrane.My research has focused on production and characterization of several protein constructs containing different domains (with or without FP or with or without TM) and the characterization of the full length HA2. Biophysical comparisons between full-length HA2 and shorter constructs including SHA2, FHA2, and SHA2-TM were performed. Biophysical characterization techniques such as CD spectroscopy, SEC, crosslinking experiments, mass spectrometry and vesicle fusion assays were used in this study. Physiologically relevant oligomeric states of these hemagglutinin constructs were identified. At pH 7.4, the physiological pH, these constructs are trimeric helical molecules in detergents. The melting temperature for full length HA2 is > 90 °C in decylmaltoside (DM) representing a highly thermostable structure. All constructs are positively charged at pH 5.0 and induce vesicle fusion with negatively-charged vesicles. However, fusion with negatively charged vesicles at pH 7.4 was negligible. With positively charged vesicles, pH-dependence was reversed leading to conclude that attractive protein/vesicle electrostatics play a role in fusion between vesicles and hemagglutinin constructs.My work will help future scientists set up crystallography experiments since this is the first time that full length HA2 has been produced in mg quantities using bacterial expression system. Furthermore, it is hypothesized that in the final stage of the fusion of full length HA2 constructs as well as full length HIV gp41 constructs there is a formation of complex in between FP and TM. But, still there is no experimental evidence for support this hypothesis. HDX experiment completed and proposed in this dissertation will help to investigate this hypothesis.

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Thesis Ph. D. Michigan State University. Chemistry 2016

Biomarkers serve as objective indicators of normal, diseased and therapeutic conditions to aid prediction, diagnosis and staging of diseases as well as monitoring and evaluating clinical responses to interventions. Metabolomics has emerged as a powerful holistic approach to assess physiological status of a biological system, and advances in metabolomics techniques promoted the discovery of biomarkers of wellness and disease. This dissertation presents the development of liquid chromatography-mass spectrometry (LC-MS) based metabolomics approaches to profile endogenous metabolites and monitor research subjects’ consumption of drugs. The analytical approaches were applied to discover potential plasma biomarkers that can help understanding and evaluating pulmonary rehabilitation (PR) exercise and osteopathic manipulative treatment (OMT) to chronic obstructive pulmonary disease (COPD). This dissertation is based on a pilot study, in which COPD patients were participants in a PR program where they performed exercises and either received OMT, sham OMT treatment, or no additional treatment. Prior to profiling endogenous metabolites, the first study described the development and validation of a LC-MS/MS method to screen fourteen non-steroidal anti-inflammatory drugs (NSAIDs) in plasma samples from these COPD patients, and data were compared to self-reported medication use assessed from questionnaires. The results showed that 24 of 26 subjects tested positive for at least one NSAID. However, only 3 participants self-reported NSAID drug use correctly. This work revealed the inaccuracy of self-reported medication information, suggesting that drug use monitoring by analytical approaches should be implemented as a routine practice to support clinical trials.Both untargeted and targeted metabolomics strategies were performed in this dissertation to assess biochemical alterations in levels of metabolites in blood plasma following PR exercise and OMT/sham/no additional treatment on COPD patients. The second study described an untargeted approach combining the power of an advanced LC-TOF-MS platform and both univariate and multivariate statistical analyses to identify potential biomarkers of the effect of exercise on the plasma metabolome. The most discriminating metabolites were identified as lipids, and most were free fatty acids, which can serve as biomarkers of exercise.In the third study, a targeted LC-MS/MS method was developed for quantification of 57 bioactive lipid mediators in human plasma samples drawn from COPD patient participants in PR with, or without extra OMT or sham treatment. Using the targeted approach, which was intended to provide a more comprehensive assessment of lipid mediators than prior publications, the responses of circulating oxylipins, endocannabinoids and polyunsaturated fatty acids (PUFAs) to PR exercise and OMT were assessed. The results demonstrated that PR exercise significantly increased PUFAs levels in the circulatory system of COPD…

