Dissertations / Theses: 'Proteomics and metabolomics'

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Shiryaeva, Liudmila. "Proteomics and metabolomics in biological and medical applications." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-43520.

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Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer. The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed. The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required. Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process. Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them.

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Salji, Mark J. "Quantitative proteomics and metabolomics of castration resistant prostate cancer." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30941/.

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Henneges, Carsten [Verfasser]. "Feature Selection and Data Mining for Proteomics and Metabolomics / Carsten Henneges." München : Verlag Dr. Hut, 2011. http://d-nb.info/1017353573/34.

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Mörén, Lina. "Metabolomics and proteomics studies of brain tumors : a chemometric bioinformatics approach." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-111309.

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The WHO classification of brain tumors is based on histological features and the aggressiveness of the tumor is classified from grade I to IV, where grade IV is the most aggressive. Today, the correlation between prognosis and tumor grade is the most important component in tumor classification. High grade gliomas, glioblastomas, are associated with poor prognosis and a median survival of 14 months including all available treatments. Low grade meningiomas, usually benign grade I tumors, are in most cases cured by surgical resection. However despite their benign appearance grade I meningiomas can, without any histopathological signs, in some cases develop bone invasive growth and become lethal. Thus, it is necessary to improve conventional treatment modalities, develop new treatment strategies and improve the knowledge regarding the basic pathophysiology in the classification and treatment of brain tumors. In this thesis, both proteomics and metabolomics have been applied in the search for biomarkers or biomarker patterns in two different types of brain tumors, gliomas and meningiomas. Proteomic studies were carried out mainly by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). In one of the studies, isobaric tags for relative and absolute quantitation (iTRAQ) labeling in combination with high-performance liquid chromatography (HPLC) was used for protein detection and identification. For metabolomics, gas-chromatography time-of-flight mass spectrometry (GC-TOF-MS) has been the main platform used throughout this work for generation of robust global metabolite profiles in tissue, blood and cell cultures. To deal with the complexity of the generated data, and to be able to extract relevant biomarker patters or latent biomarkers, for interpretation, prediction and prognosis, bioinformatic strategies based on chemometrics were applied throughout the studies of the thesis. In summary, we detected differentiating protein profiles between invasive and non-invasive meningiomas, in both fibrous and meningothelial tumors. Furthermore, in a different study we discovered treatment induce protein pattern changes in a rat glioma model treated with an angiogenesis inhibitor. We identified a cluster of proteins linked to angiogenesis. One of those proteins, HSP90, was found elevated in relation to treatment in tumors, following ELISA validation. An interesting observation in a separate study was that it was possible to detect metabolite pattern changes in the serum metabolome, as an effect of treatment with radiotherapy, and that these pattern changes differed between different patients, highlighting a possibility for monitoring individual treatment response. In the fourth study of this work, we investigated tissue and serum from glioma patients that revealed differences in the metabolome between glioblastoma and oligodendroglioma, as well as between oligodendroglioma grade II and grade III. In addition, we discovered metabolite patterns associated to survival in both glioblastoma and oligodendroglioma. In our final work, we identified metabolite pattern differences between cell lines from a subgroup of glioblastomas lacking argininosuccinate synthetase (ASS1) expression, (ASS1 negative glioblastomas), making them auxotrophic for arginine, a metabolite required for tumor growth and proliferation, as compared to glioblastomas with normal ASS1 expression (ASS1 positive). From the identified metabolite pattern differences we could verify the hypothesized alterations in the arginine biosynthetic pathway. We also identified additional interesting metabolites that may provide clues for future diagnostics and treatments. Finally, we were able to verify the specific treatment effect of ASS1 negative cells by means of arginine deprivation on a metabolic level.

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Thomas, Funmilola Clara. "Acute phase proteins, proteomics and metabolomics in the diagnosis of bovine mastitis." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6360/.

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Bovine mastitis continues to pose a major economic challenge to the dairy industry worldwide. Critical to the management and control of this condition, is the need for prompt and accurate diagnosis in field conditions, therefore a search for more sensitive and reliable biomarkers is required. In this thesis, studies focused on assessing milk samples from cows with various forms of mastitis were undertaken with a view to identifying new biomarkers for bovine mastitis. Three acute phase proteins (APP); haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) were measured in milk samples from composite milk samples of all lactating cows in a commercial dairy herd, mastitis cases, submitted to a diagnostic laboratory and following an experimental mastitis challenge of cows with Streptococcus uberis. A new enzyme linked immunosorbent assay (ELISA) was developed for measuring Hp, while commercial ELISA assay kits were used to assay M-SAA3 and CRP. Other mastitis related parameters evaluated in the samples included the somatic cell counts (SCC) and the presence of pathogens. A reliable and sensitive ELISA was developed and optimized for measuring milk Hp. A cut off value for Hp of 7.9 μg/ml was established for milk with SCC less than 200,000 cells/ml. Pathogen-specific variations were observed in the concentration of each APP in mastitic milk. It was observed that the environmental pathogens showed higher concentrations of APP compared to other pathogens, from the study of mastitis milk samples submitted to the diagnostic laboratory. Also, it was possible to distinguish between samples from subclinical and clinical mastitis and between samples from subclinical and healthy udders using each of the APP (P<0.05). Haptoglobin, M-SAA3 and CRP showed corresponding variation with stage of infection during the course of experimental mastitis, and specifically CRP was observed to rise earlier than other two APP. Furthermore, characterization of the profile of these APP in the immediate post-calving milk samples was carried out to determine how valuable they would be in recognizing new mastitis infections arising at the post-partum period. It was observed that there is a general moderately-high level of APP in milk immediately following parturition which drops a few days later in healthy milk. The immunohistochemical localization of Hp in the bovine mammary gland was also assessed. It could be concluded from that study that neutrophils and the mammary epithelial cells secrete Hp into milk during mastitis. Gel and non-gel based proteomics approaches were employed to study the protein profiles and variation in mastitic milk from normal samples. Several proteins were identified that confirmed previous findings and project new mastitis markers, for example, serotransferrin, serpins, alpha-macroglobulin and neutrophil gelatinase associated lipocalins. A capillary electrophoresis mass spectrometry system (CE-MS) was also employed to elucidate the changing peptidome in milk during the course of an experimental mastitis, which lead to the generation of a panel of 77 polypeptides, which were able to significantly differentiate critical stages of mastitis. Three of these polypeptides were found in mastitic milk samples from previous peptidomic analyses thereby indicating strong biomarker value. Finally, a liquid chromatography mass spectrometry based metabolomics approach was used to study the changing profile of small metabolites in milk during the course of an experimental infection. Several pathway-based changes that highlighted metabolites of potential significance in mastitis diagnosis were recognized including lactose synthesis, nitrogen containing compounds such as betaine, L-carnitine and lipid metabolites pathways namely sn-glycerophosphocholine and choline among others. Overall, this study has shown the value of APP, milk proteomics and metabolomics in bovine mastitis diagnosis; the changing proteins and metabolites or their patterns need to be further experimentally and clinically validated as specific and sensitive markers of mastitis. Ultimately, the applicability of APP, proteins, peptides and metabolites and/or their changing patterns as mastitis biomarkers would require their adaptation to rapid (on farm) and robust measurement formats.

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Wibom, Carl. "Multivariate analyses of proteomic and metabolomic patterns in brain tumors." Doctoral thesis, Umeå universitet, Onkologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-25670.

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Glioblastoma multiforme (GBM) is the most common primary brain tumor. Given the current standard of care, the prognosis for patients diagnosed with this disease is still poor. There consequently exists a need to improve current treatments, as well as to develop new ones. Many obstacles however need to be overcome to facilitate this effort and one of these involves the development of improved methods to monitor treatment effects. At present, the effects of treatment are typically assessed by radiological means several months after its initiation, which is unsatisfactory for a fast growing tumor like GBM. It is however likely that treatment effects can be detected on a molecular level long before radiological response, especially considering many of the targeted therapies that are currently being developed. Biomarkers for treatment efficacy may be of great importance in the future individualization of brain tumor treatment. The work presented herein was primarily focused on detecting early effects of GBM treatment. To this end, we designed experiments in the BT4C rat glioma model in which we studied effects of both conventional radiotherapy and an experimental angiogenesis inhibitor, vandetanib. Brain tissue samples were analyzed using a high throughput mass spectrometry (MS) based screening, known as Surface Enhanced Laser Desorption/Ionization - Time of Flight - Mass Spectrometry (SELDI-TOF-MS). The vast amounts of data generated were subsequently analyzed by established multivariate statistical methods, such as Principal Component Analysis (PCA), Partial Least Squares (PLS), and Orthogonal Partial Least Squares (OPLS), developed for analysis of large and complex datasets. In the radiotherapy study we detected a protein spectrum pattern clearly related to tumor progression. We notably observed how this progression pattern was hampered by radiotherapy. The vandetanib study also revealed significant alterations of protein expression following treatment of different durations, both in tumor tissue and in normal brain contralateral to the tumor. In an effort to further elucidate the pathophysiology of GBM, particularly in relation to treatment, we collected extracellular fluid (ECF) samples from 11 patients diagnosed with inoperable GBM. The samples were collected by means of stereotactic microdialysis, both from within the contrast enhancing tumor and the brain adjacent to tumor (BAT). Samples were collected longitudinally from each patient in a time span of up to two weeks, during which the patient received the first five fractions of radiotherapy. The ECF samples were then analyzed by Gas Chromatography Mass Spectrometry (GC-MS) to screen them with respect to concentrations of low molecular weight compounds (metabolites). Suitable multivariate analysis strategies enabled us to extract patterns of varying metabolite concentrations distinguishing between samples collected at different locations in the brain as well as between samples collected at different time points in relation to treatment. In a separate study, we also applied SELDI-TOF-MS and multivariate statistical methods to unravel possible differences in protein spectra between invasive and non-invasive WHO grade I meningiomas. This type of tumor can usually be cured by surgical resection however sometimes it grows invasively into the bone, ultimately causing clinical problems. This study revealed the possibility to differentiate between invasive and non-invasive benign meningioma based on the expression pattern of a few proteins. Our approach, which includes sample analysis and data handling, is applicable to a wide range of screening studies. In this work we demonstrated that the combination of MS screening and multivariate analyses is a powerful tool in the search for patterns related to treatment effects and diagnostics in brain tumors.

