Many mixtures important to research consist of hundreds or even thousands of individual components of interest. These types of mixtures are far too complex to separate by a single chromatographic dimension in any reasonable amount of time. However, if a multidimensional approach is used, where a complex mixture is separated by an initial dimension, simpler fractions of that separation are collected and each of those fractions are analyzed individually, highly complex mixtures can be resolved in relatively short amounts of time. This dissertation serves as a guide to multidimensional chromatography, in particular, two-dimension liquid chromatography. There are many aspects of multidimensional separations that have been investigated to show its aspects, drawbacks and potential ability to separate highly complex mixtures. Measurements for the performance of multidimensional chromatography, the effects of the first and subsequent dimensions and the approaches to pairing dimensions are shown with experimental examples. Fundamental and practical features of multidimensional chromatography are explained as well as theoretical discussions on current and future multidimensional chromatography performance. Experimentally, very high peak capacities were obtained (ca. 7000) and an algorithm to predict how to best optimize a two-dimensional separation based on the time used and performance was created for designing experiments.

Work related musculoskeletal disorders are the dominating causes of reported ill-health in industrialized countries. These chronic pain conditions are one of the most costly public health problems in Europe and North America. When work related musculoskeletal disorders are considered to be of muscular origin and the trapezius muscle is affected, the common appellation is trapezius myalgia. Since little is known about the genesis or how it is maintained, it is of great importance to better understand the pathophysiology of trapezius myalgia; doing so will better enable recommendations for prevention, treatment and rehabilitation. Several hypotheses have been presented based on biochemical alterations in the muscle, suggesting increased signaling of inflammatory substances and altered metabolism. Previous research has not been able to present the comprehensive picture of the muscle in pain. Thus there is a demand for more comprehensive research regarding the biochemical milleu of the chronic trapezius muscle. Proteomic and metabolomic methods allow non-targeted simultaneous analyses of a large number of proteins and metabolites. The main emphasis in this thesis is on a proteomic method, two-dimensional differential gel electrophoresis (2D-DIGE). The method is validated to human skeletal muscle biopsy research with laboratory specific settings. In the baseline study, there were 14 metabolic, contractile, structural and regulatory proteins that differed significantly in abundance when trapezius and vastus lateralis muscles were compared. Using the validated 2D-DIGE method and the baseline study, a comparison between healthy and myalgic muscles was made. Biopsies from female cleaners with and without myalgia were compared to obtain results from women with the same type of work exposure. In the multivariate model, 28 identified unique proteins separated healthy and myalgic muscle and were grouped according to function: metabolic (n=10), contractile (n=9), regulatory (n=3), structural (n=4), and other (n=2). Finally, a second screening method, metabolomics, was introduced to analyze differences in metabolite content as a complement to and verification of the proteomic results. Gas chromatography-mass spectrometry (GC-MS) was performed on muscle interstitial fluid samples obtained with microdialysis, and differences in the abundance of extracellular metabolites were revealed.  The 2D-DIGE method is a reliable method to analyze human skeletal muscle. The outcomes of the proteomic analyses were dependant on the statistical approach. Systematic differences in protein and metabolite content were detected using a multivariate approach. Univariate analyses were used to analyze individual proteins for their significance. The significant proteins in the baseline study were predominately related to muscle fiber type which correlated with the differences in fiber type content between trapezius and vastus lateralis. The proteomic and metabolomics studies where myalgic and healthy muscles were compared provide us with new clues and new aspects regarding the pathophysiology of the myalgic muscle. Technically advanced methods employed in the thesis enabled an explorative screening of proteins of relevance for the pathophysiology of the myalgic muscle. The results of these analyses may contribute to the formulation of future hypothesis that need to be further evaluated.

