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Nové postupy v metabolomické analýze biotekutin / New procedures in metabolomic analysis of biofluidsŘimnáčová, Lucie January 2017 (has links)
The aim of this thesis has been the research on reactivity of protic metabolites with chloroformates and its application for GC-MS analysis of biofluids. The research was conducted in three separate studies and the results are three new, original methods for GC-MS determination of low- molecular protic metabolites in biological material especially in biofluids. The first study explores the discovery of fast derivatization of alicyclic hydroxyl groups by fluoroalkyl chloroformates (FCFs) under anhydrous conditions [1]. FCF fully converts the hydroxyl group into a corresponding carbonate and the step can easily be coupled with liquid-liquid microextraction (LLME) of the arising derivatives into organic phase. The reaction of the alicyclic OH group with FCFs was tested on 12 clinically relevant steroids and 4 tocopherols. The analytical properties of determined analytes were described and the method was validated for the GC-MS determination of 6 diagnostic sterols and 4 tocopherols in human serum and amniotic fluid. The new method was further successfully used for determination of sterols and tocopherols in tissues of the bug Pyrrhocoris apterus [2]. The second study was focused on the reactions of protic, particularly urinary metabolites with FCFs, mainly heptafluorobutyl chloroformate (HFBCF)....
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Use of metabolomic studies to understand the chemical role of ETHE1 in Arabidopsis thalianaWipulaguna, M.A. Anushika Shiromi 03 December 2014 (has links)
No description available.
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Systems Biology in an Imperfect World: Modeling Biological Systems with Incomplete InformationPokrzywa, Revonda Maria 11 November 2009 (has links)
One of the primary goals of systems biology is to understand the complex underlying network of biochemical interactions which allow an organism to respond to environmental stimuli. Models of these biological interactions serve as a tool to both codify current understanding of these interactions as well as a starting point for scientific discovery. Due to the massive amount of information which is required for this modeling process, systems biology studies must often attempt to construct models which reflect the whole of the system while having access to only partial information. In some cases, the missing information will not have a confounding effect on the accuracy of the model. In other cases, there is the danger that this missing information will make the model useless.
The focus of this thesis is to study the effect which missing information has on systems level studies within several different contexts. Specifically, we study two contexts : when the missing information takes the role of incomplete molecular interaction network knowledge and when it takes the role of unknown kinetic rate laws. These studies yield interesting results. We show that when metabolism is isolated from gene expression, the effects are not limited to those reactions under strong control by gene expression. Thus, incomplete understanding of molecular interaction networks may have unexpected effects on the resulting analysis. We also reveal that under the conditions of the current study, mass action was shown to be the superior substitute when the true rate equations for a biological system are unknown.
In addition to studying the effect of missing information in the aforementioned contexts, we propose a method for limiting the parameter search space of biochemical systems. Even in ideal scenarios where both the molecular interaction network and the relevant kinetic rate equations are known, obtaining appropriate estimates for the unknown system parameters can be challenging. By employing a method which limits the parameter search space, we are able to acquire estimates for parameter values which are much closer to the true values than those which could be obtained otherwise. / Ph. D.
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The development of cellular metabolomic platforms and their applicationsFei, Fan January 2016 (has links)
In this thesis, an analytical platform was designed and applied to various in vitro bacterial and eukaryotic cell cultures. An extraction and an analytical protocol were developed for comprehensive and simultaneous analysis of both lipid and polar metabolites for intra- and extracellular metabolomics using HILIC-LC-TOF-MS. This analytical platform was applied to four diverse research questions such as the effect of oxygen environment on growth, the interplay between gene expression and metabolism, metabolic changes that occur with age, and PAH toxicity. Specifically: (i) the effect of oxygen on the growth, physiology and metabolism of the Gram positive Streptococcus intermedius were investigated by comprehensive intra- and extracellular metabolomes and transcriptome. (ii) Metabolic insights into the role of the multipartite genome of the Gram negative bacteria Sinorhizobium meliloti and its metabolic preferences in a nutritionally complex environment. (iii) Age-associated metabolic dysregulation in murine bone marrow-derived macrophages during bacterial lipopolysaccharide-induced inflammation. (iv) Comprehensive intracellular metabolomic profiles of Sinorhizobium meliloti to sub-lethal exposure of individual or mixtures of polycyclic aromatic hydrocarbon revealed additive and dose-dependent effects. This thesis has demonstrated the versatility of the designed analytical platform and its use for diverse research in cell biology. / Thesis / Doctor of Philosophy (PhD)
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A metabolomics approach investigating the functionality of the ESX-1 gene cluster in mycobacteria / Conrad Cilliers SwanepoelSwanepoel, Conrad Cilliers January 2015 (has links)
Tuberculosis (TB) claims the lives of millions of individuals each year, and is
consequently the world’s second-most deadly infectious disease after acquired
immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010
alone. Developing countries carry the heaviest burden, with the occurrence of
multidrug-resistant (MDR) TB becoming more frequent, making more efficient
vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1
gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and
the Type Vll secretion system are thought to be responsible for the transport of
extracellular proteins across the hydrophobic, and highly impermeable, cell wall of
Mycobacterium, and consequently are thought to play a role in the virulence of this
organism. To date, our understanding of tuberculosis pathophysiology and virulence
has been described primarily using proteomic and genomic approaches.