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Hollywood, Katherine. "The development of bio-analytical techniques for the treatment of psoriasis and related skin disorders." Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/the-development-of-bioanalytical-techniques-for-the-treatment-of-psoriasis-and-related-skin-disorders(eadbfad8-6ab0-4029-b618-a1a6d715c3af).html.

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In this investigation a number of post-genomic technologies have be applied to study the dermatological disorders of psoriasis and keloid disease. In spite of considerable research focus on these diseases the pathogenesis remains unclear and currently no cure is available however, both diseases are manageable by drug intervention. It is common place that patients who are suffering from skin disorders are diagnosed and the extent of the disease assessed by a dermatologist which may be subjective due to human error. The availability and application of methods to screen patients and quantify the level of disease or response to treatment has obvious benefits in disease management. The work has incorporated a two-pronged approach combining the spectroscopic analysis of excised tissue samples and the phenotypic profiling of a rapidly proliferating cell line in response to drug intervention. The initial analysis of psoriatic skin samples by MALDI-MS provided poor results which remain relatively unexplained; however similar problems have been observed by other research groups. In a complementary approach the HaCaT cell line was exposed to increasing concentrations of three anti-psoriatic drugs namely dithranol, methotrexate and ciclosporin and the cells profiled using both metabolomic and proteomic methods. A number of metabolic pathways were highlighted including glycolysis and the TCA cycle. This has resulted in a selection of potential biomarkers which could be investigated in further work. In a small follow on study a collection of plasma samples from patients undergoing methotrexate treatment were analysed. The level of patient metadata and the number of samples was relatively limiting however, a subset of metabolites were significantly altered between responders and non-responders and with further validation could be potential biomarkers of successful treatment. The analysis of excised keloid samples was conducted using FT-IR microspectroscopy where it was possible to successfully discriminate between keloid and normal tissue. The use of imaging FTIR illustrated the complex cellular composition within a keloid scar, with increased lipid, amide and phosphate levels being observed. These measurable variations could, in the future, be incorporated into surgical procedures to allow targeted excision ensuring all keloid areas are removed. Finally a SERS-based analysis was conducted to investigate the possibility of probing dynamic enzymatic processes. This was successful and with the use of varying reporter molecules could be a beneficial tool for the analysis of metabolic processes.This project has successfully used a number of bio-analytical techniques to investigate dermatological problems. While the ultimate goal would be the application of a single analytical technique to provide answers to biological questions, it has been found that a number of complimentary techniques and statistical data handling approaches can provide a valuable insight into the problems posed.

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Pervukhin, Anton. "Molecular formula identification using high resolution mass spectrometry algorithms and applications in metabolomics and proteomics /." kostenfrei, 2009. http://d-nb.info/1001408578/34.

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Mußotter, Franz [Verfasser]. "Application of proteomics and metabolomics in molecular investigations of sensitization to contact allergens / Franz Mußotter." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1159900582/34.

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Hadrévi, Jenny. "Applying proteomics and metabolomics for studying human skeletal muscle with a focus on chronic trapezius myalgia." Doctoral thesis, Umeå universitet, Institutionen för integrativ medicinsk biologi (IMB), 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-61399.

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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.

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Agana, Bernice A. "Mass Spectrometry-Based Proteomics and Metabolomics: Understanding Protein Interactions, Proteome Complexity and Perturbations in Cellular Metabolism." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574636665012436.

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Happyana, Nizar [Verfasser], Oliver [Akademischer Betreuer] Kayser, and Robert [Gutachter] Verpoorte. "Metabolomics, proteomics, and transcriptomics of Cannabis sativa L. trichomes / Nizar Happyana. Betreuer: Oliver Kayser. Gutachter: Robert Verpoorte." Dortmund : Universitätsbibliothek Dortmund, 2014. http://d-nb.info/1101476958/34.

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Gunnaiah, Raghavendra. "Functional characterization of wheat, fusarium head blight resistance (QTL) «Fhb1» based on non-target metabolomics and proteomics." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119639.

