Understanding the complex metabolic networks present in organisms, through the use of high throughput liquid chromatography coupled mass spectrometry, will give insight into the physiological changes responding to stress. However the lack of a proper work flow and robust methodology hinders verifiable biological interpretation of mass profiling data. In this study a novel workflow has been developed. A novel Kernel based feature alignment algorithm, which outperformed Agilent’s Mass profiler and showed roughly a 20% increase in alignment accuracy, is presented for the alignment of mass profiling data. Prior to statistical analysis post processing of data is carried out in two stages, noise filtering is applied to consensus features which were aligned at a 50% or higher rate. Followed by missing value imputation a method was developed that outperforms both at model recovery and false positive detection. The use of parametric methods for statistical analysis is inefficient and produces a large number of false positives. In order to tackle this three non-parametric methods were considered. The histogram method for statistical analysis was found to yield the lowest false positive rate. Data is presented which was analysed using these methods to reveal metabolomic changes during plant pathogenesis. A high resolution time series dataset was produced to explore the infection of Arabidopsis thaliana by the (hemi) biotroph Pseudomonas syringe pv tomato DC3000 and its disarmed mutant DC3000hrpA, which is incapable of causing infection. Approximately 2000 features were found to be significant through the time series. It was also found that by 4h the plants basal defence mechanism caused the significant ‘up-regulation’ of roughly 400 features, of which 240 were found to be at a 4-fold change. The identification of these features role in pathogenesis is supported by the fact that of those features found to discriminate between treatments a number of pathways were identified which have previously been documented to be active due to pathogenesis

Issue d’un partenariat de recherche entre le CEA et les laboratoires Servier, cette thèse avait pour objectif d’évaluer l’approche métabolomique par chromatographie liquide couplée à la spectrométrie de masse (LC-MS) pour l'identification de marqueurs indirects de l'induction dans les espèces de toxicologie. Le travail de thèse a débuté par l’optimisation de la méthode d’acquisition des empreintes métaboliques tant sur le plan analytique que dans le domaine du traitement des données brutes. Un outil reposant sur les matrices d’auto corrélation a alors été développé afin de s’affranchir d’une partie de la redondance du signal obtenu par spectrométrie de masse. Dans un troisième temps, les indices de Kendrick couplés à la sélectivité méthylène ont été appliqués à l’étude de composés biologiques en spectrométrie de masse haute résolution afin de proposer une méthode alternative de visualisation des données offrant une aide à l’identification des variables. Enfin, dans une dernière partie, les efforts se sont portés sur l’identification des composés endogènes modifiés au cours du protocole d’induction. / This work is the result of a research partnership between the CEA and Les laboratories Servier. It deals with the characterization of biomarkers of metabolic enzyme induction in rat biofluids using MSbased metabolomics. The first part of this work included methodological developments regarding theacquisition and the processing of metabolic fingerprints. A tool based on autocorrelation matrices wasthen implemented to reduce the redundancy of data generated with mass spectrometry and subsequently accelerate the isolation of discriminating variables. The next step consisted in the evaluation of the combined use of Kendrick mass defects and methylene selectivity as an alternative visualization tool for large data set, which would rely on compound chemical structures. Finally, the last part of the work was dedicated to the identification of discriminating signals raised up by ametabolomic global approach from rat biofluids collected before and after an induction assay.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The study of plants on a sub-cellular level is an important, yet challenging area and its application allows for novel insight into the understanding of metabolism and its regulation. In this study I describe the development of a reverse phase liquid chromatography mass spectrometry (RPLC-MS) technique in which 29 phosphorylated and nucleotide sugars could be detected and quantified. The method was validated with the use of authentic standards and the system displayed very good linearity (Rª > 0.95), while the recovery of the standards added to the plant material before extraction was between 65 and 125%. Further, Arabidopsis thaliana wild type (Col-0) and adenylate kinase (adk1) mutant leaf discs were fed 13C labeled glucose over a period of 24 hours and harvested at defined time intervals. Non aqueous fractionation, and metabolite profiling via the above mentioned rpLC-MS method in conjunction with gas chromatography mass spectrometry (GC-MS) allowed for the detection and quantification of primary metabolites on a sub-cellular level as well as the determination of their relative isotopic label enrichments through primary carbon metabolism. Finally, a yeast complementation system was designed for the identification of tonoplast bound sucrose import proteins. The screening system identified 22 unique sequences from an Arabidopsis thaliana cDNA library. Four unknown sequences were identified, one of which displayed tonoplast membrane association upon in silico analysis. Three ATP-binding proteins were also identified as well as a sub-unit from the exocyst gene family. Further studies will include the functional characterization of the latter, as well as the development of additional cDNA libraries more suited for screening of sequences that encode sucrose importer proteins. / AFRIKAANSE OPSOMMING: Die studie van plante op a sub-sellulere vlak is ‘n belangrike maar uitdagende navorsingsarea en die toepassing daarvan dra by tot unieke insig tot ‘n beter begrip van metabolise regulasie. In die studie bespreek ek die ontwikkeling van ‘n teenoorgestelde fase vloeistof kromatografie massa spektrometrie (RPLC-MS) tegniek waarin 29 gefosforileerde en nukleotied suikers gevind en gekwantifiseer kon word. Geldigverklaring van die metode is bewerkstelling met die gebruik van oorspronklike standaarde and die systeem het baie goeie liniariteit (Rª > 0.95) getoon, terwyl die herstelbaarheid van standaarde wat bygevoeg is by die plant material voor ekstraksie tussen 65% en 125% was. Arabidopsis thaliana wilde type (Col-O) en die adenaliet kinase (adk1) mutant blaar dele is met 13C gemerkte glukose gevoed oor ‘n tydperk van 24 uur en geoes by spesifieke tydstippe. Nie-vloeibare fraksionering en metaboliet uitleg is vermag vanaf die genoemde RPLC-MS metode met behulp van gas kromotografie massa spektrometrie (GC-MS) wat die bepaling en kwantifikasie van primere metaboliete op n sub-sellulere vlak sowel as die bepaling van hul relatiewe isotropiese merker verrykers deur primere metabolisme toelaat. Verder is n gis komplementere systeem ontwerp vir die identifikasie van tonoplas gebinde sukrose invoer proteine. Die verkenningsysteem het 22 unieke volgordes opgelewer vanaf ‘n Arabidopsis thaliana kDNA biblioteek. Vier onbekende volgordes is geidentifiseer, een wat tonoplas membraan assosiasie toon met in silico analise. Drie ATP-bindings proteine is ook geidentifiseer asook ‘n sub-eenheid van die eksosyst geen familie. Verdere studies sal die funksionele karakterisering van die laaste protein insluit, asook die ontwikkeling van additionele kDNA biblioteke meer gepas vir verkenning sodiende identifiseer van volgordes wat sukrose invoer proteine vertaal.