Subsequently, using the relatively new research approach called metabolomics, and
interpreting the data using systems biology, we aimed to identify new metabolite
markers that better characterise virulence and the proteins involved, more
specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS
metabolomics research approach, we compared the varying metabolomes of M.
smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and
subsequently identified those metabolite markers differing between these strains.
Multivariate and univariate statistical analyses of the analysed metabolome were
used to identify those metabolites contributing most to the differences seen between
the two sample groups. A general increase in various carbohydrates, amino acids
and lipids, associated with cell wall structure and function, were detected in the
ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites
associated with the antioxidant system, virulence protein formation and energy
production in these mycobacteria, were also seen to differ between the two groups.
This metabolomics investigation is the first to identify the metabolite markers
confirming the role of the ESX-1 gene cluster with virulence and the underlying
metabolic pathways, as well as its associated role with increased metabolic activity,
growth/replication rates, increased cell wall synthesis and an altered antioxidant
mechanism, all of which are believed to contribute to this organism’s increased
pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015
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A metabolomics approach investigating the functionality of the ESX-1 gene cluster in mycobacteria / Conrad Cilliers SwanepoelSwanepoel, Conrad Cilliers January 2015 (has links)
Tuberculosis (TB) claims the lives of millions of individuals each year, and is
consequently the world’s second-most deadly infectious disease after acquired
immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010
alone. Developing countries carry the heaviest burden, with the occurrence of
multidrug-resistant (MDR) TB becoming more frequent, making more efficient
vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1
gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and
the Type Vll secretion system are thought to be responsible for the transport of
extracellular proteins across the hydrophobic, and highly impermeable, cell wall of
Mycobacterium, and consequently are thought to play a role in the virulence of this
organism. To date, our understanding of tuberculosis pathophysiology and virulence
has been described primarily using proteomic and genomic approaches.
Subsequently, using the relatively new research approach called metabolomics, and
interpreting the data using systems biology, we aimed to identify new metabolite
markers that better characterise virulence and the proteins involved, more
specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS
metabolomics research approach, we compared the varying metabolomes of M.
smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and
subsequently identified those metabolite markers differing between these strains.
Multivariate and univariate statistical analyses of the analysed metabolome were
used to identify those metabolites contributing most to the differences seen between
the two sample groups. A general increase in various carbohydrates, amino acids
and lipids, associated with cell wall structure and function, were detected in the
ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites
associated with the antioxidant system, virulence protein formation and energy
production in these mycobacteria, were also seen to differ between the two groups.
This metabolomics investigation is the first to identify the metabolite markers
confirming the role of the ESX-1 gene cluster with virulence and the underlying
metabolic pathways, as well as its associated role with increased metabolic activity,
growth/replication rates, increased cell wall synthesis and an altered antioxidant
mechanism, all of which are believed to contribute to this organism’s increased
pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015
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Data analysis of non-targeted mass spectrometry experimentsTengstrand, Erik January 2015 (has links)
Data processing tools are valuable to the analytical chemist as they can speed up the analysis, and sometimes solve problems that are not feasible to solve in a traditional manner. However, the complexity of many data processing tools can make their use daunting for the inexperienced user. This thesis includes two applications and two tools for data processing. The first application focuses on minimizing the manual input, reducing the time required for a simple task. The second application required more manual input, in the form of parameter selection, but process far more data. The data processing tools both include features that simplify the manual work required. The first by including visual diagnostics tools that helps in setting the parameters. The second via internal validation that makes the tool’s process more robust and reliable, and thereby less sensitive to small changes in the parameters. No matter how good or precise a data processing tool is, if it is so cumbersome that it is not used by the analytical chemists that need it, it is useless. Therefore, the main focus of this thesis is to make data processing easier. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.</p>
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Metallothionein involvement in mitochondrial function and disease : a metabolomics investigation / Jeremie Zander LindequeLindeque, Jeremie Zander January 2011 (has links)
One of the many recorded adaptive responses in respiratory chain complex I deficient cells is the
over-expression of the small metal binding proteins, metallothioneins (MTs). The antioxidant
properties of MTs putatively protect the deficient cells against oxidative damage, thus limiting
further damage and impairment of enzymes involved in energy production. Moreover, the role of
metallothioneins in supplying metal cofactors to enzymes and transcription factors in order to
promote energy metabolism was previously proposed, which could accompany their role as
antioxidants. This view is supported by the observations that MT knockout mice tend to become
moderately obese, implying a lower energy metabolic rate. Hence, the involvement of
metallothioneins in mitochondrial function and disease cannot be ignored. However, this
association is still very vague due to the diversity of their functions and the complexity of the
mitochondrion. The use of systems biology technology and more specifically metabolomics
technology was thus employed to clarify this association by investigating the metabolic differences
between wild type and MT knockout mice in unchallenged conditions as well as when
mitochondrial function (energy metabolism) was challenged with exercise and/or a high-fat diet.