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Fusarium head blight (FHB) caused by Fusarium graminearum is a dreadful disease of wheat (Triticum aestivum L.). Host resistance to FHB in wheat is quantitatively inherited. Though more than 100 QTLs have been identified, only a few have been validated. However, the resistance mechanisms governed by these QTLs are poorly understood. A type II FHB resistance QTL Fhb1 is the most consistent and largest effect QTL in wheat against FHB spread in wheat. Non-targeted metabolic and proteomic profiling of wheat near isogenic lines (NILs) with resistant and susceptible Fhb1 alleles was used to functionally characterize Fhb1 using a high resolution LC-MS. The Fhb1 from a moderately resistant cultivar Nyubai was associated with cell wall thickening, mainly at the rachis, due to deposition of hydroxycinnamic acid amides (HCAAs), phenolic glucosides and flavonoids. A hypothetical protein coding gene (GenBank: CBH32656.1) near Fhb1 locus was putatively identified as hydroxycinnamoyl transferase, which catalyzes the biosynthesis of HCAAs. Deoxynivalenol (DON) accumulation was high in both the NILs, eliminating DON detoxification as a mechanism associated with Fhb1 (Chapter III). For additional confirmation, the Fhb1 resistant allele, from a highly FHB resistant cultivar Sumai-3 was profiled. Even though the DON accumulation was low in resistant NIL, the detoxification of DON by host UDP-glycosyltransferase was moderately high in both the NILs, with no significant difference. Interestingly, unlike in the resistant NIL, constitutively present glycerophospholipids were absent in the susceptible NIL following pathogen inoculation due to degradation of membrane. Membrane degradation was caused due to programmed cell death as evidenced by DNA laddering in the susceptible NIL. A locus TAA_ctg0954b.00390.1 was identified as an Fhb1 candidate gene that contains a calmodulin binding motif and two nucleolar localization signal motifs and hence re-annotated as calmodulin binding protein (TaCaMBP_Fhb1). The TaCaMBP_Fhb1 is induced following pathogen infection, binds to Ca2+ bound calmodulin, and triggers Ca2+ signalling cascade including transcriptional activation of endonucleases that cleaves the genomics DNA and cause programmed cell death. The resistant allele of TaCaMBP_Fhb1 lacks part of the promoter region and is non-functional in triggering Ca2+ signalling. While the susceptible allele of TaCaMBP_Fhb1, with functional promoter region is capable of triggering Ca2+ signalling and programmed cell death. The necrotrophic pathogen F. graminearum feeds on the dead tissue, multiply in the host and produce more DON, following a repeated cycle in the susceptible genotype (Chapter IV). The wheat resistance mechanisms against FHB were further confirmed, based on metabolic profiling of rachis, from a resistant cultivar Sumai-3 and a susceptible cultivar Roblin, for resistance against spread of a trichothecene producing (Wild: FgTri5+) and a trichothecene non- producing (mutant: FgTri5-) isolates of F. graminearum. The wild isolate repressed several host resistance mechanisms in both the cultivars due to production of DON. The FHB resistance to spread in Sumai-3 was mainly because of increased cell wall thickening, especially at rachis, due to deposition of lignin, HCAAs and flavonoids, and partially, due to induced RR metabolites which in turn reduced the fungal biomass and toxin biosynthesis. The resistance was not attributed to DON detoxification by UDP-glycosyltransferase, as it was not significant in both the cultivars confirming our previous studies (Chapter V). The resistant alleles of two Fhb1 candidate genes, identified in this study, can be suitably stacked into genome of elite cultivars to enhance FHB resistance in wheat.
La fusariose de l'épi est une maladie fongique attaquant le blé (Triticum aestivum) induite par Fusarium graminearum. La fusariose cause de sévères pertes économiques dues à la réduction de la qualité et des rendements suite à la contamination par les mycotoxines trichothecene. La résistance du blé face à la fusariose est un trait quantitatif. Plus de 100 LCQ on été identifiés et un petit nombre a été validé. Cependant, les mécanismes de résistance gouvernés par ces LCQ sont peu connus. Fhb1 est le LCQ le plus consistent qui produit le plus grand effet face à la fusariose du blé. Une caractérisation fonctionnelle à l'aide d'un LC-MS à haute résolution de lignées isogéniques avec ou sans l'allèle susceptible Fhb1 a générée des profils de métabolites non ciblés ainsi que protéomique. Le Fhb1 d'un cultivar modérément résistant, Nyubai, a été associé avec le développement de la paroi cellulaire plus épaisse, surtout au niveau du rachis due à la déposition d'acides amides hydroxycinnamic (HCAAs), de glucosides phénolique ainsi que de flavonoïdes. Le gène codant pour une protéine hypothétique (GenBank: CBH32656.1) située près du locus Fhb1 a été identifiée comme étant possiblement une hydroxycinnamoyle transférase. Cette protéine déclencherait la biosynthèse de HCAAs. L'accumulation de DON a été plus élevée dans les deux lignes isogéniques. La détoxification de DON est un mécanisme associé avec Fhb1 (Chapitre III). Pour confirmer, l'allèle Fhb1 la résistance du cultivar Sumai-3 a été profilé. Contrairement aux lignes iso géniques, aucune présence constitutive de glycérophospholipides, n'a été détectée chez les lignées susceptibles en raison de la dégradation des membranes. La dégradation de membrane s'est avérée être causée par la mort cellulaire programmée comme le démontre le patron de dégradation de l'ADN de la variété susceptible NIL. Le locus TAA_ctg0954b.00390.1 fut identifié comme candidat pour le gène Fhb1 qui contient un domaine de liaison à la calmoduline et deux signaux de localisation nucléaire. Ce dernier fut donc annoté en tant que protéine de liaison à la calmoduline (TaCaMBP_Fhb1). TaCaMBP_Fhb1 est induit suite à l'infection du pathogène pour ensuite se lier à la calmoduline liée au Ca2+ pour ensuite initier une cascade de signaux qui inclut l'activation transcriptionnelle d'endonucléases qui clivent l'ADN génomique causant ainsi la mort cellulaire programmée. L'allèle résistante de TaCaMBP_Fhb1 présente une délétion au niveau du promoteur ce qui la rend non fonctionnel pour l'activation du signalement Ca2+ impliqué dans la mort cellulaire programmée. Le pathogène nécrotrophe F. graminearum se nourrit des tissus morts, se multiplie et produit plus de DON pour faciliter l'infection; perpétuant ainsi un cercle vicieux chez le génotype susceptible (Chapitre IV). C'est résultats on été confirmés à l'aide d'un profilage métabolique des rachis de la lignée résistante Sumai-3 et la lignée susceptible Roblin lors de l'infection avec (Wild : FgTri5+) trichothécène producteur et (Mutant :FgTri5- ) trichothécène non producteur qui sont deux isolats de F. graminearum. L'isolat producteur est parveu à inhiber plusieurs mécanismes de résistance de l'hôte dans les deux cultivars grâce à la production de DON. La résistance FHB à l'infection dans Sumai-3 était principalement lié à l'augmentation des parois cellulaires particulièrement au niveau des rachis à cause de la déposition de lignine, HCAAs et de flavonoïdes et partiellement due à l'augmentation des métabolite RR qui réduisent la biomasses des champignons ainsi que la synthèse des toxines. La résistance n'a pas été attribuée à détoxification de DON par l'UDP-glycosyltransferase, puisque les résultats étaient similaires dans les deux cultivars (Chapitre V). Les allèles résistants, des deux gènes candidats Fhb1 identifiés dans cette étude, pourraient-être ajoutés au génome de cultivars élites de blé pour augmenter leur résistance au FHB.

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Freeman, Oliver. "The pathogenesis of diabetic neuropathy : a proteomic, metabolomic and electrophysiological investigation." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/the-pathogenesis-of-diabetic-neuropathy-a-proteomic-metabolomic-and-electrophysiological-investigation(a3caaa8a-9ff7-45c7-b6b2-66c8bb6aebf1).html.

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Diabetes mellitus affects more than 382 million people worldwide and an estimated 30-50% of patients develop some form of neuropathy. Patients typically present with sensory symptoms including hypersensitivity/pain and/or loss of somatosensation. In diabetic neuropathy, the longest nerves of the peripheral nervous system (PNS) show the worst pathology and symptoms are typically felt in the distal extremities. The cause of this apparent length-dependent pathology remains unknown. Through comprehensive integration of untargeted proteomic and metabolomic analyses of the PNS in the streptozotocin rat model of diabetes, we showed that bioenergetic pathways were more dysfunctional in the distal sciatic nerve (SN) than the lumbar 4/5 dorsal root ganglia (DRG) and cranial trigeminal ganglia (TG). Whilst glucose levels increased in all tissues in diabetes, there was extensive upregulation of proteins involved in mitochondrial oxidative phosphorylation in the distal SN compared to healthy age/weight-matched controls which was not evident in the proximal DRG or TG. There were significant changes in lipid metabolites in the SN, a phenomenon which is less apparent in the DRG and not evident in the TG. We investigated the therapeutic potential of copper chelation with triethylenetetramine to reverse such changes and whilst copper chelation prevented nerve conduction velocity deficits, it did not alter aberrant nerve metabolism. To further understand the functional deficits in diabetic neuropathy, we performed in vivo microelectrode recordings from the TG and the thalamic ventral posteromedial (VPM) nucleus in control and diabetic rats in response to precise whisker stimulation. Recordings from the TG showed that the tuning of the primary afferents to graded stimuli is preserved in diabetes. Furthermore, we found that neurons within the VPM showed increased spontaneous activity in diabetes, but maintained tuning to graded whisker stimulation in their evoked firing rate. Thus, the cranial TG appear to be relatively unaffected by diabetes at a biochemical or physiological level, but diabetes may lead to pathophysiological changes within the thalamus which could alter somatosensory processing. Despite a global metabolic insult in diabetes, the molecular consequences are not consistent throughout the nervous system. We show that metabolic dysfunction occurs specifically in regions known to be more affected in neuropathy. Due to such a focal dysfunction, aberrant oxidative phosphorylation in the sciatic nerve may be a key driver to the distal pathogenesis of diabetic neuropathy.

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Fraige, Karina. "Estudo comparativo do perfil metabolômico e proteômico de uvas (Vitis vinifera) durante o processo de maturação utilizando ferramentas bioanalíticas." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/75/75135/tde-25072012-091056/.

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A análise da composição química das uvas é de grande importância para avaliar a data da colheita e a produção de vinhos de qualidade. O estudo do metabolismo das uvas é essencial para definirem-se em quais etapas os metabólitos são produzidos, assim como as proteínas e genes que regulam esses processos. Açúcares, polifenóis e ácidos orgânicos são importantes classes de metabólitos relacionados com o desenvolvimento de uvas. Os açúcares são os compostos que primeiramente indicam a data de colheita, sendo substâncias-chave em diversos processos biológicos. Os polifenóis tem se destacado por sua atividade antioxidante, além de participarem dos mecanismos de defesa da planta. Os ácidos orgânicos são responsáveis pela qualidade organoléptica e estão envolvidos em vários processos metabólicos. As proteínas não contribuem de forma significativa para o valor nutricional, mas são importantes marcadores de variedade para verificar adulterações de vinhos. O Rio Grande do Sul é responsável por grande parte das uvas produzidas no país, e recentemente o Vale do São Francisco tem se destacado na produção destas frutas. Em regiões do Sudeste um manejo de podas diferenciado vem sendo feito para a obtenção de uvas com bons índices de maturação e resistência a doenças fúngicas. Uvas produzidas em Água Vermelha e Louveira, interior de São Paulo, foram estudadas durante a maturação com relação a análises físico-químicas, perfil proteômico, por eletroforese bidimensional e espectrometria de massas, e perfil metabolômico de antocianinas, polifenóis não-antociânicos, ácidos orgânicos e açúcares por técnicas cromatográficas e eletroforéticas acopladas a detectores por arranjo de diodos e/ou espectrometria de massas. Os resultados foram analisados por ferramentas de análise multivariada e comparados com uvas maduras das regiões Sul e Nordeste. Foram observadas tendências de agrupamento das uvas verdes devido à maior acidez e das uvas maduras devido à maior concentração de antocianinas e açúcares, sendo que o perfil de antocianinas pode ser utilizado como marcador de origem varietal. Em termos do perfil proteômico foi possível estabelecer diferenças entre variedades de uvas, além de uma tendência com relação à origem geográfica. Foram identificadas 74 proteínas relacionadas principalmente, às funções de defesa e resposta a stress, metabolismo de carboidratos e metabolismo energético.
Analysis of the chemical composition of grapes is very important to evaluate harvest time and production of high quality wines. The study of grape metabolism is essential to define in which stages metabolites are produced, as well as proteins and genes that regulate these processes. Sugars, polyphenols and organic acids are important classes of metabolites related with grape development. Sugars are compounds that primarily indicate harvest time, and they are key substances in various biological processes. Polyphenols have been noted for its antioxidant activity, also for taking part in mechanisms of plant defense. Organic acids are responsible for organoleptic quality, and they are evolved in diverse metabolic processes. Proteins do not contribute significantly to the nutritional value, but they are important variety markers to verify adulterations of wines. Rio Grande do Sul is responsible for most of the grapes produced in Brazil, and recently Vale do São Francisco has received attention because of the production of these fruits. In some regions of Southeast a different pruning handle has started to obtain grapes with good levels of ripeness and resistance in developing fungal diseases. Grapes cultured in Água Vermelha and Louveira, São Paulo State, were studied during ripening in relation to physical-chemical analysis, proteomic profile, by two-dimensional electrophoresis and mass spectrometry; the metabolomic profile of anthocyanins, non-anthocyanin polyphenols, organic acids and sugars by chromatographic and electrophoretic techniques coupled to diode array and/or mass spectrometry detectors. The results were analyzed by multivariate analysis and compared with those of mature grapes from South and Northeast regions. The data show clustering of green grapes due to higher acidity and clusters of mature grapes due to higher anthocyanin and sugars concentrations, and the profile of anthocyanins can be used as a varietal marker. Considering the proteomic profile, it was possible to group different varieties of grapes with a trend in relation to geographical origin being also observed. It was identified 74 proteins related, mainly, to defense and stress response, carbohydrate metabolism and energetic metabolism.

