In this thesis, "culture" refers to the collection of subjective human traits, such as preferences an opinions, that a given, geographically bounded population has at a given moment in time.... Show moreIn this thesis, "culture" refers to the collection of subjective human traits, such as preferences an opinions, that a given, geographically bounded population has at a given moment in time. Representative samples of individuals from such populations are studied, focusing on individual opinions expressed on various topics, present in multivariate empirical data that had been previously collected, mainly via social surveys. We propose and exploit new methods for analyzing such data, relying on mathematical notions specific to statistical mechanics and information theory, but also on agent-based models/simulations of opinion/cultural dynamics driven by social influence. These methods provide new insights about how human culture is organized. They provide indications that cultural structure has universal properties, independent of the geographical region and of the set of survey questions. Furthermore, these properties suggest that culture is shaped around a small number of "rationalities", while also having a certain hierarchical organization that is robust to social influence dynamics. Finally, we propose a method of filtering the noise in the data, which seems to allow for the identification of cultural modules that are not visible otherwise. However, we also show that visible modules may well be just artifacts of survey design. Show less
Inflammation is an immune reaction of the body to the external stimuli such as toxins or pathogens, and is characterized by redness, swelling, pain, and heat. The process of inflammation is... Show moreInflammation is an immune reaction of the body to the external stimuli such as toxins or pathogens, and is characterized by redness, swelling, pain, and heat. The process of inflammation is regulated by several pro-inflammatory and antiinflammatory cytokines. Tumor necrosis factor-_ (TNF-_) is a major pro-inflammatory cytokine involved in the inflammatory response.Elevated TNF-_ expression has been found to be associated with the development of diabetes, atherosclerosis, septic shock, and tumorigenesis. Thus inhibition of TNF-_ at any step of inflammatory pathways provides an attractive treatment for inflammatory diseases as well as for series of other common diseases.Plants provide an alternative sources of medicines used traditionally by people worldwide since thousands of years ago. The aim of this thesis was to develop methods for the rapid identification of active compounds in plant extracts by correlating NMR metabolomics and bioassay results by means of multivariate data analysis. This work demonstrates the great potential of NMR spectroscopy in combination with chemometrics for the screening of large set of crude extracts, to study the effects of different variables on the activity, and identifying sets of active compounds in complex mixtures like plant extracts. Show less
The chapters that comprised this thesis cover a broad range of subjects from analytical method development to clinical application of metabolic profiling. They are united by the facts that all of... Show moreThe chapters that comprised this thesis cover a broad range of subjects from analytical method development to clinical application of metabolic profiling. They are united by the facts that all of these studies aimed at analysis of biological fluids and that the presented methods and approaches may ultimately become parts of a robust metabolomics workflow that might be used in a future personalized medicine. Show less
Undoubtedly, grapes and wine are globally the most important fruit and food commodities, respectively. The first objective of this research is to optimize an extraction protocol suitable for grape... Show moreUndoubtedly, grapes and wine are globally the most important fruit and food commodities, respectively. The first objective of this research is to optimize an extraction protocol suitable for grape metabolic profiling followed by the application of that protocol to perform metabolic characterization of grape cultivar, wine types, and vintage using NMR spectroscopy in combination with chemometrics methods. This approach was also used to study different physiological processes in grapevine like ripening of berry and resistance against fungal pathogen. Metabolic characterization of different white wines to highlight the metabolites responsible for sensory attributes was also carried out. Another task of this research was to correlate the metabolic profiling data from grapes and wine with the bioactivity data using various multivariate regression models. Show less