Lung cancer is the cancer form that has the highest mortality worldwide and inaddition the survival of lung cancer is very low. Only 15% of the patients are alivefive years from set diagnosis. More research is needed to understand the biologyof lung cancer and thus make it possible to discover the disease at an early stage.Early diagnosis leads to an increased chance of survival. In this thesis 179 lungcancer- and 116 control samples of blood serum were analyzed for identificationof metabolomic biomarkers. The control samples were derived from patients withbenign lung diseases.Data was gained from GC/TOF-MS analysis and analyzed with the help ofthe multivariate analysis methods PCA and OPLS/OPLS-DA. In this thesis it isinvestigated how to pre-treat and analyze the data in the best way in order todiscover biomarkers. One part of the aim was to give directions for how to selectsamples from a biobank for further biological validation of suspected biomarkers.Models for different stages of lung cancer versus control samples were computedand validated. The most influencing metabolites in the models were selected andconfoundings with other clinical characteristics like gender and hemoglobin levelswere studied. 13 lung cancer biomakers were identified and validated by raw dataand new OPLS models based solely upon the biomarkers.In summary the identified biomarkers are able to separate fairly good betweencontrol samples and late lung cancer, but are poor for separation of early lungcancer from control samples. The recommendation is to select controls and latelung cancer samples from the biobank for further confirmation of the biomarkers.NyckelordLung cancer is the cancer form that has the highest mortality worldwide and inaddition the survival of lung cancer is very low. Only 15% of the patients are alivefive years from set diagnosis. More research is needed to understand the biologyof lung cancer and thus make it possible to discover the disease at an early stage.Early diagnosis leads to an increased chance of survival. In this thesis 179 lungcancer- and 116 control samples of blood serum were analyzed for identificationof metabolomic biomarkers. The control samples were derived from patients withbenign lung diseases.Data was gained from GC/TOF-MS analysis and analyzed with the help ofthe multivariate analysis methods PCA and OPLS/OPLS-DA. In this thesis it isinvestigated how to pre-treat and analyze the data in the best way in order todiscover biomarkers. One part of the aim was to give directions for how to selectsamples from a biobank for further biological validation of suspected biomarkers.Models for different stages of lung cancer versus control samples were computedand validated. The most influencing metabolites in the models were selected andconfoundings with other clinical characteristics like gender and hemoglobin levelswere studied. 13 lung cancer biomakers were identified and validated by raw dataand new OPLS models based solely upon the biomarkers.In summary the identified biomarkers are able to separate fairly good betweencontrol samples and late lung cancer, but are poor for separation of early lungcancer from control samples. The recommendation is to select controls and latelung cancer samples from the biobank for further confirmation of the biomarkers.Nyckelord

Along with genes and proteins, metabolites play important roles in sustaining life. There remains much to be learned about the in vivo roles of metabolites. Metabolomics is a comparative tool to study global metabolite levels in samples under various conditions. This dissertation describes the development and application of metabolomics strategies for discovery of biologically active or novel metabolites with priori knowledge about genes, proteins, or phenotypes. The power of metabolomics for discovery of novel metabolites from genes is demonstrated through the work with the pyochelin (pch) gene cluster. Comparison of the extracellular metabolomes of pch gene cluster mutants to the wild-type Pseudomonas aeruginosa (strain PA14) identified 198 ions regulated by the pch genes. In addition to known metabolites, a pair of novel metabolites were characterized as 2-alkyl-4,5-dihydrothiazole-4-carboxylates (ATCs). Subsequent assays revealed that ATCs bind iron and that their production is regulated by iron levels and dependent on pchE gene in the pch gene cluster. Metabolomics can also facilitate discovery of active metabolites from proteins, as shown in the work with orphan nuclear receptor Nur77. We applied a metabolomics platform for detected protein-metabolite interactions to identify lipids that bind to Nur77. Using this approach, we discovered that the Nur77 ligand-binding domain (Nur77LBD) enriched unsaturated fatty acids (UFAs) in tissue lipid mixtures. Subsequent biophysical and biochemical assays indicate that UFAs bind to Nur77LBD to cause changes in the conformation and oligomerization of the receptor. Last, analogous to classic fractionation experiments, metabolomics can also be applied to discover active metabolites from phenotypes. Using combination of genetics, biochemistry, and metabolomics, we identified three phenazine compounds produced by Pseudomonas aeruginosa that are toxic to the nematode Caenorhabditis elegans. 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of PCA and pyocyanin are strictly pH-dependent at non-overlapping pH ranges. The diversity within a class of metabolites can be used to modulate bacterial toxicity in different environmental niches. / Chemistry and Chemical Biology

No description available.