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Huang, Zhengyan. "Differential Abundance and Clustering Analysis with Empirical Bayes Shrinkage Estimation of Variance (DASEV) for Proteomics and Metabolomics Data." UKnowledge, 2019. https://uknowledge.uky.edu/epb_etds/24.

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Mass spectrometry (MS) is widely used for proteomic and metabolomic profiling of biological samples. Data obtained by MS are often zero-inflated. Those zero values are called point mass values (PMVs). Zero values can be further grouped into biological PMVs and technical PMVs. The former type is caused by the absence of components and the latter type is caused by detection limit. There is no simple solution to separate those two types of PMVs. Mixture models were developed to separate the two types of zeros apart and to perform the differential abundance analysis. However, we notice that the mixture model can be unstable when the number of non-zero values is small. In this dissertation, we propose a new differential abundance (DA) analysis method, DASEV, which applies an empirical Bayes shrinkage estimation on variance. We hypothesized that performance on variance estimation could be more robust and thus enhance the accuracy of differential abundance analysis. Disregarding the issue the mixture models have, the method has shown promising strategies to separate two types of PMVs. We adapted the mixture distribution proposed in the original mixture model design and assumed that the variances for all components follow a certain distribution. We proposed to calculate the estimated variances by borrowing information from other components via applying the assumed distribution of variance, and then re-estimate other parameters using the estimated variances. We obtained better and more stable estimations on variance, means abundances, and proportions of biological PMVs, especially where the proportion of zeros is large. Therefore, the proposed method achieved obvious improvements in DA analysis. We also propose to extend the method for clustering analysis. To our knowledge, commonly used cluster methods for MS omics data are only K-means and Hierarchical. Both methods have their own limitations while being applied to the zero-inflated data. Model-based clustering methods are widely used by researchers for various data types including zero-inflated data. We propose to use the extension (DASEV.C) as a model-based cluster method. We compared the clustering performance of DASEV.C with K-means and Hierarchical. Under certain scenarios, the proposed method returned more accurate clusters than the standard methods. We also develop an R package dasev for the proposed methods presented in this dissertation. The major functions DASEV.DA and DASEV.C in this R package aim to implement the Bayes shrinkage estimation on variance then conduct the differential abundance and cluster analysis. We designed the functions to allow the flexibility for researchers to specify certain input options.

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Navarro, Sanz Miriam. "Proteomic and metabolomic approaches to study diabetic retinopahty." Doctoral thesis, Universitat Rovira i Virgili, 2018. http://hdl.handle.net/10803/670958.

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L’objectiu general d’aquesta tesi doctoral va ser desenvolupar, analitzar i validar noves eines bioinformàtiques que converteixin les dades crues de metabolòmica (adquirides per espectrometria de masses) en coneixement biològic amb la finalitat d’estudiar les alteracions en el proteoma i metaboloma de cèl·lules humanes del pigment retinal exposades a condicions de hiperglucèmia i/o hipòxia. Per assolir aquest objectiu general, aquesta tesi s'ha estructurat en dos blocs principals: ⁻ Objectius metodològics: (i) Hem analitzat les bases de dades espectrals de masses de metabolòmica basades en LC / MS. (ii) Finalment, hem generat i millorat la caracterització de les dades de metabolómica de LC / MS centrant-nos en l'anotació de MS1 i MS2. ⁻ Objectius biològics: (iii) Hem proposat un nou mètode que detecta i analitza els canvis en les xarxes d'interacció proteïna-proteïna (PPI) per condicions d’hiperglucèmia i / o hipòxia. (iv) Hem presentat un flux de treball nou que és capaç de predir i validar les alteracions metabòliques degudes a condicions d’hiperglucèmia i / o hipòxia integrant a la vegada les dades d'expressió de proteïnes en les xarxes metabòliques.
El objetivo general de esta tesis doctoral fue desarrollar, analizar y validar nuevas herramientas bioinformáticas que conviertan los datos crudos de metabolómica (adquiridos por espectrometría de masas) en conocimiento biológico con el fin de estudiar las alteraciones en el proteoma y metaboloma de células humanas del pigmento retinal expuestas a condiciones de hiperglucemia y / o hipoxia. ⁻ Objetivos metodológicos: (i) Hemos analizado las bases de datos espectrales de masas de metabolómica basada en LC / MS. (ii) Finalmente, hemos generado y mejorado la caracterización de los datos de metabolómica de LC / MS centrándonos en la anotación de MS1 y MS2. ⁻ Objetivos biológicos: (iii) Hemos propuesto un nuevo método que detecta y analiza los cambios en las redes de interacción proteína-proteína (PPI) por condiciones hiperglucemia y / o hipoxia. (iv) Hemos presentado un flujo de trabajo novedoso que es capaz de predecir y validar las alteraciones metabólicas debidas a condiciones hiperglucemia y / o hipoxia integrando a la vez los datos de expresión de proteínas en las redes metabólicas.
The general objective of this doctoral thesis was to develop, analyse and validate new bioinformatic tools for converting raw MS-based metabolomics data into biological knowledge, in order to study alterations in the proteome and metabolome of human retinal pigment epithelium cells exposed to hyperglycemic and/or hypoxic conditions. To reach this general objective, this thesis has been structured in two main blocks: ⁻ Methodological aims: (i) We have analysed mass spectral databases for LC/MS-based untargeted metabolomics. (ii) Finally, we have generated and improved the characterization of LC/MS metabolomics data focusing on MS1 and MS2 annotation. ⁻ Biological aims: (iii) We have proposed a novel method that detects and analyses changes in protein-protein interaction (PPI) networks by hyperglycemic and/or hypoxic conditions. (iv) We have presented a novel workflow which is able to predict and validate metabolite alterations due to hyperglycemic and/or hypoxic conditions integrating protein expression data in metabolic networks.

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Abdullah, Sewa Faraj. "Proteomics approaches to polyketide synthases interfaces by mass spectrometry and NMR spectroscopy and the application of chemometrics to metabolomics." Thesis, University of Bristol, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.682232.

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Proteomics is a rapidly growing discipline dealing with structure, molecular interactions, conformational dynamics, modifications and the functions of proteins. Mass spectrometry (MS) and (nuclear magnetic resonance) (NMR) have been used comprehensively to study protein interactions. Acyl carrier protein (ACP) interacts with more than 30 partner proteins during either fatty acid or polyketide biosynthesis. In order to be fully activated ACP gains a 4'-phosphopantetheinyl (4'-PP) group from coenzyme A using acyl carrier protein synthase (AcpS) via posttranslational modification. Protein-protein interactions of the ACP from the actinorhodin (act) polyketide synthase (PKS) complex and AcpS were investigated using oxidative footprinting with hydroxyl radicals generated from the Fenton reaction. Chemical modification of acidic residues was also used to investigate the interaction between these proteins using l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) this acted as a zero length cross linker to induce modification with Me-Glycine. MS was used to identify the modified residues and peptides and the extent of modification. Several residues were found to be protected in the complex between the two proteins and these may participate in the interaction interface between ACP and ACPS. Isotopically labelled ACP was expressed and purified and multidimensional NMR experiments were recorded to investigate this interaction interface identified using oxidative footprinting techniques. Chemical shift perturbations for ACP residues were calculated, and these revealed that many residues were affected by oxidation of ACP. Oxidation of methionine to methionine sulfoxide was confirmed. Metabolomics is a discipline which deals with metabolites in a biological system. It provides a wealth information for disease diagnosis, drug discovery, toxicology and genetic modification. Attempts have been made in this thesis to utilize metabolomics in biometrics. Mice were used as a model to attempt to determine individuals' age by their scent. In this part of the project chemometric methods were used to discriminate mice using a gas chromatography-mass spectrometry dataset of volatile organic compounds obtained from their urine. Principal component regression (PCR), partial least squares regression (PLSR) and support vector regression (SVR) were used to determine mouse age. Mice could be discriminated by their age using SVR without overfitting the data.