Refrigerated Stability of Diluted Succinylcholine, Pancuronium, and Atracurium. T. Archibald1, S. Brown1, A. Gonzalez-Estrada2 1College of Pharmaceutical Sciences, Bill Gatton College of Pharmacy, East Tennessee State University, Johnson City, TN2Quillen College of Medicine, Allergy and Clinical Immunology, East Tennessee State University, Johnson City, TN The purpose of this study is to investigate the stored stability of dilutions of neuromuscular blocking agents (NMBAs), namely succinylcholine, pancuronium, and atracurium, for skin prick/intradermal testing. Concentrations of NMBAs were monitored by liquid chromatography-mass spectrometry (LC-MS/MS) for a period of 14 days. Dilutions of NMBAs were prepared in saline by factors of 10x, 100x, 1,000x, 10,000x, and 100,000x as sensitivity of the assay allowed. Each drug was prepared with an n = 5 for each dilution, using a different newly opened product for each series. Diluted drug products were stored in a laboratory refrigerator until sampling. On sampling days (day 0, 1, 2, 4, 7, and 14), one milliliter aliquots of each dilution were removed, filtered, and analyzed against freshly prepared set of reference dilutions. The results are expressed as beyond use date (BUD), defined as recovery of drug versus the reference (90-110%). Based on the LC-MS/MS data, the BUD for succinylcholine diluted by 10x and 100x is 48 and 24 hours, respectively. The1000x dilution is also stable for 24 hours.Higher dilutions of succinylcholine (10,000x to100,000x) should be used immediately following preparation (within less than 24 hours), as the potency of these dilutions had decreased below 90% at the 24 hr sampling. .Pancuronium diluted by 10x and 100x, had a BUD of 48 hours, and the1,000x dilution was stable for 24 hours.As with the succinylcholine, the 10,000x and 100,000x dilutions expressed potency of <90% at 24 hours. .Atracurium diluted to 10x had a BUD of 96 hours, the100x dilution is stable for 24 hours yet higher dilutions (1,000x to 10,000x) do not persist beyond 24 hours. . The 100,000x dilution of atracurium was unknown, given than the signal intensity was too weak to monitor by our LC-MS/MS method. With increasing dilution factors, the stability of these drugs in saline decreases, associated with an increasing deviation between samples and freshly prepared references. The most stable dilutions for each of the drugs tested were 10x and 100x. Stability of these drugs is likely compromised by hydrolysis of the ester bonds in the drug molecules.