The two main causes of child mortality and morbidity in Africa are malaria and invasive bacterial diseases. In addition, co-infections in sub-Saharan Africa are the rule rather than the exception. However, not much is known about the host-pathogen interaction during a concomitant infection or how it affects the outcome of disease. In order to study the immunological responses during malaria and bacterial co-infections, we established a co-infection mouse model. In these studies we used two pathogenic bacteria found in malaria co-infected patients: Streptococcus pneumoniae and Relapsing fever Borrelia duttonii. Hosts co-infected with malaria and Borrelia showed greatly increased spirochetal growth but low parasite densities. In addition, the co-infected hosts presented symptoms of experimental-cerebral malaria, in an otherwise unsusceptible mouse model. This was found to be a consequence of a dysregulated immune response due to loss of timing and control over regulatory mechanisms in antigen presenting cells thus locking the host in an inflammatory response. This results in inflammation, severe anemia, internal organ damage and pathology of experimental cerebral malaria. On the other hand, in the malaria - S. pneumoniae co-infection model we found that co-infected hosts cleared the bacterium much more efficiently than the single infected counterpart. This efficiency of clearance showed to be neutrophil dependent. Furthermore, in vitro studies revealed that neutrophils isolated from malaria-infected hosts present an altered migratory effect together with a significantly increased capacity to kill S. pneumoniae. This suggests that a malaria infection primes neutrophils to kill S. pneumoniae more efficiently. Furthermore, a study was carried out on plasma samples from Rwandan children under the age of five, on which a full metabolomics profile was performed. We showed that these children could be divided in different disease categories based on their metabolomics profile and independent of clinical information. Additionally, the mild malaria group could further be divided in two sub-groups, in which one had a metabolomic profile resembling that of severe malaria infected patients. Based on this, metabolite profiling could be used as a diagnostic tool to determine the distinct phase, or severity of a malaria infection, identify risk patients and provide helpful and correct therapy.

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. Very little is currently known about the molecular mechanisms required for pathogenicity of S. nodorum, despite its major impact on Australian agriculture. S. nodorum is a polycyclic pathogen. Rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate within 2-3 weeks. Several cycles of infection are needed to build up inoculum for the damaging infection of flag leaves and heads, sporulation is therefore a critical component of the infection cycle of S. nodorum; our aim is to determine the genetic and biochemical requirements for sporulation for development of control of the pathogen. Disease progression of S. nodorum on wheat cv. Amery was monitored by light microscopy to determine the time point when pycnidia development began. Early pycnidia development was evident 12 days post-infection. This information was used to guide a genomics and a metabolomics based approach to determine the requirements for sporulation in S. nodorum. The genomics approach utilised two cDNA libraries created from sporulating and non-sporulating cultures. EST frequency was used to determine highly expressed genes under the two developmental states. Gene expression from the most highly represented genes during sporulation were confirmed using quantitative PCR. A gene encoding an arabitol 4-dehydrogenase (Abd1), was mutagenised, in its absence sporulation was reduced by approximately 20%. The metabolomics approach isolated metabolites from both in planta infection and in vitro growth. Rapid changes in the abundance of metabolites were detected during the onset of sporulation. Key fungal metabolites identified include mannitol and trehalose. The concentration of both mannitol and trehalose increased dramatically in concert with pycnidia formation. Both mannitol and trehalose have also been linked to pathogenicity in filamentous fungi. Creation of deletion mutants of the gene encoding trehalose 6-phosphate synthase showed the synthesis of trehalose is required for full sporulation of S. nodorum in planta and in vitro.

