Polycyclic aromatic hydrocarbons (PAH) are important environmental pollutants generally formed during incomplete combustion of organic matter containing carbon and hydrogen. Introduced into the human body by adsorption through the skin, ingestion or inhalation, the biotransformation processes of PAH lead to the formation of multiple metabolites. Due to the short elimination lifetime from the body, the quantitative determination of monohydroxy-PAH (OH-PAH) in urine samples provides accurate information on recent exposure to environmental PAH. Urine analysis of OH-PAH with established methodology relies on sample clean-up and pre-concentration followed by chromatographic separation and quantification. Although chromatographic techniques provide reliable results in the analysis of OH-PAH, their experimental procedures are time consuming and expensive. Additional problems arise when laboratory procedures are scaled up to handle thousands of samples under mass screening conditions. Under the prospective of a sustainable environment, the large usage of organic solvents is one of the main limitations of current chromatographic methodology. It is within this context that new analytical approaches based on easy-to-use and cost-effective methodology become extremely relevant. This dissertation focuses on the development of screening methodology for the routine analysis of PAH metabolites in numerous samples. It explores the room-temperature fluorescence properties of six metabolites originating from parent PAH included in the Environmental Protection Agency priority pollutants list. 1- hydroxyfluorene, 1-hydroxypyrene, 6-hydroxychrysene, 9-hydroxyphenanthrene, 3- hydroxybenzo[a]pyrene and 4-hydroxybenzo[a]pyrene are used as model biomarkers to investigate the analytical potential of new methods based on solid-phase extraction (SPE) and iii room-temperature fluorescence (RTF) spectroscopy. Quantitative determination of metabolites is carried out either in the eluent extract[1, 2] or on the surface of extraction membranes[3, 4] . The direct determination – i.e., no chromatographic separation - of the six metabolites is based on the collection of excitation-emission matrices and synchronous fluorescence spectra.

Huntington disease (HD) is an inherited neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Metabolic dysfunction is a feature of HD that is recapitulated in HD mouse models. Our lab has shown that circadian feeding rhythms are disrupted in humanized HD mice and restored by suppression of brain HTT. Furthermore, when circadian feeding rhythm is artificially restored, in addition to normalization of metabolic function, liver and striatal HTT is temporarily reduced, demonstrating that HTT is involved in gut-brain feedback. The gut microbiome, which can regulate gut-brain feedback, has been implicated in the pathogenesis of other central nervous system disorders and we hypothesize it also plays a role in HD. The objective of this study is to investigate alterations in relative abundance of HD gut microbiota using existing plasma metabolomics data to identify candidate bacteria. If distinct microbiota profiles are demonstrated, this would provide the basis for future unbiased studies to investigate the complete HD microbiome.

No description available.

<p>The world is facing a public health threat due to increasing emergence of antibiotic resistance in pathogens. <em>Streptomyces </em>the soil-dwelling, Gram-positive, filamentous bacteria belonging to the family actinomycetes, are proven to be rich sources of natural antibiotics. Genome sequencing of <em>Streptomyces coelicolor, </em>a model organism of this genus, has revealed that in addition to the five antibiotics characterized so far, it possesses abundant genetic architecture of unexpressed biosynthetic or cryptic clusters for secondary metabolite production. The reason for their silence appears to be the poor understanding of their specific activation stimuli. In <em>Streptomyces coelicolor,</em> a pleiotropic regulator belonging to the two-component system family, <em>afsQ1</em>, has shown to activate the production of actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA). The aim of this research was to employ the genetically engineered <em>afsQ1</em> allele (named <em>afsQ1*</em>), which mimics the phosphorylated active form and obviates the need for specific external stimulus, and screen for novel antibiotic production. In this study, <em>afsQ1* </em>was introduced in various wild actinomycete isolates from the Wright Actinomycetes Collection (WAC) by conjugation and the resulting mutants were screened for antibiotic production. Two out of six WAC strains showed <em>afsQ1*- </em>induced antimicrobial activity. Interestingly, we were able to purify two antibiotic compounds, namely 1082 [M+2H]²⁺<strong> </strong>and 782 [M+H]⁺<strong> </strong>from the strain WAC00263. 