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Hamzeiy, Hamid [Verfasser], and Christoph [Akademischer Betreuer] Turck. "Advancing computational methods for mass spectrometry-based proteomics, metabolomics, and analysis of multi-omics datasets / Hamid Hamzeiy ; Betreuer: Christoph Turck." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2021. http://d-nb.info/1229835806/34.

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Poljak, Anne Medical Sciences Faculty of Medicine UNSW. "Oxidative, inflammatory and vascular factors in Alzheimer's disease." Publisher:University of New South Wales. Medical Sciences, 2008. http://handle.unsw.edu.au/1959.4/41273.

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In spite of impressive recent progress, the aetiopathogenesis of Alzheimers disease (AD) remains incompletely understood. The distinctive neuropathological features of AD, in particular the plaques and tangles, have been the particular focus of most aetiological theories. It is well accepted that AD is a multifactorial disease, with alterations to a variety of brain structures and cell types, including neurons, glia and the brain vasculature. Studies of risk factors have revealed a diversity of genetic variables that interact with health, diet and lifestyle-related factors in the causation of AD. These factors influence the structure, aggregation and function of a set of proteins that are increasingly the focus of research. The work in this thesis has focused on the pathophysiological aspects of some of these proteins in a number of cellular compartments and brain. Several assays have been established and techniques utilized in the completion of this work, including; differential detergent fractionation of brain tissue, 1D and 2D PAGE, western blotting with chemiluminescence detection, ELISA assays of Abeta 1-40 and 1-42, quantitative ECNI GCMS of o- and m-tyrosine as well as metabolites of the kynurenine pathway, quantitative MALDI-TOF assay of hemorphins and LCMSMS based proteomics, to identify proteins with altered expression levels in AD relative to control brain tissue. A variety of regional differences have been observed in the biochemistry of the AD cortex which are probably the outcome of local response variations to AD pathology. One of the most consistent threads throughout this work has been an apparent resilience of the occipital lobe relative to the other brain regions, as reflected in lower overall levels of oxidative stress and increased levels of proteins associated with metabolic processes, neuronal remodeling and stress reduction.

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He, Ruifeng, Fernanda Salvato, Jeong-Jin Park, Min-Jeong Kim, William Nelson, Tiago Balbuena, Mark Willer, et al. "A systems-wide comparison of red rice (Oryza longistaminata) tissues identifies rhizome specific genes and proteins that are targets for cultivated rice improvement." BioMed Central, 2014. http://hdl.handle.net/10150/610086.

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BACKGROUND:The rhizome, the original stem of land plants, enables species to invade new territory and is a critical component of perenniality, especially in grasses. Red rice (Oryza longistaminata) is a perennial wild rice species with many valuable traits that could be used to improve cultivated rice cultivars, including rhizomatousness, disease resistance and drought tolerance. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development and function in this plant.RESULTS:We used an integrated approach to compare the transcriptome, proteome and metabolome of the rhizome to other tissues of red rice. 116 Gb of transcriptome sequence was obtained from various tissues and used to identify rhizome-specific and preferentially expressed genes, including transcription factors and hormone metabolism and stress response-related genes. Proteomics and metabolomics approaches identified 41 proteins and more than 100 primary metabolites and plant hormones with rhizome preferential accumulation. Of particular interest was the identification of a large number of gene transcripts from Magnaportha oryzae, the fungus that causes rice blast disease in cultivated rice, even though the red rice plants showed no sign of disease.CONCLUSIONS:A significant set of genes, proteins and metabolites appear to be specifically or preferentially expressed in the rhizome of O. longistaminata. The presence of M. oryzae gene transcripts at a high level in apparently healthy plants suggests that red rice is resistant to this pathogen, and may be able to provide genes to cultivated rice that will enable resistance to rice blast disease.

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Lee, Regent. "Biomarkers of coronary atherosclerotic plaque rupture." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:7f0136bf-ad55-4dc8-bcc3-1d55cb269ef8.

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Coronary atherosclerotic plaque rupture is a critical event during atherosclerosis disease progression. Clinical consequences of atherosclerotic plaque rupture vary from asymptomatic to acute arterial thrombosis, yet the mechanisms underpinning such divergent biological response remain poorly understood. Novel biological signatures of plaque rupture will confer further insights into the dynamic responses triggered by plaque rupture event(s), and may provide alternative strategies for modulation of this prevalent disease. This thesis aims to investigate the events that accompany coronary plaque rupture and their relations to subsequent, downstream systemic effects. In a prospective clinical study of patients undergoing non-emergency percutaneous coronary intervention (PCI), stenting induced plaque disruption was used as a model of plaque rupture in vivo. Optical coherence tomography (OCT) imaging of the plaque lesion was performed prior to stenting, followed by serial blood sampling from targeted anatomical sites in reference to the plaque disruption event. Coronary plaque debris were also retrieved in a controlled manner. Analysis of candidate markers demonstrated a role of matrix metalloproteinase 9 (MMP9) in the systemic response to plaque disruption. Local and systemic elevations of MMP9 were observed promptly after plaque disruption. The systemic release of MMP9 was independent to the myocardial injury and systemic inflammation as a result of PCI. OCT analysis further suggested that plaque morphology may be a determining factor in the subsequent MMP9 release, as the disruption of lipid rich plaque(s) resulted in more acute elevation 'of ~1'MP9 when compared to disruption of non-lipid lesions. Changes of systemic MMP9 served as an index of response to the stent induced plaque disruption. Subjects with divergent MMP9 responses to the plaque disruption event were put forward for further comprehensive investigation during the discovery phase, using mass spectrometry techniques to investigate the lipidomic, metabolomics, and proteomic signatures of plaque disruption. Coronary atherosclerotic plaque disruption was associated with immediate changes in the plasma lipidomics and metabolomics profiles, which had distinct relations to the subsequent systemic MMP9 release. Coronary plaque debris provided a "catalogue" of proteins which could be acutely liberated into systemic circulation. Several such novel proteins were detected in circulation after plaque disruption, which triggered disparate responses in known canonical pathways. Evidence from this thesis implicates the role of liver X receptor / retinoic acid receptor pathway (LXR/RXR) as a key mediator to the divergent systemic responses after plaque disruption, and pinpoints a direction for future investigations.

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Alberice, Juliana Vieira. "Avaliação analítica de potenciais biomarcadores para câncer de bexiga em urina." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/75/75135/tde-28082014-152517/.

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O câncer de bexiga é uma neoplasia urogenital que acomete homens e mulheres, sendo que somente no Brasil 8.600 novos casos ao ano são diagnosticados. Cistoscopia transuretral é a conduta padrão no diagnóstico e acompanhamento do câncer de bexiga. Entretanto, tal procedimento é extremamente invasivo e doloroso além de ter elevado custo e não garantir todos os resultados. Assim, busca-se por marcadores moleculares que possam auxiliar no diagnóstico e progressão do câncer de bexiga, bem como diminuir a necessidade de exames invasivos no acompanhamento de pacientes tratados. Nesse sentido, a urina tem papel de destaque como fonte de biomarcadores devido principalmente ao seu caráter não invasivo.
Nesse trabalho foram utilizadas duas abordagens \'ômicas\': proteômica e metabolômica, para a busca de biomarcadores em urina para o diagnóstico e prognóstico do câncer de bexiga, respectivamente. Com a abordagem proteômica buscou-se apenas por biomarcadores para o diagnóstico da doença e, utilizando as técnicas de eletroforese 2-DE, OFFGEL e MS, juntamente com análise estatística multivariada, foi possível identificar 32 proteínas que apresentam-se como potenciais marcadores para o câncer de bexiga. A abordagem metabolômica foi empregada para a busca de biomarcadores para reincidência e progressão da doença. As técnicas analíticas utilizadas nessa abordagem, LC-MS e CE-MS, mostraram-se complementares e, dos resultados obtidos com ambas e avaliados com análise estatística multivariada foi possível indicar 19 metabólitos para reincidência e 23 metabólitos para progressão do câncer de bexiga.
Assim, neste trabalho explorou-se como as ciências \'ômicas\', a qual abrange técnicas analíticas de high-throughput, estatística multivariada e ferramentas de bioinformática auxiliando na descoberta de potenciais biomarcadores não invasivos para o diagnóstico e prognóstico do câncer de bexiga. Portanto, o presente estudo foi de grande importância e relevância à medida que ilustrou como tais técnicas podem potencialmente auxiliar no diagnóstico e prognóstico de doenças e contribuir para tratamentos personalizados no futuro, indicando a potencialidade de estudos dessa natureza.
Bladder cancer is an urogenital cancer affecting men and women, and just in Brazil 8,600 new cases are diagnosed annually. Transurethral cystoscopy is a standard conduct in the diagnosis and monitoring of bladder cancer. However, this procedure is extremely invasive, painful, expensive and does not guarantee the best results. Thus, the searching for molecular markers may assist in the diagnosis and monitoring of bladder cancer, as well as decreasing the need for invasive tests in the monitoring of patients treatment. In this way, urine shows an important role as a source of biomarkers, mainly due to its non-invasive nature.
In this work we used two \'omics\' approaches: proteomics and metabolomics, to search for biomarkers in urine for the diagnosis and progression of bladder cancer, respectively. The proteomics approach was explored for biomarkers for diagnosing disease. Using 2-DE, OFFGEL electrophoresis, and MS techniques, as well multivariate statistical analysis, they were identified 32 proteins that may be pointed as potential markers for bladder cancer. The metabolomics approach was used to search for biomarkers for progression and recurrence of the disease. The analytical techniques used for this approach, LC-MS and CE-MS, were complementary to each other and the results evaluated with multivariate statistical analysis indicated that 19 metabolites for recurrence and 23 metabolites for progression of bladder cancer could possibly be used for validation studies.
Thus, we demonstrated how the \'omics\' sciences, which include high- throughput analytical techniques, multivariate statistical analysis, and bioinformatics tools, aid in the discovery of potential biomarkers for noninvasive diagnosis, evaluate recurrence and monitor progression of bladder cancer. Therefore, this study was of high relevance to demonstrate the potential of such techniques to contribute to studies of personalized medicine.