Στην παρούσα εργασία, για πρώτη φορά, τίθεται η πιο πρόσφατη εισαχθείσα τεχνολογία των –omics, η μεταβολομική, στην υπηρεσία της μοριακής βιολογίας για την μελέτη της θερμοκρασιακής μεταβολικής πλαστικότητας. Με τον όρο θερμοκρασιακή μεταβολική πλαστικότητα αναφερόμαστε στην ιδιότητα ενός γονότυπου να παράγει ποικίλους φαινότυπους σε επίπεδο μεταβολισμού, ως απόκριση στις θερμοκρασιακές συνθήκες στις οποίες υπόκειται. Σκοπός, λοιπόν της παρούσας ερευνητικής εργασίας ήταν η μελέτη της επίδρασης της πρώιμης θερμοκρασίας ανάπτυξης στο μεταβολικό πρότυπο ενήλικων θηλυκών και αρσενικών zebrafish σε γονάδες, εγκέφαλο και μυϊκό ιστό. Στα πλαίσια του σκοπού αυτού, τρεις πληθυσμοί zebrafish τοποθετήθηκαν στους 22οC, 28οC και 32οC από 0-20 ημερών, κατά την 20η ημέρα και οι τρεις πληθυσμοί αναπτύχθηκαν σε κοινή θερμοκρασία 28οC μέχρι την πάροδο εφτά μηνών, οπότε και πραγματοποιήθηκε δειγματοληψία. Ακολούθησε η ανάκτηση των μεταβολικών προτύπων, μετά την αριστοποίηση του πρωτοκόλλου για κάθε ιστό, χρησιμοποιώντας αέριο χρωματογράφο-φασματόμετρο μάζας. Τα αποτελέσματα της πολυπαραμετρικής στατιστικής ανάλυσης έδειξαν να μη διαφαίνεται θερμοκρασιακή μεταβολική πλαστικότητα στις γονάδες και στο μυϊκό ιστό, μιας και η ομαδοποίηση των μεταβολικών προτύπων έγινε ως προς το φύλο. Συγκεκριμένα και στις δυο κατηγορίες ιστών παρατηρήθηκε μεγάλη μείωση της μεταβολικής ενεργότητας στα θηλυκά ως προς τα αρσενικά zebrafish. Εξαιρετικής σημασίας, όμως, είναι το γεγονός ότι, για πρώτη φορά, αποδείχθηκε η επίδραση της πρώιμης θερμοκρασίας ανάπτυξης, στο μεταβολικό πρότυπο ενήλικων θηλυκών και αρσενικών zebrafish μέσω της δραματικής μείωσης της ενεργότητας του κεντρικού μεταβολισμού των εγκεφάλων 22ο C και 32ο C ως προς τους 28ο C. / With the term temperature induced metabolic plasticity one refers to the feature of genotype to produce different phenotypes on a metabolic level, due to the temperature conditions of its environment. The aim of this thesis is to study the effect of temperature on the metabolic profile during the early growth stage of adult male and female zebrafish in three tissues: gonads, brain and muscle. For this end three populations of zebrafish were bread at 22οC, 28οC and 32οC from 0 to 20 post-fertilization (pf) days. After the 20th pf day all populations were bread at 28οC for seven months. The metabolic profiles of all tissues were acquired using gas chromatography-mass spectrometry; in this thesis the protocol of metabolic profile acquisition was optimized for each tissue separately. Multivariate statistical analysis of the profiles showed no temperature plasticity in gonads and muscles. However, for the first time worldwide it was shown temperature induced plasticity in the brains metabolism of adult male and female zebrafish. Characteristically, a decline of the metabolic activity was observed in the 22ο C and 32ο C grown zebrafish compared to the 28ο C grown.