Thesis Ph. D. Michigan State University. Chemistry 2019

The entry of influenza virus into a host cell is mediated by the viral protein hemagglutinin (HA), which forms an initial complex that contains three HA1 subunits in association with a bundle containing the ectodomains of three HA2 subunits. Each ectodomain has a N-terminal 250303-residue fusion peptide (FP) and 1603030-residue soluble ectodomain (SE). HA2 also has a 250303-residue transmembrane domain (TM) and a 100303-residue endodomain. Entry begins with HA1 binding to target cell sialic acids, followed by endocytosis of the virus and endosome maturation with pH reduction to < 6. The HA1 subunits move away from the HA2 bundle, which then undergoes a large structural rearrangement to a final hyperthermostable state with a trimer-of-SE hairpins. The pH reduction is also correlated with HA2-mediated joining/fusion of the virus and endosome membranes, and consequent deposition of the viral nucleocapsid in the cytoplasm. The FP and TM are the only HA2 segments that are deeply inserted in the fused membrane. Residues 38-105 from the SE hairpins form an interior parallel helical bundle, while 154-176 are strands within the grooves of the bundle exterior. The FP forms a separate hairpin structure with antiparallel and close packing of two FP helices. My research focuses on better understanding the HA2 structure and fusion mechanism, which may aid the development of antiviral drugs and vaccine. One main project in this dissertation compares WT-HA2 with G1E(FP) and I173E(SE strand) mutants. WT-HA2 induces vesicle fusion at pH 5.0, whereas fusion is greatly reduced for both mutants. Circular dichroism for HA2 and FHA2 2261 FP + SE constructs show dramatic losses in stability for the mutants, including a melting temperature reduced by 40 °C for I173E-FHA2. This evidences destabilization of SE hairpins via dissociation of strands from the helical bundle, which is also supported by larger monomer fractions for mutant vs. WT proteins. The G1E mutant may have disrupted FP hairpins, with consequent non-native FP binding to dissociated SE strands. It is commonly proposed that free energy released by the HA2 structural rearrangement catalyzes HA-mediated fusion. The present study supports an alternate mechanistic model in which fusion is preceded by FP insertion in the target membrane and formation of the final SE hairpin. Lower fusion by the mutants is due to loss of hairpin stability and consequent reduced membrane apposition of the virus and target membranes. Another project describes the hydrogen-deuterium exchange-mass spectrometry (HDX-MS) on HA2. Fusion models often depict a FP/TM complex in the final HA2 state with membrane traversal by both domains, and a role for this complex in membrane pore expansion. The HDX-MS data are inconsistent with this complex, and show respective high FP and low TM aqueous exposures. The data support independent FP and TM with respective membrane interfacial and traversal locations. The data also support low aqueous exposure of the 22-38…

Thesis Ph. D. Michigan State University. Chemistry 2013.

Plants cannot escape from herbivory and other environmental stresses including cold and drought, and have evolved sophisticated defense systems to survive. Improving food production has become one of the most urgent problems facing humankind with 7 billion population and limited land available. Plants also serve as the base of the earth's sustainable fuel supply, and synthesize a diverse suite of natural compounds that help defend them against stress, but also are potential pharmaceutics. Improving our understanding of plant defense systems is a key factor in using plants as natural resources to provide solutions to these problems. To defend themselves, plants synthesize oxidized fatty acids, or oxylipins, to regulate gene expression in response to stress. In response to wounding and certain stresses, many plants synthesize the cyclopentenone oxylipin 12-oxo-phytodienoic acid (OPDA) as a precursor of the master regulatory hormone jasmonic acid (JA). JA is then conjugated to isoleucine in cytoplasm to produce the universal defense gene regulator JA-isoleucine. OPDA has been shown to be an independent but not fully understood metabolite that regulates plant defenses. Reactive electrophiles such as OPDA are subject to conjugation to the tripeptide glutathione. This process is catalyzed by an assortment of glutathione transferase enzymes, and is expected to deactivate the biological functions of electrophiles. However, understanding of the functions of specific GSTs (>50 in model plant Arabidopsis from sequenced genome), particularly in plants, is limited.The research described in this dissertation has aimed to reveal biological functions of GSTs in defense response to mechanical wounding in Arabidopsis. The basic strategy has combined information about GST protein levels with profiling of metabolites in a GST knockout mutant to correlate metabolic phenotypes with genotypes, for deduction of gene functions. 8 GSTs were identified in wild type Arabidopsis leaves, and one of them, AtGSTU5, was shown to be highly accumulated after mechanical wounding. Several Arabidopsis knockout mutants, including AtGSTu5, were grown for wounding experiments and phenotype assessment using non-targeted metabolite profiling. The glutathione conjugate of OPDA was quantified and shown to be accumulated after mechanical wounding in leaves of wild type and other tau-family GST knockout mutants of Arabidopsis, but not in the knockout mutant AtGSTu5. This finding suggests that AtGSTU5 is responsible for in vivo glutathione conjugation of OPDA. A new LC-TOF-MS protocol was developed to explore the range of endogenous glutathione conjugates in extracts of Arabidopsis leaves. A family of novel glutathione conjugates is also discovered and proposed to be derived from OPDA-containing galactolipids. In the identification and quantitation of plant oxylipins, tandem mass spectrometry (MS/MS) data are often the primary source of metabolite structural information, but ion fragmentation pathways are…