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Wu, Jikang Dr. "Mass Spectrometry-Based Metabolomics and Protein Native Structure Characterization to Improve Intervention in Salmonellosis and Proteomics-based Biomarker Characterization in Invasive Aspergillosis." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1534775122796275.

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Emami, Khoonsari Payam. "Proteomics Studies of Subjects with Alzheimer’s Disease and Chronic Pain." Doctoral thesis, Uppsala universitet, Klinisk kemi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-331748.

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Alzheimer’s disease (AD) is a neurodegenerative disease and the major cause of dementia, affecting more than 50 million people worldwide. Chronic pain is long-lasting, persistent pain that affects more than 1.5 billion of the world population. Overlapping and heterogenous symptoms of AD and chronic pain conditions complicate their diagnosis, emphasizing the need for more specific biomarkers to improve the diagnosis and understand the disease mechanisms. To characterize disease pathology of AD, we measured the protein changes in the temporal neocortex region of the brain of AD subjects using mass spectrometry (MS). We found proteins involved in exo-endocytic and extracellular vesicle functions displaying altered levels in the AD brain, potentially resulting in neuronal dysfunction and cell death in AD. To detect novel biomarkers for AD, we used MS to analyze cerebrospinal fluid (CSF) of AD patients and found decreased levels of eight proteins compared to controls, potentially indicating abnormal activity of complement system in AD. By integrating new proteomics markers with absolute levels of Aβ42, total tau (t-tau) and p-tau in CSF, we improved the prediction accuracy from 83% to 92% of early diagnosis of AD. We found increased levels of chitinase-3-like protein 1 (CH3L1) and decreased levels of neurosecretory protein VGF (VGF) in AD compared to controls. By exploring the CSF proteome of neuropathic pain patients before and after successful spinal cord stimulation (SCS) treatment, we found altered levels of twelve proteins, involved in neuroprotection, synaptic plasticity, nociceptive signaling and immune regulation. To detect biomarkers for diagnosing a chronic pain state known as fibromyalgia (FM), we analyzed the CSF of FM patients using MS. We found altered levels of four proteins, representing novel biomarkers for diagnosing FM. These proteins are involved in inflammatory mechanisms, energy metabolism and neuropeptide signaling. Finally, to facilitate fast and robust large-scale omics data handling, we developed an e-infrastructure. We demonstrated that the e-infrastructure provides high scalability, flexibility and it can be applied in virtually any fields including proteomics. This thesis demonstrates that proteomics is a promising approach for gaining deeper insight into mechanisms of nervous system disorders and find biomarkers for diagnosis of such diseases.

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Borges, Janaina de Santana. "Análise comparativa do proteoma e metaboloma de raízes de dois clones de E. grandis x E. camaldulensis, sendo um tolerante e um susceptível a condições de estresse hídrico." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-26062013-143137/.

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A crescente demanda por produtos madeireiros no mercado nacional e internacional requer produção constante de madeira, sendo o gênero Eucalyptus uma alternativa para atender esta demanda. A seleção do local para plantio deste gênero requer estudos relacionados às características de adaptabilidade da espécie. Para regiões com déficit hídrico é necessária a seleção de uma espécie ou clone resistente a esta característica. Muitos autores mostram a potencialidade de produção do Eucalyptus camaldulensis e do híbrido E. grandis x E. camaldulensis para regiões áridas do Brasil, em relação a outras espécies de eucalipto. A adaptação de uma espécie a determinado ambiente, esta relacionada a muitas características genéticas que influenciam, por exemplo, o proteoma e o metaboloma desta espécie. As células de um organismo possuem o mesmo genoma, mas apresentam as mais variadas funções e morfologias, e isto está relacionado ao fato de existir diferenças no padrão de expressão de proteínas e metabólitos destas células. As áreas de proteômica e metabolômica auxiliam no entendimento de processos biológicos e fornecem um panorama sobre o estado das plantas em determinado momento e em resposta a determinadas condições/estresses ambientais. Assim o principal objetivo deste trabalho é realizar uma análise comparativa do proteoma e metaboloma de raízes de dois clones de E. grandis x E. camaldulensis, sendo um tolerante e um susceptível ao déficit hídrico, após os indivíduos terem sido submetidos a diferentes regimes hídricos, sendo 100% da capacidade de campo utilizada nas plantas controle e 30% nas plantas tratamento. Os resultados gerados mostraram a existência de proteínas relacionadas ao estresse hídrico e a existência de metabólitos secundários diferencialmente expressos nas plantas controle e tratamento. Esta pesquisa possui um caráter inovador por ser um dos primeiros trabalhos relacionados à área de proteômica e metabolômica de raiz de eucalipto sob estresse hídrico. Novos estudos relacionados a esta área são bem vindos, podendo contribuir na identificação de genes tolerantes ao estresse hídrico e que poderão ser utilizados no futuro na engenharia genética de plantas.
The increasing demand for wood products in the domestic and international markets requires constant wood production and the Eucalyptus genus is an alternative to meet this demand. The site selection for planting this genus requires studies related to characteristics of adaptability of the species to be used. For regions with drought stress, for example, a drought resistant clone or species must be selected. Many authors have shown the potential of Eucalyptus camaldulensis and hybrids of this species with E. grandis for production in arid regions of Brazil. The adaptation of a species to a particular environment may be related to many genetic features which have an impact, for example, on the proteome and metabolome of this species. The cells in an organism have the same genome, but exhibit the most varied functions and morphologies, which are related to differences in the expression pattern of proteins and metabolites of those cells. The areas of proteomics and metabolomics can assist in the understanding the biological processes and supply an overview about plants status at any given time and in response to certain conditions / environmental stresses. The aim of this work is to perform a comparative analysis of the roots´s proteome and metabolome of two E. grandis x E. camaldulensis clones, one tolerant and another susceptible to drought, after the individuals have been subjected to different water regimes, 100% of field capacity for the control plants and 30% for the treated ones. The results showed the presence of proteins related to drought stress and the presence of secondary metabolites differentially expressed in the control when compared to treatment plants. This research has an innovative feature to be one of the first works involving proteomics and metabolomics studies of eucalyptus roots under water stress. New researches related to this field are welcome and may help to identify genes tolerant to drought stress that may be used in the future for genetic engineering plants.

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Vanee, Niti. "HIGH THROUGHPUT DATA FRAMEWORK BASED CHARACTERIZATION AND EVALUATIONS OF THERMOBIFIDA FUSCA FOR INDUSTRIAL APPLICATIONS." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3274.