Tocopheryl Polyethylene Glycol Succinate 1000 (TPGS 1000) can inhibit P-glycoprotein (P-gp); TPGS 1000 was not originally designed to inhibit an efflux pump. Recent work from our laboratories demonstrated that TPGS activity has a rational PEG chain length dependency. In other recent work, inhibition mechanism was investigated and appears to be specific to the ATPase providing P-gp energy. Based on these observations, we commenced rational surface-active design. The current work summarizes new materials tested in a validated Caco-2 cell monolayer model; rhodamine 123 (10 μM) was used as the P-gp substrate. These results demonstrate that one may logically construct non-ionic surfactants with enhanced propensity to inhibit in vitro efflux. One new surfactant based inhibitor, Tocopheryl Polypropylene Glycol Succinate 1000 (TPPG 1000), approached cyclosporine (CsA) in its in vitro efflux inhibitory potency. Subsequently, TPPG 1000 was tested for its ability to enhance the bioavailability of raloxifene - an established P-gp substrate - in fasted male rats. Animals dosed with raloxifene and TPPG 1000 experienced an increase in raloxifene oral bioavailability versus a control group which received no inhibitor. These preliminary results demonstrate that one may prepare TPGS analogs that possess enhanced inhibitory potency in vitro and in vivo.

No description available.

The development of a biologic protein involves different stages and becomes a highly complex process which can be costly and time consuming to scale up for industrial production. Therefore, optimization is a necessary part of the production process development to lower the production expenses.An on-going project is working on upscaling the production of a protein derived from mussel adhesive proteins (MAPs) which has great properties to be used as a pharmaceutical drug or in medical devices. The protein is expressed in a bacterial host cell and the necessary post translational modifications (PTMs) are done in-vitro using enzymes. The work presented in this report was done to optimize both the protein production in lab scale bioreactors and the enzymatic reaction using an immobilized prolyl-4-hydroxylase (P4H) which does a post translational modification on prolyl-residues. Additionally, an interaction study was conducted to better understand the hydroxylation using the prolyl-4-hydroxylase.For the bioreactor optimization four initial trials were performed testing different growth and induction temperatures and also comparing exponential to linear feeding. From these trials it appeared that having 30 ℃ growth overnight and induction at the same temperature in combination with an exponential feeding rate gave the best results. The modifications done by the prolyl-4-hydroxylase were analysed by LC-MS and suggest that longer incubation time and more immobilized protein gives more modifications in the tested ranges and the possibilities of reusing the immobilized proteins looks promising. No conclusive data was discovered for the optimal substrate concentration. The interaction study revealed the importance of reagents used for catalysis with the enzyme to be present for interaction to occur, however more work needs to be done to discover an accurate KD for the interaction.

(1) Introduction: Sulfonates, which can be diet- or host-derived, are a class of compounds detected in the gut, are involved in host–microbiome interactions and have several health effects. Our aim was to develop a method to quantify five of the sulfonates in the intestine and apply it in a simplified human microbiome model. These were taurine, its metabolic precursor cysteate and one of its degradation products isethionate, as well as sulfoquinovose and one of its most relevant degradation products 2,3-dihydroxy-1-propanesulfonate. (2) Methods: An extraction and sample preparation method was developed, without the need for derivatization. To detect and quantify the extracted sulfonates, a multiplexed LC-MS/MS-MRM method was established. (3) Results: The accuracy and precision of the method were within GLP-accepted parameters. To apply this method in a pilot study, we spiked either taurine or sulfoquinovose into an in vitro simplified human microbiota model with and without Bilophila wadsworthia, a known sulfonate utilizer. The results revealed that only the culture with B. wadsworthia was able to degrade taurine, with isethionate as an intermediate. After spiking the communities with sulfoquinovose, the results revealed that the simplified human microbiome model was able to degrade sulfoquinovose to 2,3-dihydroxypropane-1-sulfonate, which was probably catalyzed by Escherichia coli. In the community with B. wadsworthia, the 2,3-dihydroxypropane-1-sulfonate produced was further degraded by B. wadsworthia to sulfide. (4) Conclusions: We successfully developed a method for sulfonate quantification and applied it in a first pilot study.

The naturally occurring dipeptide carnosine (-alanyl-L-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography–mass spectrometry. In addition, we studied carnosine’s effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.