1082 [M+2H]²⁺,<strong> </strong>a potentially novel antimicrobial peptide, exhibited activity against a wide range of Gram-positive bacteria including resistant pathogens such as vancomycin-resistant <em>Enterococcus</em> ATCC# 51299, a clinical isolate of methicillin resistant <em>Staphylococcus aureus</em>, and a clinical isolate of <em>S. aureus</em> BM3002. Moreover, it also showed activity against an opportunistic Gram-negative multi-drug resistant pathogen <em>Acinetobacter baumannii</em> B0098426R and a virulent strain of the fungus <em>Cryptococcus neoformans </em>H99<em>. </em>The second newly expressed molecule, 782 [M+H]⁺<strong> </strong>was not as potent as 1082 [M+2H]²⁺,<strong> </strong>so<strong> </strong>far only exhibited antimicrobial activity against the Gram-positive laboratory strains <em>Bacillus subtilis</em> #168 and <em>Micrococcus luteus</em>. These results reiterate that the technique of heterologous expression of the pleiotropic regulator, <em>afsQ1*</em>, in diverse actinomycetes is an excellent tool to induce novel antimicrobial production.</p> / Master of Science (MSc)

Non-targeted metabolite profiling by liquid chromatography-mass spectrometry (LC-MS) was used to determine the metabolite responses of Arabidopsis roots to auxin or ethylene. Crosstalk between these hormones regulates many important physiological processes in plants, including the initiation of lateral root formation and the response to gravity. These occur in part through alterations in the levels of flavonoids, specialized plant metabolites that have been shown to act as negative regulators of auxin transport. However, much remains to be learned about auxin and ethylene responses at the level of the metabolome. LC-MS analysis showed that a number of ions changed in response to both hormones in seedling roots. Although classes of specialized metabolites such as flavonols and glucosinolates change in abundance in response to both auxin and ethylene, there was little overlap with regard to the specific metabolites affected. These data will be integrated with information from transcriptomic and proteomic experiments to develop framework models that connect phytohormones and specialized metabolism with specific physiological processes. Previous studies by imaging techniques have shown that flavonols increase in response to both auxin and ethylene in the root elongation zone, but LC-MS showed that flavonols decreased in abundance in response to these hormones. Therefore a method was developed for targeted metabolite profiling of flavonols in individual root tips by flow injection electrospray mass spectrometry. This method uncovered spatial differences in metabolic profiles that were masked in analyses of whole roots or seedlings, and verified that flavonols increase in response to these hormones in root tips. / Master of Science

Organophosphates (OPs) were initially designed as insecticides and later engineered as dangerous nerve agents that threaten our livelihood and safety. One of the treatments for OP exposure is the administration of an oxime to reactivate inhibited acetylcholinesterase (AChE). The currently approved oxime therapy in the US, pralidoxime (2-PAM), is unable to reactivate inhibited AChE in the brain due to its low ability to cross the blood-brain barrier (BBB) which has led our laboratory to develop novel substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937) that penetrate the BBB more effectively. Our lead oxime candidate, Oxime 20, has proven efficacious in reactivating inhibited AChE both in vitro and in vivo and is in the preliminary steps of drug development which require metabolism studies such as pharmacokinetics (PK), protein-binding (PB) and metabolite identification. PK parameters were explored for Oxime 20 and found to have an average half-life of 11.6 hours and an average Tmax of 0.083 hours in rats and an average half-life of 15 hours and an average Tmax of 0.11 hours in minipigs. As compared to 2-PAM, our oxime has displayed a 2-4 times longer half-life and a 3 times faster Tmax which allows it to be distributed at a faster rate and stay in circulation for longer. Furthermore, Oxime 20 was found to be >84% plasma protein-bound as compared to 2-PAM which was <8% protein-bound. These PB characteristics align with the PK parameters as highly protein-bound drugs tend to have a longer half-life than low protein-bound drugs. Finally, our oxime displayed a potentially safe metabolism in the presence of microsomes with the generation of two more polar metabolites as compared to Oxime 20, a hydroxyl metabolite and a carboxylic acid metabolite. With these findings, Oxime 20 continues to show promise and excellent characteristics for drug development and potentially will be the next suitable and effective option for treatment of OP exposure either by itself or in combination with 2-PAM.