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Cellulolytic organisms are being heavily studied for the production of biofuels, given that lignocellulosic biomass would be a cheap, abundant, and renewable starting material for chemical production. A challenge with cellulolytic microorganisms is that they are typically poorly characterized and often difficult to genetically manipulate. Our group focuses characterization and engineering of a thermophilic aerobic, cellulolytic actinobacterium, Thermobifida fusca. The wider range of optimal temperature and pH for the growth condition, besides the secretion of several group of cellulases, have made this microbe a potentially efficient host system for industrially application. After the development of first ever successful genetic manipulation protocol by for T. fusca in 2011 in our group the quest continues to better understand and further explore this microbe with such remarkable capabilities. Available genome annotation of the bacteria gives a preliminary clue towards the exploration of its biological system. Genome-scale metabolic reconstruction provides one such framework to populate all the available piece of information to mimic the biological systems to the closest functional state. Further, this skeletal base network can be made more realistic by applying the constraint that controls the flux through various reactions in the pathway network thereby providing the optimal solution space for operation. For the purpose of curation of this in silico model, we aim to integrate the experimental datasets (proteomic and metabolomics) and optimize the agreement between the in silico and in vivo conditions at a steady state condition. Once the model considerably imitates the original biological network, it will be used for the fundamental understanding of the microbial system for the application towards production biofuel and high yields of compound of pharmaceutical interest. The ultimate objective of this project is to design the candidate strain for the cellulolytic production of Natural products. Natural products play an important role in manufacturing of several active pharmaceutical ingredients (APIs). APIs or precursors of APIs can be produced in living organisms with the major challenge of designing and optimizing metabolic pathways to obtain the compounds of interest. In this capacity, living organisms can act as renewable catalysts with high product specificity to produce APIs with potential cost savings over purely synthetic chemistry synthesis routes. This is an effort to understand and design industrially usable microorganism T. fusca to act as a host system for the purpose of production of these compounds. The present project focuses on, in silico characterization and experimental validation of T. fusca, with particular focus on the terpenoids backbone biosynthesis (TBB) pathways using a genome-scale metabolic model, transcriptomics, proteomics and metabolite analysis. The DXP pathway leads to the production of terpenoids precursors that have applications in nutraceutics and pharmaceutics. This study generates the metabolic model, iTFU975 for T. fusca based on the proteomics dataset as the starting point. Further the model and the experimental dataset together helps to characterize the secondary metabolites pathways and compounds in the network associated with the production of terpenoids. In conclusion, this is an effort to characterize the natural products biosynthesis in T. fusca by establishing a bridge between the analytical methodologies and computational efficiencies on “-omics” knowledge to prove the diverse applicability of Systems Biology.

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Zasada, Christin. "Experimental and mathematical analysis of the central carbon metabolism in cancer and stem cells." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18309.

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Die Entstehung von Tumoren und damit einhergehenden Veränderungen wurden insbesondere im letzten Jahrzehnt kontrovers diskutiert. Bisher standen nur wenige Datensätze mit ausreichender Datendichte zur Verfügung um eine umfassende Untersuchung der Regulation des Stoffwechsels durchzuführen. Die in dieser Arbeit zusammengefassten Projekte adressieren verschiedene Aspekte der Stoffwechselregulation und beschreiben die Verknüpfung von Zellkulturexperimenten mit innovativen Hochdurchsatz-Technologien, komplexer Datenanalyse und Computer-basierter Modellierung zur Bestimmung der Stoffwechselflüsse in eukaryotischen Zellen. Die Kombination von GC-MS und LC-MS basierten Technologien ermöglicht die quantitative Analyse des zentralen Kohlenstoffwechsels. Markierungsexperimente mit stabilen Isotopen (pSIRM) erlauben die dynamische Analyse der Stoffwechselaktivität. In verschiedenen Projekten wurden das Proteom und Metabolom von Krebszellen, humanen Stammzellen (hESCs), induzierten pluripotenten Stammzellen (iPS) und deren dazugehörigen differenzierten Vorläufer- oder Nachfolgerzellen bestimmt. Die multivariate, statistische Analyse der Daten ermöglichte die Differenzierung verschiedener Zelltypen basierend auf der Kombination aller quantitativ bestimmten Daten. Quantitative Bestimmungen der Poolgrössen, Isotopeninkorporationen, sowie der extrazellulären Raten in neuronalen, pluripotenten Vorläuferzellen (Luhmes d0) und Neuronen (Luhmes d6) ermöglichte die Bestimmung der Stoffwechselflusskarte beider Zelltypen unter Verwendung der instationären metabolischenen Flussanalyse (INST-MFA). Die Etablierung einer Qualitätskontrolle für GC-MS basierte Daten (MTXQC), sowie die Zuordnung der GC-MS Fragmente zur Molekülstruktur, ermöglichten den Ausbau des Netzwerkes des zentralen Kohlenstoffwechsels und die Implementierung der Daten für die metabolische Flussanalyse.
Metabolic reprogramming of the central carbon metabolism (CCM) is highly debated during the last decade. It describes the rearrangement of nutrient consumption for providing energy and building blocks for cellular proliferation and maintenance. So far, only sparse data are available for an in-depth analysis of metabolic reprogramming events. The herein summarised projects address metabolic programming from different perspectives and show the implementation of cell culture experiments, cutting-edge high-throughput technologies, bioinformatics, and computational modelling into one workflow providing the determination of metabolic flux maps of mammalian cells. The combination of GC-MS and LC-MS-based methodologies enable the quantitative analysis of proteins and metabolites of the CCM. Pulsed stable isotope-resolved metabolomics (pSIRM) experiments allow monitoring the fate of nutrients within the network of the CCM. The time-dependent and position-specific incorporation of carbon-13 leads to an indirect measurement of the metabolic flux, the only one functional readout of a cell. High-throughput technologies were applied in four projects to gain insights in metabolic reprogramming in cancer cell lines, human embryonic stem cells (hESCs), induced pluripotent stem (iPS) cells and their derived fibroblasts. A global principal component analysis demonstrated the discrimination of phenotypes by different classes of quantitative data. The comparison of metabolic and protein levels confirms the presence of the Warburg effect in both cell types. Though, the executing enzymes vary regarding their isoenzyme identity and expression levels. Methodological improvements provided the implementation of GC-MS derived data for INST-MFA. The mapping of GC-MS derived fragments to the molecule structure enables an extension of the CCM network. Robustness of the input data has been improved by the development of a R-scripting based quality control tool (MTXQC).

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Sahlin, Sara. "The next-generation of aquatic effect-based monitoring? : A critical review about the application,challenges and barriers with omics in field." Thesis, Linnéuniversitetet, Institutionen för biologi och miljö (BOM), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-87517.

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Traditional water monitoring encounter limitations due to the large number of contaminants present in our waters possible giving raise to mixture effects. This thesis aimed to investigate how the emerging omics approaches (transcriptomics, proteomics and metabolomics) can be used as an effect-based monitoring approach to assess and predict adverse effects in the freshwater environment. Moreover, this thesis analysed challenges and barrier with omics. A systematic literature search was conducted using Scopus and Web of Science to find case-studies using omics in field studies and reviews regarding challenges and barriers. The results in this thesis suggest that the use of fish species (either collected in the wild or in situ set-ups), transcriptomics and investigations of WWTP recipient was the most common way to apply omics. In order to interpret omics-data multiple studies conducted chemical monitoring in conjunction, investigated additional traditional biomarkers and/or used omics to identify altered biological or functional pathways that possible could lead to adverse effects at higher levels. According to the challenges and barriers identified in this thesis, the future of omics in environmental monitoring rely on the possibility to characterise and quantify natural variability, define appropriate critical effect sizes (i.e. thresholds of critical effects) and define baseline data. Moreover, it is necessary to develop frameworks and standardisations for omics-approaches (e.g. study-designs) to promote the interpretation of the results. Future research is also needed to develop and increase the understanding of how the proteomics and metabolomics can be applied. By improving the use of omics a more holistic water monitoring can be achieved including screenings for biological responses and the ability to detect early warnings which will enhance the prioritisation and site management of polluted water bodies, including those with limited prior knowledge regarding potential contaminants.

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Franco, Flávia de Moraes. "Caracterização do proteoma nuclear e do perfil metabólico primário de folhas da cana-de-açúcar (Saccharum spp) sob condição de déficit hídrico e recuperação." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-09082016-170155/.