Les TSA représentent un groupe de troubles neurodéveloppementaux défini par des déficits des interactions sociales, de la communication et des comportements restreints et répétitifs. A ce jour le diagnostic de l’autisme se fait uniquement sur la base de symptômes cliniques. Il n’existe aucun biomarqueur des TSA. Ce travail s’inscrit donc (1) dans la recherche de biomarqueurs prédictifs dans les TSA et (2) dans la mise en évidence des dysfonctions métaboliques cérébrales dans un modèle de rat (rat valproate, VPA). Pour mettre en évidence des biomarqueurs urinaires prédictifs nous avons analysé simultanément les données issues de plusieurs technologies analytiques afin d’améliorer la robustesse et la capacité prédictive des modèles statistiques. L’autre partie de la thèse consistait à caractériser et à comparer au cours du développement l’évolution du métabolome du rat VPA. Nous avons mis en évidence des perturbations des voies de la neurotransmission, énergétique, du stress oxydatif etc. Même si on ne peut pas transposer les résultats obtenus chez le rat, à l’homme, le modèle VPA permet néanmoins de mieux comprendre les perturbations physiologiques cérébrales induites par ce médicament. / ASDs are a group of neurodevelopmental disorders defined by deficits in social interaction, communication and restricted and repetitive behaviors. To date, the diagnosis of autism is made only on the basis of clinical symptoms. There is no biomarker of ASD. The aim of this work is (1) the search of predictive biomarkers in ASD and (2) the better understanding of brain metabolic dysfunctions in a rat model (valproate rat, VPA). To highlight urinary predictive biomarkers we analyzed together data from different analytical technologies in order to improve the robustness and predictive power of statistical models. The second part of the thesis was to characterize and compare the cerebral metabolome of VPA rat during development. We showed disturbances of neurotransmission, energy, oxidative stress pathways. Even if results obtained in rats cannot be transposed to humans, the VPA model still allows a better understanding the brain physiological disturbances induced by the drug.

Metabolomic Insights into the Pharmacological and Genetic Inhibition of Cyclooxygenase-2 William T. E. Briggs The cyclooxygenase (COX)-2 inhibitors, or “coxibs,” are excellent anti-inflammatory agents, but their reputation has been tarnished by the adverse cardiovascular (CV) events, including heart failure (HF), with which they are associated. Whilst the risk of HF represents the greatest adverse CV event signal seen with these compounds, it is also perhaps the least well understood and has often been explained away as a consequence of the thrombotic risk with which the coxibs are also associated. One recent hypothesis, put forward by Ahmetaj-Shala et al., suggests that asymmetric dimethylarginine (ADMA) may serve as a mechanistic bridge between COX-2 inhibition and HF. However, the ADMA-COX-2 hypothesis was developed based on findings in a constitutive mouse model of COX-2 knock-out (KO), which is compromised by severe developmental cardio-renal pathology, and pharmacological studies which may not accurately reflect coxib use in clinical practice. Various studies have explored the metabolic changes induced by coxib treatment. However, these studies have been limited in scope and have tended to focus on specific pathways or certain tissues/bio-fluids. This has left large regions of the metabolome, in the context of coxib-treatment, unexplored. Given that metabolic remodelling is a key feature of HF, changes in these metabolites may hold the key to understanding the pathogenesis of coxib-induced HF. L-Carnitine shuttles activated long-chain fatty acids (FAs) across the inner mitochondrial membrane to the mitochondrial matrix, where they are oxidised by β-oxidation. This is especially important in the heart, which derives the majority of its energy from the metabolism of FAs. Changes in carnitine metabolism are also seen in HF. It is therefore biologically plausible that derangements in carnitine metabolism may contribute to the pathogenesis of coxib-induced HF. This thesis employs a combination of targeted and untargeted metabolomic techniques, stable isotope labelling and quantitative reverse transcription polymerase chain reaction (RT-qPCR) to i) profile the metabolic changes induced by celecoxib and rofecoxib, in the mouse; ii) specifically interrogate the effect of celecoxib, rofecoxib and global COX-2 gene deletion on carnitine synthesis, metabolism and shuttling, and iii) explore the advantages and disadvantages of the inducible post-natal global (IPNG) COX-2 KO (COX-2-/-) mouse, an alternative to the constitutive COX-2-/- mouse used by Ahmetaj-Shala et al. The results of this thesis demonstrate that i) celecoxib and rofecoxib have similar metabolomic consequences in the mouse; ii) carnitine metabolism may be affected by celecoxib, rofecoxib and dietary composition, via a peroxisome proliferator-activated receptor-alpha (PPAR-α) mediated effect on hepatic carnitine synthesis and iii) the IPNG COX-2-/- mouse neither exhibits the severe developmental cardio-renal pathology nor the altered ADMA metabolism observed in the constitutive COX-2-/- mouse. These findings contradict those of Ahmetaj-Shala et al., oppose the ADMA-COX-2 hypothesis and highlight a potential role for carnitine metabolism and diet in coxib induced HF.