Sagebrush (Artemisia spp.) is an important source of food and cover for many animals, especially during winter months. Understanding how wildlife species respond to sagebrush management actions can help improve conservation planning. Dixie harrow is a method of improving spring/summer habitat for many herbivores by reducing sagebrush cover to stimulate the growth of grasses and forbs. These treatments, however, may influence the quantity and quality of sagebrush available to greater sage-grouse (Centrocercus urophasianus; hereafter, sage-grouse) during winter. We evaluated the effects of Dixie harrow on sage-grouse resource selection during winter (Chapter 1) and on the nutritional value of sagebrush (Chapter 2). We were unsure what effect Dixie harrow would have on the nutritional value of sagebrush, but hypothesized that sage-grouse would select for untreated areas because they contained a higher quantity of food and cover. We captured 81 sage-grouse and fit them with GPS transmitters. Using 6,728 winter locations, we modeled third-order resource selection. Further, we collected samples of sagebrush plants that sage-grouse had eaten from (n = 54), samples of sagebrush plants passed by but not eaten from (n = 54), as well as samples from random locations inside (n = 60) and outside Dixie harrow treatments (n = 60). Contrary to our hypothesis, sage-grouse selected for Dixie harrow treatments during winter. We found that sage-grouse selectively browsed sagebrush plants with increased nutritional value, and that sage-grouse browsed plants inside treatments more frequently than outside the treatments, but Dixie harrow treatments had no measurable effect on the nutritional value of sagebrush. Based on our results, Dixie harrow treatments performed at the southern extent of the sage-grouse range will create habitat that sage-grouse prefer during winter, but we were unable to ascertain why sage-grouse select for Dixie harrow treatments during winter.

Ce travail décrit l’analyse de la distribution des flux de carbones au sein de deux souches de Streptomyces coelicolor A3(2) : la souche sauvage nommée M145 et son mutant M1146 incapable de produire les antibiotiques actinorhodine, undecylprodigiosine, et l’antibiotique dépendant du calcium. Metabolite Balance Analysis et Isotopomer Balance Analysis sont mis en œuvre pour proposer un modèle de distribution des flux de carbones de S. coelicolor en phase exponentielle de croissance. Les souches M145 et M1146 sont cultivées dans un milieu minimum limitant en azote et leurs comportements métaboliques sont comparés. Dans la souche non productrice M1146, un taux de croissance plus élévé, un flux plus important dans la voie des pentoses phosphates, un flux plus faible au niveau du cycle de Krebs ainsi qu’une activité respiratoire plus faible sont mis en évidence. Cela traduit le coût énergétique important associé à la production d’actinorhodine par M145. De plus, ce travail propose un rôle important de la nicotinamide nucléotide transhydrogénase pour le maintien de l’homéostasie du NADPH lors de la production d’actinorhodine par M145. Comme il existe de bonnes corrélations entre les données expérimentales et celles issues de la modélisation au niveau des bilans carbones, des bilans de pouvoir réducteur et des échanges gazeux, il sera intéressant d’utiliser cette modélisation avec la technique de Flux Balance Analysis pour prédire les variations de la distribution des flux de carbones dans des mutants de S. coelicolor pour lesquels des gènes auraient été sur-exprimés ou délétés. / This work describes an analysis of carbon flux distribution in two strains of Streptomyces coelicolor A3(2), namely the wild type strain M145 and its derivative M1146 that is no longer able to produce the antibiotics actinorhodin, undecylprodigiosin and the calcium dependent antibiotic. Metabolite Balance Analysis and Isotopomer Balance Analysis were used to propose a model for carbon flux distribution in S. coelicolor during the exponential phase of growth. Strains M145 and M1146 were grown under nitrogen limitation in minimal medium and their metabolic behaviour were compared. In the non-producing strain M1146, a higher growth rate, a higher flux via the pentose phosphate pathway, a decreased flux through the TCA cycle and a decreased respiratory activity were evidenced. This highlighted the high energetic cost for actinorhodin production in M145. In this paper, we also propose a key role for the nicotinamide nucleotide transhydrogenase in NADPH homeostasis in M145 during actinorhodin production. As there are good correlations between experimental data and the model in terms of carbon balance, reducing power balance and gas exchanges, this model will be of great interest for Flux Balance Analysis to predict carbon-flux distribution changes in S. coelicolor strains in which gene are deleted or overexpressed.