If lignocellulosic biomass is to serve as the carbon feedstock for future energy and chemical manufacture, it will be necessary to develop chemistry tools to disassemble it into fragments for further upgrading. Of particular interest is the lignin fraction of the biomass, as it is the most carbon- and energy-rich component and currently underutilized. This complex polymer consists of highly oxygenated and methoxy-substituted phenylpropane building blocks, mainly guaiacyl and syringyl moieties, held together by C-O or C-C linkages. This dissertation examines the use of Electrocatalytic Hydrogenation (ECH) at a Raney nickel cathode to depolymerize, hydrogenate, and deoxygenate lignin to biofuel and meanwhile investigates the reaction mechanisms of the ECH surface reactions of lignin monomer and dimer models. Along with new technology development, the safety of personnel should always be considered. Due to the expected major changes of chemical structures of the components of lignin biofuels, their potential impact on the human health is unexplored. Thus, this project seeks to analyze the risk associated with the newly emerging cyclic compounds in biofuel by comparing the toxicities for two of the representative compounds: cyclohexanol and cyclohexane.

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Thesis Ph. D. Michigan State University. Chemical Engineering 2014.

Large scale production of economically viable biofuels can only be achieved from widely available resources, notably lignocellulosic biomass. This feedstock is largely composed by complex carbohydrates, which can be enzymatically hydrolyzed and converted to fuels and chemicals by fermentative organisms. Lignocellulosic biomass is recalcitrant to enzymatic degradation and therefore, a pretreatment step is required for achieving acceptable sugar yields. Recent research has improved our fundamental understanding about the physico-chemical events occurring during ammonia pretreatment of biomass that correlate with enzymatic hydrolysis yields. From this understanding, a novel Extractive Ammonia (EA) pretreatment was developed and is presented herein for the first time. This technology allows the conversion of naturally occurring cellulose I (CI) to cellulose III (CIII) and the selective extraction of lignin from the plant cell wall with liquid ammonia. These extractives are collected in a separate stream as a valuable byproduct. While CIII is known to improve enzymatic hydrolysis rates up to two fold compared to native CI, lignin is acknowledged as a major inhibitor for both enzymes and microbes. Other key events include ester-bonds cleavage via ammonolysis and hydrolysis reactions during EA. Ester bonds play an important role in cross-linking lignin and carbohydrates and therefore, their cleavage disrupts the complex cell wall architecture, allowing carbohydrates to be more easily accessible by enzymes.The effect of pretreatment variables on CIII conversion was studied using isolated cellulose from commercial sources (Avicel). These studies revealed that samples of higher CIII crystallinity can significantly increase enzymatic activity, which contradicts the current paradigm based on CI results, which show that higher CI crystallinity reduces enzymatic activity of cellulases. In the present work, EA performance was evaluated using corn stover (CS) as primary biomass feedstock. An empirical model was created using statistical design of experiments to predict EA pretreatment effectiveness on lignin extraction, ester-bond cleavage and sugar yields as a function of pretreatment conditions. The results show that EA allows extraction of up to ~ 50 % of the lignin present in corn stover, while cleaving about 70 % of the ester bonds. These effects, in synergy with cellulose III formation, allow maximum monomeric glucose and xylose conversions of 93 % and 79 %, respectively, using 15 mg/g glucan of enzyme for 24 h, at 1% glucan loading enzymatic hydrolysis. At 15 % to 20% solid loadings, EA allows up to 2.7 fold reduction of enzyme loading during enzymatic hydrolysis compared to AFEXTM-CS. The benefits of EA on fermentation were also explored using the novel RaBIT process, which is capable of doubling biofuel productivity while decreasing enzyme loading by 30 % compared with traditional SHF. By coupling EA and RaBIT technologies was possible to efficiently…

Thesis Ph. D. Michigan State University. Chemistry 2012.

Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. With the advantages in sensitivity, speed, and the capability of analyzing large/complex protein systems, mass spectrometry combined with protein labeling has found increasing utility for the characterization of protein surface. However, a common problem associated with the use of chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the `needle in a haystack' problem). To address this challenge, an experimental strategy was developed involving the synthesis and application of a novel `fixed charge' sulfonium ion containing amine-specific protein modification reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-electrospray (ESI)-MS, automated collision induced dissociation (CID)-MS/MS, and data dependent neutral loss mode MS3 in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, Cellular Retinoic Acid Binding Protein II (CRABP II). After reaction with different reagent : protein ratios and digestion with Glu-C, modified peptides were selectively identified and the number of modifications within each peptide were determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e. proton mobility) of the peptide. The observation of these characteristic neutral losses were then used to automatically `trigger' the acquisition of an MS3 spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II. With the initial success demonstrated on a model protein, the experimental strategy was extended to reveal the mechanisms corresponding to oxidation induced inactivation of calcineurin (CN), from a structural perspective. CN is a Ca2+/calmodulin (CaM) activated phosphatase that participates in a wide variety of physiological processes. CaN is also reported to be inactivated by H2O2- or superoxide-induced oxidation both in vivo and in vitro. However, the mechanism is still under debate. Here, the relative rates of H2O2 induced oxidation of methionine residues within CN were first determined using a multi enzyme digestion strategy coupled with analysis using capillary HPLC-ESI-MS and CID/ETD-MS/MS. Then the developed experimental strategy, i.e., combining protein modification by DMBNHS with data dependent multistage tandem mass spectrometry, was applied to characterize changes in CN…

"Aerosols are an ever-present part of our daily environment and have extensive effects on both human and environmental health. Particles in the inhalable range (1-10 æm diameter) are of particular concern because their deposition in the lung can lead to a variety of illnesses including allergic reactions, viral or bacterial infections, and cancer. Understanding the transport of inhalable aerosols across both short and long distances is necessary to predict human exposures to aerosols. To assess the transport of hazardous aerosols, surrogate tracer particles are required to measure their transport through occupied spaces. These tracer particles must not only possess similar transport characteristics to those of interest but also be easily distinguished from the background at low levels and survive the environmental conditions of the testing environment. A previously-developed DNA-tagged particle (DNATrax), composed of food-grade sugar and a DNA oligonucleotide as a "barcode" label, shows promise as a new aerosol tracer. Herein, the use of DNATrax material is validated for use in both indoor and outdoor environments. Utilizing passive samplers made of materials commonly found in indoor environments followed by quantitative polymerase chain reaction (qPCR) assay for endpoint particle detection, particles detection was achieved up to 90 m from the aerosolization location and across shorter distances with high spatial resolution. The unique DNA label and PCR assay specificity were leveraged to perform multiple simultaneous experiments. This allowed the assessment of experimental reproducibility, a rare occurrence among aerosol field tests. To transition to outdoor testing, the solid material provides some protection of the DNA label when exposed to ultraviolet (UV) radiation, with 60% of the DNA remaining intact after 60 minutes under a germicidal lamp and the rate of degradation declining with irradiation time. Additionally, exposure of the DNATrax material using formulations of two different food-grade sugars (maltodextrin and erythritol) to humidity as high as 66% had no significant effect on the DNA label's degradation or the particle's aerodynamic diameter, confirming particle stability under such conditions. In summary, confirmation of the DNATrax particles' size and label integrity under variable conditions combined with experiment multiplexing and high resolution sampling provides a powerful experimental design for modeling aerosol transport through occupied indoor and outdoor locations." – Pages ii-iii.