The metabolic differences between these mice were also studied when complex I of the respiratory
chain was inhibited with rotenone. The metabolome content of different tissues and bio-fluids were
examined in an untargeted fashion using three standardized analytical platforms and the data
mined using modern metabolomics and related statistical methods. Clear metabolic differences
were found between the wild type and MT knockout mice during unchallenged conditions. These
metabolic differences were persisted and were often amplified when mitochondrial metabolism was
specifically challenged through exercise, high-fat intake or complex I inhibition. The data pointed to
an overall reduced metabolic rate in the MT knockout mice and possible insulin resistance after the
interventions which imply (and confirm) the involvement of MTs in promoting energy metabolism in
the wild type mice. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012
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A multidisciplinary study of human exposure to arsenic and other trace elementsCascio, Claudia January 2011 (has links)
Arsenic (As) is a carcinogenic agent that is present in varying levels in environmental matrices including water and food. Long term As exposure can lead to skin lesions, peripheral neuropathy, diabetes, renal system effects and cardiovascular diseases. Bio-monitoring of human urine, toenail, serum and cerebrospinal fluid was carried out in this thesis to assess the exposure to arsenic and other trace elements. A multidisciplinary approach based on Inductively Coupled Plasma Mass Spectrometry (ICP-MS), HPLC-ICP-MS and Proton-Nuclear Magnetic Resonance Spectroscopy (1H-NMR) in conjunction with a questionnaire based survey was employed. The impact of rice consumption (a well-known vector of arsenic in the general population) on human urinary As levels was assessed. Results obtained show that the Bangladeshi (UK-B) community in the United Kingdom, who consume ca. 30-fold more rice than the white Caucasians (UK-C), are exposed to a higher level of arsenic. ICP-MS and HPLC-ICP-MS revealed a significant increase in dimethylarsinic acid (DMA) and inorganic arsenic (iAs) species in UK-B compared to UK-C, while cationic compounds were lower in UK-B than in UK-C. DMA and iAs levels in the Bangladeshis were positively correlated to rice consumption. Rice is likely to be responsible for the increase in levels of DMA and iAs in urine of UK-B. The link between this and the disproportional occurrence of diabetes and cardiovascular diseases (CVD) in UK-B needs to be investigated. Another important finding of this study is that the DMA to monomethyl arsenic (MA) ratio, which is often used as an indication of arsenic methylation capacity, should be applied with caution in populations consuming large quantities of rice because variation in the quantity and type of rice eaten may alter the urinary DMA levels and thereby the DMA/MA ratio. Urinary arsenic, selenium, copper and zinc were monitored for a group of Bangladeshis, Pakistanis, Indians and Caucasians living in the UK. The most striking finding was the increase in urinary copper in the UK-B group compared to other ethnicities and to reference values reported for the general UK population. Among the possible reasons for this could include dietary exposure via ethnic food consumption or a change in copper metabolism in the Bangladeshis. High serum copper levels have been correlated to CVD in the US population. In this context, further work is recommended to investigate if there is a relationship between urinary copper and the disproportionately high incidence of CVD in UK Bangladeshis. An approach based on 1H-NMR was used to detect changes in human urinary metabolomic profile as a function of As exposure through different routes. For this, the urine of UK-B, UK-C and a group residing in Bangladesh (BD-B) were monitored. The effects of other factors were explored, including arsenic urinary profile, chewing pan, ethnicity, rice consumption, selenium and diabetes. The three populations show distinctive metabolomic profiles. Urinary arsenic speciation was used in evaluating the effects of arsenic on the metabolomic profile for the UK group. This revealed that the %DMA positively correlates to %N,N-dymethylglycine, %alanine and %betaine. Comparative analysis of the 1H NMR spectra revealed that the BD-B urinary profiles were depleted in the number and quantity of metabolites. Visible signs of lower protein intake and undernourishment emerged from the urinary metabolomic profile of BD-B including a 2.5 decrease in creatinine levels compared to UK-B. Urinary creatinine and the metabolomic profile provide evidence for undernourishment in the BD-B population group that was not evident from previous studies on dietary protein intake in this population performed using food frequency questionnaires. Public health officials might consider also using bio-monitoring studies for nutrient intake rather than solely relying on estimations from food frequency questionnaires. The results reveal the complexity of the subject and pave the way for future studies, highlighting the need for awareness about diet and other specific confounding factors. Multiple Sclerosis (MS) is considered a multifactorial disease and