The advent of large-scale and high-throughput technologies has recently caused a shift in focus in contemporary biology from decades of reductionism towards a more systemic view. Alongside the availability of genome sequences the exploration of organisms utilizing such approach should give rise to a more comprehensive understanding of complex systems. Domestication and intensive breeding of crop plants has led to a parallel narrowing of their genetic basis. The potential to improve crops by conventional breeding using elite cultivars is therefore rather limited and molecular technologies, such as marker assisted selection (MAS) are currently being exploited to re-introduce allelic variance from wild species. Molecular breeding strategies have mostly focused on the introduction of yield or resistance related traits to date. However given that medical research has highlighted the importance of crop compositional quality in the human diet this research field is rapidly becoming more important. Chemical composition of biological tissues can be efficiently assessed by metabolite profiling techniques, which allow the multivariate detection of metabolites of a given biological sample.<br><br> Here, a GC/MS metabolite profiling approach has been applied to investigate natural variation of tomatoes with respect to the chemical composition of their fruits. The establishment of a mass spectral and retention index (MSRI) library was a prerequisite for this work in order to establish a framework for the identification of metabolites from a complex mixture. As mass spectral and retention index information is highly important for the metabolomics community this library was made publicly available. Metabolite profiling of tomato wild species revealed large differences in the chemical composition, especially of amino and organic acids, as well as on the sugar composition and secondary metabolites. Intriguingly, the analysis of a set of <i>S. pennellii</i> introgression lines (IL) identified 889 quantitative trait loci of compositional quality and 326 yield-associated traits. These traits are characterized by increases/decreases not only of single metabolites but also of entire metabolic pathways, thus highlighting the potential of this approach in uncovering novel aspects of metabolic regulation. Finally the biosynthetic pathway of the phenylalanine-derived fruit volatiles phenylethanol and phenylacetaldehyde was elucidated via a combination of metabolic profiling of natural variation, stable isotope tracer experiments and reverse genetic experimentation. / Die Einführung von Hochdurchsatzmethoden zur Analyse von biologischen Systemen, sowie die umfangreiche Sequenzierung von Genomen haben zu einer Verlagerung der Forschung „im Detail“ zu einer ganzheitlicheren Betrachtungsweise auf Systemebene geführt. Aus einer jahrhundertlangen, intensiven Züchtung und Selektion von Nutzpflanzen resultierte gleichzeitig eine Abnahme der genetischen Varianz. Daraus resultierend sind Nutzpflanzen anfälliger gegenüber Stressfaktoren, wie Pathogenen, hohen Salzkonzentrationen oder Trockenheit, als ihre Wildarten. Das Potential konventioneller Züchtung scheint somit heute an seine Grenzen gekommen zu sein. Daher versucht man mittels moderner Molekulartechnik, wie zum Beispiel Marker-gestützte Selektion, Gene oder ganze Genombereiche von Wildarten mit hoher genetischer Variation in Nutzpflanzen einzukreuzen, vornehmlich mit dem Ziel einer Ertrags- bzw. Resistenzsteigerung. Neueste medizinische Studien belegen, dass die Ernährung eine wesentliche Rolle für die menschliche Gesundheit spielt. Besonders wichtig sind hierbei die gesundheitsfördernden Substanzen in pflanzlichen Nahrungsmitteln. Aus diesem Grund kommt der Erforschung der biochemischen Zusammensetzung von biologischen Proben eine immer größere Bedeutung zu. Diese Untersuchung kann elegant durch Metabolitenprofile, welche die multivariate Analyse komplexer biologischer Proben erlauben, durchgeführt werden.