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Thesis Ph. D. Michigan State University. Biochemistry and Molecular Biology 2016

The motivation of this work is to comprehend and overcome challenges in understanding and in design of optimal heterologous metabolic pathways that lead to production of biofuels and other valued biochemicals in microbial hosts. I specifically address the problem that within a designed pathway, introduced enzymes are often inefficient leading to a reduction of metabolic flux and consequently product yield. This enzymatic underperformance can be attributed to either poor catalytic fitness or poor soluble expression in the host. To help develop technologies that remedy these inefficiencies, the field of metabolic engineering was surveyed for current approaches that identify an optimal pathway variant and the limitations thereof. I identified numerous inadequacies in current isogenic and high-throughput pathway screening and optimization methods. Specifically, the amount of time and the number of unique variants tested in current methods is limiting. With the advent of high-throughput deep sequencing technologies, large population-based studies are now feasible which reduce the amount of time and increase the total number of unique variants tested. Therefore, this work set out to utilize this promising new approach to test unique enzyme variants in a pathway. I developed a new deep sequencing approach to study the enzyme levoglucosan kinase (LGK) from L. starkeyi that was introduced into E. coli. LGK converts levoglucosan into glucose-6-phosphate which is then used for microorganism growth. A growth selection was developed such that growth on levoglucosan as a sole carbon source was dependent on an active LGK enzyme, and the change in growth was correlated to the change in enzymatic activity. This method was able to quantify the effect of over 8,000 single point mutations on specific levoglucosan flux. The datasets were able to predict whether a beneficial mutation improved stability or catalytic efficiency. Combining computational modelling with these datasets aided the creation of nine enzyme designs. One enzyme design incorporating 38 mutations was crystallized to learn the structural basis of the beneficial mutations. The best enzyme design had a 15-fold improvement in growth rate and 24-fold improvement in pathway activity. Developing this deep sequencing method illuminated a number of problems and opportunities: 1) growth selections are difficult to design and may not be feasible for enzymes in secondary metabolism, 2) improving the soluble expression of an enzyme is potentially an easy avenue to increase specific flux however, 3) stabilizing mutations often have small trade-offs in catalytic fitness. Therefore, the second project set out to extend the original deep sequencing method to improve soluble expression of enzymes without trading-off catalytic fitness in the absence of a growth selection. Using three solubility screens: yeast surface display, GFP fusion, and Tat export, I screened two enzymes, TEM-1 beta-lactamase and…

Thesis Ph. D. Michigan State University. Biochemistry and Molecular Biology 2015.

Glandular trichomes on the plant surface are specialized epidermal cells which can produce and store a variety of plant specialized metabolites. Specialized phytochemicals produced by trichome are extremely diverse among Solanaceae families and a lot of them have been characterized with the advance of plant omics. Due to their benefits for plant defense system and commercial values, biosynthesis and accumulation of trichome metabolites haven been intensively studied. In this study, analytical chemistry and genetic approaches were integrated to explore the remaining questions about mechanism for the production of specialized metabolites in Solanum trichomes. In particular, this study included three studies addressing the dynamics, distribution and development of specialized metabolism in glandular trichomes from tomato and its relatives. This first study presented the development of analytical methods to profile localized metabolites in biological tissues through chemical imaging, and application of this method to investigations of glandular trichomes. Using this mass spectrometry imaging method, single-cell metabolic profiling and spatially preserved chemical information was achieved in tomato leaf trichomes for several type of trichome specialized metabolites, including acylsugar, flavonoids and terpenes. This work extended imaging mass spectrometry to specialized epidermal cells without using dissection or tissue fixation for the first time. The second study described the isotope labeling methodology for tracing specialized metabolites flux in trichomes. In general, an integrated approach combining whole plant 13CO2 labeling and data-independent LC-MS quantitative profiling was developed to monitor the metabolic dynamic flux and turnover rate of protective plant specialized metabolites. This analytical strategy is applicable to a wide range of specialized metabolites. The third study applied the isotopic labeling strategy to discover the dynamics of trichome specialized metabolites accumulation and the effect of developmental stage of tomato leaves. Using flux analysis method, we measured fluctuations in labeling enrichment and metabolite accumulation during different tomato leaf developmental stages, and demonstrated that total acylsugar accumulation reaches a steady state at early stage of development. Evidence was generated to support the concept that degradation of undetected metabolic intermediates provides a key regulatory point in the acylsugar metabolic network offers a new viewpoint for engineering plant metabolic networks for high levels of end product acylsugars. This novel regulation mechanism extends our understanding of catabolic activities of specialized metabolites.