Despite the rich diversity in molecular structures and biological functionality, the complex matrix of the compounds with a broad dynamic range has limited the use of plants as an important source... Show moreDespite the rich diversity in molecular structures and biological functionality, the complex matrix of the compounds with a broad dynamic range has limited the use of plants as an important source for drug development. Adressing this issue, several papers have described the use of the more holistic approach targeting on a wide range of metabolite present in plant extract, i.e. metabolomics. This new approach involves the use of various analytical methods followed by appropriate multivariate data analysis. The aim is to shorten the bioassay guided isolation route, particularly in the identification and dereplication step This thesis describes a new strategy to improve dereplication and identification steps in drug discovery process from plants. The integration of comprehensive extraction coupled to NMR metabolomics and multivariate data analysis is found to be a potential new approach to uncovering bioactive compounds from crude plant extracts. Show less
Summary of the Thesis: Vegetables have always been considered as healthy food. So also Brassica vegetables are well known all over the world as a common food due to the presence of health affecting... Show moreSummary of the Thesis: Vegetables have always been considered as healthy food. So also Brassica vegetables are well known all over the world as a common food due to the presence of health affecting compounds (Chapter 2). A vast amount of data is available for health promoting compounds in Brassicaceae vegetables. These health promoting affects are due to a range of phytochemicals including primary (carbohydrates, amino acids and organic acid) and secondary metabolites (phenolics and glucosinolates), along with vitamins and minerals. These metabolites are interconnected through different biosynthetic pathways and are affected by different external stimuli. Plant metabolic responses are specific for different kinds of stress, but use in part similar metabolic pathways (Chapter 3). Certain internal or external factors play an important role for the metabolite profile of vegetables, thus changing the nutritional value for human (Chapter 4). These factors are related to the plant response to external stress factors and helping the plant to survive. These factors includes bacteria (Chapter 5), metals ions ( Chapter 6) and post harvest storage conditions (Chapter 7). The aim of this thesis was to study the Brassica phytochemicals and their response to stress factors by using a holistic analytical approach. Show less
It has been shown by this thesis that plant metabolomics is a promising tool for studying the interaction between B. rapa and pathogenic fungi. It gives a picture of the plant metabolites during... Show moreIt has been shown by this thesis that plant metabolomics is a promising tool for studying the interaction between B. rapa and pathogenic fungi. It gives a picture of the plant metabolites during the interaction. Brassica rapa has many defense related compounds such as glucosinolates, IAA, phenylpropanoids and flavonoids and even some primary metabolites including amino and organic acids which might play role in resistance against pathogens. These metabolic pathways undergo rerouting during the infection and the required precursors for the defense compounds might be formed. The rerouting of fluxes at the level of chorismate seems to play a role in resistance of B. rapa to pathogenic fungi. Resistance involves a very high level of complexity. Understanding resistance thus requires studies of different combinations of cultivars and microorganisms. By taking a systems biology approach and measuring on the level of transcriptome, proteome and metabolome and analyzing the large data sets from interactions of different cultivars with different fungi in combination with data on susceptibility by means of multivariate analysis might give a better insight in the defense response of the plant. As we used different cultivars in our study, the fungal infection and also treatment with signal molecules induced secondary metabolites that branch from the chorismate pathway including phenylpropanoids, flavonoids, IAA and indole glucosinolates. Even with using different cultivars from B. rapa, all cultivars showed enhanced accumulation of secondary metabolites derived from the chorismate pathway. Phenylpropanoids, benzoyl glucosinolates, flavonoids and indole glucosinolates should be the target compounds for genetic engineering research. By using genetic engineering techniques, one can breed new cultivars that are characterized by higher levels of these secondary metabolites. Show less
The female cones of hop (Humulus lupulus L.) are added to beer, providing taste and flavour and contributing to the stability of foam. The main constituents of hop related to these properties are... Show moreThe female cones of hop (Humulus lupulus L.) are added to beer, providing taste and flavour and contributing to the stability of foam. The main constituents of hop related to these properties are generically known as alpha-acids. During the brewing process, these acids are isomerized, resulting in the formation of three pairs of trans-/cis-iso-alpha–acids. The aim of this thesis project was to develop a method for the analysis (Chapter 2 and 7) as well as the purification of iso-alpha-acids (Chapter 3-5). Moreover, the stability of iso-α-acids need to be improved (Chapter 6) since these compounds degrade easily in the light and in the presence of oxygen. We have shown that β-CD is effective to separate cis-isomers from trans-isomers and at the same time increases the stability of the complexed compounds. The pure individual iso-alpha-acids can now be produced from pure alpha-acids, obtained from a CPC separation, followed by isomerization and beta-CD separation of the two isomers. These compounds can be used as reference standard for analytical purposes to replace the DCHA-salts of trans-iso-alpha-acids. It also opens the possibility to use pure iso-alpha-acids in brewery research. An HPLC system using these pure individual iso-alpha-acids as calibration standard was developed as well. The application of 2D J resolved NMR has been developed and seems promising to detect a wide range of compounds in beer. Show less
The introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in... Show moreThe introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in the life science area. In this thesis we explored the possibilities to apply these holistic technologies to investigate the effects of known and potential anti-inflammatory compounds on macrophages. For this purpose we made use of a monocyte-like human histocytic lymphoma cell line U937. U937 cells can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. The two differentiation stages, monocyte and macrophage, were compared by using oligonucleotide microarrays and 2-D gel electrophoresis in combination with principal component analysis (PCA). This differentiation study is described in Chapter 2. The differential expression of three protein biomarkers, gamma interferon inducible lysosomal thiol reductase (GILT), cathepsin D and adipocyte-fatty acid binding protein (A-FABP) were biologically validated by Western blot and real time polymerase chain reaction (real time PCR). GILT and A-FABP were also found to be differentially expressed at the mRNA level as indicated by the results of the microarray experiment. Moreover, the transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. From the results presented in Chapter 2 can be concluded that the U937 cell line is an excellent model system for the blood-derived macrophage and that microarrays and 2-D gel electrophoresis are suitable methods to identify biomarkers for differentiation. Chapter 3 describes the use of a systems biology approach to categorize anti-inflammatory drugs based on their mRNA, protein and lipid expression pattern, as determined by oligonucleotide microarrays, 2-D gel electrophoresis and a LC-MS method for lipids, in combination with principal component discriminant analysis (PC-DA). The results described in this chapter demonstrate that different classes of anti-inflammatory compounds show distinct and characteristic mRNA, protein, and lipid expression patterns, which can be used to categorize known anti-inflammatory drugs, as well as to discover and classify new leads. The latter was exemplified by the categorization of zilpaterol, a poorly characterized ____-agonist. Exposure to zilpaterol gives rise to an almost identical expression pattern as that observed after exposure to the well-characterized __2-agonists clenbuterol and salbutamol, suggesting that zilpaterol is indeed a ____-agonist. In addition, this study revealed potential biomarkers for the different anti-inflammatory drugs under investigation. The categorization of the anti-inflammatory drugs on the basis of proteomics data alone was not successful. The most likely explanation for this is that by the analysis of whole cell lysates, only highly abundant proteins can be visualized, while the low abundant proteins, which are often involved in important metabolic pathways, are not. Therefore, a more focused approach was used to investigate the mechanism of action of zilpaterol, which is described in Chapter 4. In Chapter 4, U937 macrophages were stimulated with LPS to induce an inflammatory response. This response was inhibited by the addition of zilpaterol (LZ) and this inhibition was antagonized by the _2-adrenergic receptor antagonist propranolol (LZP). Two-dimensional difference gel electrophoresis (DIGE) in combination with Student__s t-test and two multivariate data analysis tools (PCA and partial least squares discriminant analysis PLS-DA) were used to examine the secreted proteome induced by the three treatments. This revealed 8 potential protein biomarkers. The protein spots were identified using nano LC-MS-MS. Only two of the identified proteins, namely macrophage inflammatory protein-1_ (MIP-1_) and macrophage inflammatory protein-1_ (MIP-1_) are known to be secreted proteins. The inhibition of MIP-1_ by zilpaterol and the involvement of the _2-AR and cyclic adenosine-3__,5__-cyclic monophosphate (cAMP) were confirmed using a specific immuno-assay. The experiments described in this chapter demonstrate the importance of pre-fractionation of complex protein samples before performing proteomics studies. The categorization of zilpaterol in Chapter 3 as a _2-adrenegic receptor agonist was further explored in Chapter 5. In this chapter we investigated the binding affinity of zilpaterol to the _1- and _2 receptor by using a receptor binding assay. Furthermore, we examined the role of the _1- and _2 adrenoceptor in the inhibition of the LPS induced tumor necrosis factor-alpha (TNF-_) production and the induction of cAMP by U937 macrophages. For this purpose we made use of a selective _1-receptor antagonist (atenolol), a selective _2-antagonist (ICI 118551) and a non-selective _-antagonist (propranolol). Finally, the inhibitory effect of zilpaterol on the TNF-_ production was investigated in LPS-treated male Wistar rats. The results obtained in this way clearly show that zilpaterol is a _2-adrenergic agonist and a inhibitor of the LPS-induced TNF-_ production by macrophages both in vivo and in vitro. The three _2-agonists specific biomarkers, Granulocyte Chemotactic Protein-2 (GCP-2/CXCL6), Oncostatin M (OSM), and Vascular Endothelial Growth Factor (VEGF) that were identified in Chapter 3, were further examined in Chapter 6. The three markers were significantly up-regulated both in U937 macrophages and in blood-derived macrophages exposed to a _2-agonist (clenbuterol and zilpaterol) in the absence or presence of LPS, as determined by a specific enzyme-linked immunosorbent assays (ELISA). Moreover, this up-regulation was also accomplished by other cyclic AMP elevating agents (forskolin, prostaglandins E2, and dibutyryl cAMP), suggesting a role of cAMP in the up-regulation of GCP-2/CXCL6, VEGF and OSM. We hypothesize that these proteins may be involved in some of the adverse effects in the treatment of asthma with _2-adrenergic receptor agonists. In the second part of this thesis we focussed on a multi-component drug, namely Cannabis sativa. In Chapter 7, the immuno-modulating effects of unheated and heated Cannabis extracts were investigated. This study revealed that unheated Cannabis extracts and its major non-psychoactive compound _9-tetrahydrocannabinolic acid (THCa) were able to inhibit the LPS induced TNF-_ production both in U937 macrophages and in blood-derived macrophages. The inhibitory effect on TNF-_ was not mediated by the cannabinoid receptors CB1 and CB2. Furthermore, this study showed that unheated Cannabis extracts and THCa exert their inhibitory effect on the TNF-_ production via a mechanism that is different from that of heated Cannabis extract and its main constituent the psychoactive compound _9-tetrahydrocannabinol (THC). The inhibition of TNF-_ release by unheated Cannabis extract and THCa was prolonged over a relatively long period of time. By contrast, although THC and heated extracts initially inhibit the release of TNF-_, after longer incubation times they seem to increase TNF-_ production to levels that are even higher than in the absence of THC or Cannabis extract. This difference in response of the U937 macrophages to THC and THCa was also observed in an experiment in which we examined the effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibited the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. Finally, we studied the effect of THCa and unheated Cannabis extract in a pilot study using an Experimental Autoimmune Encephalomyelitis (EAE) mouse model. Unheated Cannabis extract and THCa had a favourable effect on the clinical and histological signs of EAE. However, these results are preliminary and not clearly significant, therefore further investigation is necessary. Chapter 8 describes the categorization of unheated and heated Cannabis extracts using the same model system as described in Chapter 3. The mRNA patterns obtained from U937 macrophages exposed to LPS in the absence or presence of different anti-inflammatory drugs and unheated and heated Cannabis extracts were analysed using PC-DA. The study revealed that heated and unheated Cannabis extracts give rise to different expression patterns, which is in agreement with the observations made in Chapter 7 that they exert their TNF-_ inhibitory effect via different pathways. Moreover, their expression patterns did not overlap with that of other classes of anti-inflammatory compounds known to inhibit the TNF-_ production. These results suggest that the Cannabis extracts can not be assigned to one of the above mentioned classes of inflammatory inhibitors. Further investigation is necessary to unravel the exact mechanism of action of unheated and heated Cannabis extracts. In conclusion, the studies in this thesis show that the application of systems biology approaches are very useful in the categorization of anti-inflammatory compounds based on their mRNA and lipid expression patterns and to find specific biomarkers for these compounds. The categorization based on the protein expression pattern was less successful. This is most probably due to the fraction of proteins that was analysed on the gel. With proteomics techniques only a small fraction of proteins can be analysed simultaneously. Pre-fractionation, enrichment techniques and different analytical methods are therefore necessary to analyse a wide range of proteins with diverse physiological properties and dynamic range. The datasets obtained by transcriptomics, proteomics and metabolomics were analysed using statistical and pattern recognition tools. The datasets often contained a limited number of samples with respect to the large number of variables. It is therefore important to use these techniques as an explorative tool only and to validate the potential biomarkers found by additional individual measurements. Taken together, the use of systems biology for the investigation of anti-inflammatory drugs yielded very promising results, even though only a small part of the systems biology circle was used. Show less