<br><br> In dieser Arbeit wurde zur Untersuchung der biochemischen Zusammensetzung von Tomatenwildarten und interspezifischen <i>S. pennellii</i> Tomatenintrogressionslinien (IL) eine GC/MS basierte Metabolitenanalyseplattform verwendet. Hierzu war es zunächst notwendig eine Massenspektrenbibliothek, zur Annotierung von Massenspektren und Retentionsindices von, in pflanzlichen Proben vorkommenden, Metaboliten anzulegen. Die Analyse der Tomatenwildarten ergab große Unterschiede gegenüber der Kulturtomate im Hinblick auf den Gehalt an Amino- und organischen Säuren, sowie der Zuckerzusammensetzung und den Gehalt an Sekundärmetaboliten. Die darauf folgende Analyse der ILs, von den jede ein genau definiertes genomisches Segment von <i>S. pennellii</i> beinhaltet, bestätigte diese enorme Variation mit 889 metabolischen und 326 ertragsassozierten-Veränderungen in den ILs. Die metabolischen Veränderungen zeichneten sich durch abnehmende bzw. steigende Gehalte von einzelnen Metaboliten, aber auch durch eine koordinierte Änderung aus. In dieser Arbeit wurde weiterhin der Biosyntheseweg der Volatilenstoffe Phenylethanol und Phenylacetaldehyd mit Hilfe einer IL untersucht. Hierbei konnten durch stabile Isotopenmarkierung und eines „reverse genetics“-Ansatzes Gene bzw. Enzyme identifiziert werden, die für die Dekarboxylierung des Eduktes Phenylalanin verantwortlich sind. Diese Arbeit beschreibt erstmals die umfassende Analyse von biochemischen Komponenten auf Genombasis in Tomatenintrogressionslinien und zeigt damit ein Werkzeug auf zur Identifizierung von qualitativen biochemischen Merkmalen in der modernen molekularen Züchtung.

Ein genereller Ansatz zur Charakterisierung von biologischen Systemen bietet die Untersuchung des Metaboloms, dessen Analyse als „Metabolomics“ bezeichnet wird. “Omics”- Technologien haben das Ziel, ohne Selektionskriterien möglichst alle Bestandteile einer biologischen Probe zu detektieren (identifizieren und quantifizieren), um daraus Rückschlüsse auf nicht vorhersehbare und somit neuartige Korrelationen in biologischen Systemen zu ziehen. Ein zentrales Dogma in der Biologie besteht in der Kausalität zwischen Gen – Enzym – Metabolite. Perturbationen auf einer Ebene rufen systemische Antworten hervor, die in einem veränderten Phänotyp münden können. Metabolite sind die Endprodukte von zellulären regulatorischen Prozessen, deren Abundanz durch die Resonanz auf genetische Modifikationen oder Umwelteinflüsse zurückzuführen ist. Zudem repräsentieren Metabolite ultimativ den Phänotyp eines Organismus und haben die Fähigkeit als Biomarker zu fungieren. Die integrale Analyse verschiedenster Stoffwechselwegen wie Krebszyklus, Pentosephosphatzyklus oder Calvinzyklus offeriert die Identifikation von metabolischen Mustern. In dieser Arbeit wurden sowohl das targeted Profiling via GC-TOF-MS als auch das untargeted Profiling via GC-TOF-MS und LC-FT-MS als analytische Strategien genutzt, um biologische Systeme anhand ihrer Metabolite zu charakterisieren und um physiologische Muster als Resonanz auf endogene oder exogene Stimuli zu erkennen. Dabei standen die metabolische, phänotypische und genotypische Plastizität von Pflanzen im Fokus der Untersuchungen. Metabolische Varianzen eines Phänotyps reflektieren die genotyp-abhängige Resonanz des Organismus