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Thesis Ph. D. Michigan State University. Plant Biology 2015

The branched-chain amino acids (BCAAs) leucine, isoleucine and valine are among nine essential amino acids that humans and other animals must obtain from their diets, and can be nutritionally limiting in plant foods. Rapid development of transcript profiling technologies has enabled research on plant metabolism that offers potential to improve crop nutritional quality by allowing researchers to apply correlative analysis in hypothesis generation followed by experimental validation. Despite genetic evidence of its importance in regulating seed amino acid levels, the full BCAA catabolic network is not completely understood in plants, and limited information is available regarding its regulation. In this study, a combination of transcript and mutant analyses was performed to study the pathway and regulation of BCAA catabolism in Arabidopsis thaliana. Transcript coexpression analyses revealed positive correlations among BCAA catabolic genes in stress, development, diurnal/circadian and light datasets. BCAA catabolism genes show coordinated oscillation in diurnal and circadian treatments, and their expression patterns are altered in clock and phytochrome B mutants, providing evidence for the regulation of BCAA catabolism by the circadian clock and light. Functional divergence is suggested by transcript profile comparison between four pairs of BCAA catabolic enzyme paralogs, and the paralogs do not increase their transcript levels upon the loss of their duplicated copies in the dark. In addition, mutants defective in putative branched-chain ketoacid dehydrogenase subunits accumulate higher levels of BCAAs in mature seeds, providing genetic evidence for their function in BCAA catabolism. BCAA catabolism genes are highly expressed during the night on a diel cycle and during prolonged darkness, and mutants undergo senescence early and over-accumulate leaf BCAAs during prolonged darkness. These results extend the previous evidence that BCAAs can be catabolized and serve as respiratory substrates at multiple steps. Furthermore, comparison of amino acid profiles between mature seeds and dark-treated leaves revealed differences in amino acid accumulation when BCAA catabolism is perturbed. Together, these results demonstrate the consequences of blocking BCAA catabolism during both normal growth conditions and under energy-limited conditions.

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Thesis Ph. D. Michigan State University. Chemistry 2014.

Models play a vital role in predicting environmental fates of pollutants, which is critical for effective remediation. However, many fate and transport models for complex mixtures, e.g. petroleum products, do not incorporate the individual compounds, which are responsible for toxicity and environmental persistence. In this research, a diesel/water microcosm mimicked an environmental fuel spill with simulated weathering by evaporation and irradiation. Temporal changes in composition were assessed by gas chromatography-mass spectrometry (GC-MS) and time of flight mass spectrometry (ToF-MS) with atmospheric pressure chemical ionization (APCI). During evaporation, first-order kinetic rate constants were calculated for selected compounds and employed to develop predictive models, based on GC retention indices. Models were initially developed for compounds from individual classes (normal alkane, branched alkane, alkyl benzene, and polycyclic hydrocarbon) and later expanded to include compounds from all classes (comprehensive model). Using the comprehensive model, the rate constants were predicted with an average error of 10%, whereas the class specific models resulted in less error (4 - 8%). A model was also developed that incorporated varying temperature (5 to 35 °C), allowing for the prediction of the rate constants over environmentally relevant temperatures (16 % error). Using the rate constant, the fraction remaining of individual compounds was determined. The fraction remaining of individual compounds was used to calculate the fraction remaining of the total fuel (± 6%), and was in good agreement with currently available evaporation models. The variable-temperature model successfully applied to predict the fraction remaining of other petroleum products, demonstrating applicability beyond diesel fuel. The variable-temperature model was also used to predict chromatographic profiles of a fuel after evaporation, estimated the length of time a fuel has been evaporated using the predicted chromatogram, and estimate the time to reach a specific percent evaporated for an individual compound or for the entire fuel. First-order kinetic rate constants were also determined for diesel fuel irradiated with simulated sunlight for 10 hours by GC-MS and APCI-ToF-MS. The decay of hydrocarbons and formation of oxygenated compounds began within the first hour of irradiation. Using GC-MS, a two-fold increase in the rate constant was observed during irradiation (0.004 - 1.211 h^-1) than predicted from the variable-temperature evaporation model (0.000 - 0.379 h^-1). Compounds unlikely to evaporate also decayed, indicating they were precursors to photooxidation. In the APCI-ToF-MS, rate constants were determined for decay of hydrocarbons (0.003 - 0.210 h^-1) and formation of oxygenated compounds (0.002 - 1.173 h^-1). The kinetic rate constants developed in this work provided valuable information about changes in individual…