Activated sludge treatment systems rely on a microbial consortium structurally organized in bioflocs to treat pollutants present in wastewater. The treatment process efficiency in these systems can be severely affected by toxic chemicals present in the influent wastewater. The effects of chemical toxins at the treatment process level are determined by the mechanisms that occur at the biofloc and cellular levels, which can be physical, chemical and physiological in nature. We believe that the overall process effects of chemical toxins on activated sludge systems likely result from a combination of all three types of mechanisms and that they are interdependent, in the sense that specific bacterial stress response mechanisms (physiological mechanisms that protect the cell from toxic conditions) may lead to physical/chemical alterations at the floc level, and vice-versa. Ultimately, understanding the mechanisms that occur at the floc and metabolic scales will help to design more robust and efficient treatment systems, and to develop tools to prevent and mitigate the effects of toxic chemicals on activated sludge systems. In this research, we set out to establish the link between the effects of chemical toxins on activated sludge cultures at the process, floc and metabolic scales. First, the effects of shock loads of different toxic sources (1-chloro-2,4-dinitrobenzene (CDNB), cadmium, 1-octanol, 2,4-dinitrophenol (DNP), weakly complexed cyanide, pH 5, 9 and 11, and high ammonia levels) on activated sludge process parameters (biomass growth, respiration rate, flocculation, chemical oxygen demand (COD) removal, dewaterability and settleability) were studied. For all chemical shocks except ammonia and pH, concentrations that caused 15, 25 and 50% respiration inhibition were used to provide a single pulse chemical shock to sequencing batch reactor (SBR) systems containing a nitrifying (10 day solids retention time – SRT) and a non-nitrifying (2 day SRT) biomass. We found that cadmium and pH 11 shocks were the conditions that most detrimentally affected all the processes, followed by CDNB. DNP and cyanide primarily led to effects on respiration, while pH 5, 9, octanol and various ammonia concentrations did not impact the treatment process to a significant extent. Additionally, there was a clear correlation between biomass deflocculation and increases in the effluent soluble COD of the shocked reactors for different chemical sources. With this study, we were able to establish a source-effect matrix linking classes of chemical toxins to their potential inhibitory effects on activated sludge processes, thereby contributing to a better understanding of the potential effects of toxic industrial discharges into biological treatment systems. The findings of the first phase of the research, specifically the correlation between chemical-induced deflocculation and increases in soluble COD, served as a motivation to explore the role of floc structure in the response of activated sludge cultures to toxic compounds, and to conduct a more in-depth analysis of the supernatant (soluble phase) of toxin-exposed activated sludge. In one study, we evaluated the respiration inhibition induced by octanol, cadmium, N-ethylmaleimide (NEM), cyanide and DNP on activated sludge biomasses with different floc structures but similar physiological characteristics, with the objective of assessing the role of the extracellular polymeric substances (EPS) in flocs as a protection barrier against chemical toxins. Mechanical shearing was applied to fresh mixed liquor to produce biomasses with different floc structure properties and specific oxygen uptake rate assays were conducted on the sheared and unsheared mixed liquors. The results showed that the respiration inhibition by octanol and cadmium was more intense in sheared mixed liquor (which had less EPS material available in the flocs and smaller floc sizes) than in the unsheared biomass. Conversely, the respiration inhibition induced by NEM and cyanide was similar for the different mixed liquors tested. These results allowed us to conclude that the EPS matrix functions as a protective barrier for the bacteria inside activated sludge flocs to chemicals that it has the potential to interact with, such as hydrophobic (octanol) and positively-charged (cadmium) compounds, but that the toxicity response for soluble, hydrophilic toxins (NEM and cyanide) is not significantly influenced by the presence of the polymer matrix. In the final study that was conducted, we used the metabolomics-based technique metabolic footprinting to assess if the soluble phase of mixed liquor exposed to different chemical toxins exhibited a toxin-specific biochemical composition. We hypothesized that toxin-specific effects could be distinguished through footprint patterns of those soluble samples. The impact of cadmium, DNP and NEM shock loads on the composition of the soluble fraction of activated sludge mixed liquor was analyzed by liquid chromatography-mass spectrometry (LC-MS). The results from this study indicated that there was a significant release of biomolecules (proteins, carbohydrates and humic acids) from the floc structure into the bulk liquid due to chemical stress. More importantly, using a multivariate statistical method called discriminant function analysis with genetic algorithm variable selection (GA-DFA), we were able to show that the soluble phase samples from the different reactors could be differentiated, thereby indicating that the footprints generated by LC-MS were different for the four conditions tested and, therefore, toxin-specific. These footprints, thus, contain information about specific biomolecular differences between the samples, and we found that only a limited number of m/z (mass to charge) ratios from the mass spectra data was needed to differentiate between the control and each chemical toxin-derived samples. In addition, since the experiments were conducted with mixed liquor from four distinct wastewater treatment plants, the discriminating m/z ratios may potentially be used as universal stress biomarkers. These results are promising and indicate that LC-MS may be used for the discovery of activated sludge stress biomarkers, to allow the development of new toxin detection technologies for prevention of upset events in activated sludge systems. / Ph. D.