auf umweltbedingte Parameter (abiotischer und biotischer Stress, Entwicklung) und können mit sensitiven Metabolite Profiling Methoden determiniert werden. Diese Anwendungen haben unter anderem auch zum Begriff des „Environmental Metabolomics“ geführt. In Kapitel 2 wurde der Einfluss biotischer Interaktionen von endophytischen Bakterien auf den Metabolismus von Pappelklonen untersucht; Kapitel 3 betrachtet die metabolische Plastizität von Pflanzen im Freiland auf veränderte biotische Interaktionsmuster (Konkurrenz/Diversität/Artenzusammensetzung); Abschließend wurde in Kapitel 4 der Einfluss von spezifischen genetischen Modifikationen an Peroxisomen und den daraus resultierenden veränderten metabolischen Fluss der Photorespiration dargestellt. Aufgrund der sensitiven Analyse- Technik konnten metabolische Phänotypen, die nicht zwingend in einen morphologischen Phänotyp mündeten, in drei biologischen Systemen identifiziert und in einen stoffwechselphysiologischen Kontext gestellt werden. Die drei untersuchten biologischen Systeme – in vitro- Pappeln, Grünland- Arten (Arrhenatherion-Gesellschaft) und der Modellorganismus (Arabidopsis) – belegten anschaulich die Plastizität des Metabolismus der Arten, welche durch endogene oder exogene Faktoren erzeugt wurden. / A general approach to characterise biological systems offers the analysis of the metabolome, named “metabolomics”. “Omics”- technologies are untargeted approaches without any selection criteria which aim to detect every potential analyte in a sample in order to draw conclusions about new correlations in biological systems. A central dogma in biology is the causality between gene – enzyme – metabolite. Perturbations on one level are reflected in systemic response, which possibly result in a changed phenotype. Metabolites are end products of its gene expression and metabolism, whose abundance is determined as a resonance of genetic modifications or environmental disturbance. Furthermore metabolites represent the ultimate phenotype of an organism and are able to act as a biomarker. The integral analysis of distinct metabolic pathways like TCA, Pentose phosphate and Calvin cycle consequently leads to the identification of metabolic patterns. In this work targeted profiling via GC-TOF-MS as well as untargeted profiling via GC-TOF-MS and LC-FT-MS were used as analytical strategies to characterise biological systems on the basis of their metabolites and to identify physiological patterns as resonance of endogenic or exogenic stimuli. The focus of the investigations concentrates on the metabolic, phenotypic and genotypic plasticity of plants. Metabolic variance of a phenotype is reflected in the genotypic dependence response of an organism on environmental parameters which may be detected via sensitive metabolic profiling methods. In chapter 2 the influence of biotic interaction of endophytic bacteria on the metabolism of their poplar host was analyzed; chapter 3 explores the metabolic plasticity of field-grown grassland species as a consequence of biotic interaction pattern (competition / diversity / species composition); In conclusion, chapter 4 illustrates the influence of specific genetic modifications on peroxisomes and the consequent changed metabolic flux in the photorespiration pathway. Due to the sensitive analytic methods, metabolic phenotypes in all three biological systems could be identified and classified in a physiological context. The three biological systems – in vitro poplar plants, field-grown grassland species and the model organism Arabidopsis – demonstrate the plasticity of the metabolism of species in response to stimuli.