its cause remains unknown. A case-control study on a MS cluster from the volcanic region of Mt. Etna (a natural emitter of geogenic trace elements in the environment) was undertaken. Urine and toenails were monitored for trace elements along with food consumption and life-style habits. Levels of a range of trace elements were reported for the first time for a population living in the Mt. Etna region. No significant differences were found in trace element levels in urine and toenails of MS patients and controls. However, urinary levels of nickel, manganese and selenium were higher than those reported in the literature for the general population from Italy, Germany and the UK. These findings and observations might suggest a role for nickel in the pathology of MS. However, larger studies on the possible role of nickel on MS, and trace elements in general, should be performed. Cerebrospinal fluid (CSF) and some serum from MS patients and controls from the Mt. Etna region were also monitored in this study using ICP-MS. There were significant differences in the trace elemental profile of CSF of MS volunteers and controls, including an increase in arsenic and zinc in the CSF of MS patients. Lead, aluminium, cadmium and molybdenum were significantly increased in the CSF of MS patients as well. In contrast, selenium was lower in MS patients compared to controls. The enrichment of certain trace elements in the CSF of MS patients could be the result of an impairment of the blood brain barrier and tight junction disruption due to MS and its progression, resulting in serum protein leakage and trace elements across the blood–brain barrier. Studies are necessary in the future to identify the chemical species present in the CSF and also determine their role in biological processes including their harmful effects on the brain.
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Metabolic profiling and imaging of CHO cells for fusion protein productionSzula, Ewa January 2017 (has links)
Fc-fusion proteins (e.g. EPO-Fc) are the most often created fusion proteins due to their beneficial biological and pharmacological properties. The economic success of Fc-fusion proteins and other biopharmaceuticals production however, greatly depends on a robust, low-cost and highly effective protein mammalian cell extraction system . Understanding of how cells respond to a protein production environment based on metabolic profiles provides new goals for bioengineering of cell lines for best performance in biomanufacturing. Furthermore, insights on how individual cell metabolism and therefore phenotype, respond to cell microenvironment allows the underlying biological mechanisms to be explored in greater detail. This study focused on the application of mass spectrometry (MS) technologies, combining the analysis of metabolic profiles of cells extracts by GC-MS and MALDI-MS and spatial visualisation and distribution of metabolites within cells producing the fusion protein by MALDI-MSI and SIMS imaging. The analysis of external and internal metabolome profiles of cells producing the protein showed an extended effect of EPO-Fc fusion protein production on cell metabolism. The findings indicate that changes observed in EPO-Fc producing cells are related to enhanced protein and lipid synthesis highlighting that these cells are in a state of increased metabolic activity with the protein exocytosis into growth medium. Moreover, the composition of lipid bilayer of induced cells seemed to be different to non-induced cells. These findings were confirmed with the analysis of EPO-Fc induced cells using MS metabolic imaging. Multivariate analysis highlighted a number of metabolites that were significantly influenced by the protein expression when compared to control cells. The major metabolic changes in induced cells were those related to lipid metabolism. The information about metabolic changes in tetracycline-induced cells obtained from the analysis of cell populations was further supported with the analysis based on single-cell studies. Single-cell based studies also proved that investigations of individual cells provide additional insights about changes in metabolism of induced cells that can be referred to a unique, single cell and its phenotype. The analysis of CHO cells revealed a high level of heterogeneity within a cell population. Different cell phenotype and hence, metabolite content allowed for correlation between cell locations and their metabolite characteristics, specific for each type of cells. This project has successfully shown combination of bio-analytical techniques to investigate external and internal metabolome changes related to a fusion protein production in mammalian cells. Additionally, the significance of single cell approaches in metabolomics has also been highlighted, providing insights into the sub-cellular distribution of metabolites in cells producing EPO-Fc and information on the level of heterogeneity within a cell population. A multidimensional approach for metabolic profiling and future technological improvements of single-cell platforms are required to provide improved data acquisition and data analysis in order to better understand unknown processes involved in cell metabolism.
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