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A cana-de-açúcar é uma das principais culturas em países tropicais. O Brasil, além de ser o maior produtor mundial, é também líder em produção de açúcar e álcool. Atualmente, a maior parte da cana-de-açúcar cultivada no Brasil encontra-se em condições , como resultado, a cultura é sujeita ao déficit hídrico em alguns estágios. Portanto, é essencial entender as respostas fisiológicas e moleculares da planta à disponibilidade de água. Nesse contexto, as análises de metabolômica e proteômica objetivam identificar diferentes vias metabólicas e proteínas relacionadas ao mecanismo de defesa e de recuperação. Plantas de Saccharum spp com sete meses de idade foram submetidas a diferentes condições hídricas, déficit e reidratação, e amostras controle foram mantidas irrigadas. A identificação do perfil metabólico primário foi realizada através da cromatografia gasosa combinada com espectrometria de massas (GC-MS). Para identificação das proteínas nucleares, as amostras complexas foram digeridas, e posteriormente, sequenciadas por (LC-MS). As análises estatísticas entre os tratamentos (PLS-DA) mostraram diferenças significativas tanto para metabólitos quanto para as proteínas. Um total de 86 metabólitos foram identificados, onde 8 compostos foram preferencialmente abundantes no estresse, e 10 na recuperação e, portanto, podem ser utilizados como marcadores. Alguns desses compostos participam de vias metabólicas comuns, como biossíntese de alcaloides derivados da ornithine, lysine e nicotinate e de biossíntese de fenilpropanoides. Metabólitos que não participam dessas vias mas foram, pelo menos, duas vezes mais abundantes nos tratamentos quando comparados ao controle também foram discutidos, para o déficit destacam-se galacturonic acid-1-phosphate, pyroglutamic acid e creatinine e para recuperação methyl dihydrogen phosphate, phosphoric acid e 2-hydroxypyridine. Um total de 761 proteínas foram identificadas, sendo 21 nucleares e responsivas ao déficit hídrico, e 32 nucleares e relacionadas ao processo de recuperação. As classes funcionais das proteínas relacionadas ao déficit são de tradução e processo de oxidação-redução, e das proteínas da recuperação são de tradução e proteólise envolvida no processo catabólico proteico. A combinação de diferentes técnicas nesse estudo revela uma dinâmica regulatória complexa no mecanismo de tolerância da cana-de-açúcar ao déficit hídrico.
Sugarcane is one of the main crops in tropical countries. Brazil, besides being the world\'s largest producer of this crop, is also a leader in sugar and ethanol production. Nowadays, most of the sugarcane growing in Brazil is under rain-fed conditions, as a result, the culture is subject to water deficit at certain stages. Thus, it is essential to understand the physiological and molecular plant responses to water availability. In this context, the metabolomic and proteomic analyses aims to identify different metabolic pathways and proteins related to the mechanisms of tolerance and recovery. Samples of seven month old plants of Saccharum spp were subjected to different water conditions, deficit and rehydratation, whereas control samples were kept irrigated. The identification of primary metabolite profile was performed by Gas-Chromatography combined with Mass- Spectrometry (GC-MS). To identify nuclear proteins, the complex samples were digested and then sequenced by LC-MSE. Statistical analyses among treatments PLS-DA showed significant differences in both metabolites and proteins of Saccharum spp in different conditions. A total of 86 metabolites were identified, where 8 are preferably abundant in water stress and 10 in recovery, thus, they can be used as markers. Some of these compounds are present in common pathways like biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid and biosynthesis of phenylpropanoids. Metabolites that do not participate in these pathways, but that were at least two times more abundant in treatments when compared to control, were also discussed. They were galacturonic acid-1-phosphate, pyroglutamic acid and creatinine that were related to deficit condition and methyl dihydrogen phosphate, phosphoric acid and 2-hydroxypyridine to recovery. A total of 761 proteins were identified, of which 21 were nuclear and drought responsive and 32 were nuclear and recovery responsive. The functional classes of water stress proteins are translation and oxidation-reduction process and of recovery proteins are translation and proteolysis involved in cellular protein catabolic process. The combination of different techniques in this study revealed a complex regulatory dynamics in the mechanism of sugarcane water stress tolerance that have not been discussed in the literature.

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Swensen, Adam Clayton. "Investigation of Dynamic Biological Systems Using Direct Injection and Liquid Chromatography Mass Spectrometry." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6574.

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In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.

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Jones, Christina Michele. "Applications and challenges in mass spectrometry-based untargeted metabolomics." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54830.

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Metabolomics is the methodical scientific study of biochemical processes associated with the metabolome—which comprises the entire collection of metabolites in any biological entity. Metabolome changes occur as a result of modifications in the genome and proteome, and are, therefore, directly related to cellular phenotype. Thus, metabolomic analysis is capable of providing a snapshot of cellular physiology. Untargeted metabolomics is an impartial, all-inclusive approach for detecting as many metabolites as possible without a priori knowledge of their identity. Hence, it is a valuable exploratory tool capable of providing extensive chemical information for discovery and hypothesis-generation regarding biochemical processes. A history of metabolomics and advances in the field corresponding to improved analytical technologies are described in Chapter 1 of this dissertation. Additionally, Chapter 1 introduces the analytical workflows involved in untargeted metabolomics research to provide a foundation for Chapters 2 – 5. Part I of this dissertation which encompasses Chapters 2 – 3 describes the utilization of mass spectrometry (MS)-based untargeted metabolomic analysis to acquire new insight into cancer detection. There is a knowledge deficit regarding the biochemical processes of the origin and proliferative molecular mechanisms of many types of cancer which has also led to a shortage of sensitive and specific biomarkers. Chapter 2 describes the development of an in vitro diagnostic multivariate index assay (IVDMIA) for prostate cancer (PCa) prediction based on ultra performance liquid chromatography-mass spectrometry (UPLC-MS) metabolic profiling of blood serum samples from 64 PCa patients and 50 healthy individuals. A panel of 40 metabolic spectral features was found to be differential with 92.1% sensitivity, 94.3% specificity, and 93.0% accuracy. The performance of the IVDMIA was higher than the prevalent prostate-specific antigen blood test, thus, highlighting that a combination of multiple discriminant features yields higher predictive power for PCa detection than the univariate analysis of a single marker. Chapter 3 describes two approaches that were taken to investigate metabolic patterns for early detection of ovarian cancer (OC). First, Dicer-Pten double knockout (DKO) mice that phenocopy many of the features of metastatic high-grade serous carcinoma (HGSC) observed in women were studied. Using UPLC-MS, serum samples from 14 early-stage tumor DKO mice and 11 controls were analyzed. Iterative multivariate classification selected 18 metabolites that, when considered as a panel, yielded 100% accuracy, sensitivity, and specificity for early-stage HGSC detection. In the second approach, serum metabolic phenotypes of an early-stage OC pilot patient cohort were characterized. Serum samples were collected from 24 early-stage OC patients and 40 healthy women, and subsequently analyzed using UPLC-MS. Multivariate statistical analysis employing support vector machine learning methods and recursive feature elimination selected a panel of metabolites that differentiated between age-matched samples with 100% cross-validated accuracy, sensitivity, and specificity. This small pilot study demonstrated that metabolic phenotypes may be useful for detecting early-stage OC and, thus, supports conducting larger, more comprehensive studies. Many challenges exist in the field of untargeted metabolomics. Part II of this dissertation which encompasses Chapters 4 – 5 focuses on two specific challenges. While metabolomic data may be used to generate hypothesis concerning biological processes, determining causal relationships within metabolic networks with only metabolomic data is impractical. Proteins play major roles in these networks; therefore, pairing metabolomic information with that acquired from proteomics gives a more comprehensive snapshot of perturbations to metabolic pathways. Chapter 4 describes the integration of MS- and NMR-based metabolomics with proteomics analyses to investigate the role of chemically mediated ecological interactions between Karenia brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. This integrated systems biology approach showed that K. brevis allelopathy distinctively perturbed the metabolisms of these two competitors. A. glacialis had a more robust metabolic response to K. brevis allelopathy which may be a result of its repeated exposure to K. brevis blooms in the Gulf of Mexico. However, K. brevis allelopathy disrupted energy metabolism and obstructed cellular protection mechanisms including altering cell membrane components, inhibiting osmoregulation, and increasing oxidative stress in T. pseudonana. This work represents the first instance of metabolites and proteins measured simultaneously to understand the effects of allelopathy or in fact any form of competition. Chromatography is traditionally coupled to MS for untargeted metabolomics studies. While coupling chromatography to MS greatly enhances metabolome analysis due to the orthogonality of the techniques, the lengthy analysis times pose challenges for large metabolomics studies. Consequently, there is still a need for developing higher throughput MS approaches. A rapid metabolic fingerprinting method that utilizes a new transmission mode direct analysis in real time (TM-DART) ambient sampling technique is presented in Chapter 5. The optimization of TM-DART parameters directly affecting metabolite desorption and ionization, such as sample position and ionizing gas desorption temperature, was critical in achieving high sensitivity and detecting a broad mass range of metabolites. In terms of reproducibility, TM-DART compared favorably with traditional probe mode DART analysis, with coefficients of variation as low as 16%. TM-DART MS proved to be a powerful analytical technique for rapid metabolome analysis of human blood sera and was adapted for exhaled breath condensate (EBC) analysis. To determine the feasibility of utilizing TM-DART for metabolomics investigations, TM-DART was interfaced with traveling wave ion mobility spectrometry (TWIMS) time-of-flight (TOF) MS for the analysis of EBC samples from cystic fibrosis patients and healthy controls. TM-DART-TWIMS-TOF MS was able to successfully detect cystic fibrosis in this small sample cohort, thereby, demonstrating it can be employed for probing metabolome changes. Finally, in Chapter 6, a perspective on the presented work is provided along with goals on which future studies may focus.

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Mounayar, Rana. "Oral fat sensitivity in humans : links with salivary composition." Thesis, Dijon, 2013. http://www.theses.fr/2013DIJOS074.

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La perception du gras chez l’homme est un phénomène complexe du fait de sa nature multi-sensorielle impliquant la perception de la texture, la perception aromatique mais également la perception gustative. Cette dernière a été suggérée après l’identification de récepteurs aux acides gras au niveau des bourgeons gustatifs. Par ailleurs, des études récentes ont montré que la sensibilité au gras est variable entre individus. Des facteurs génétiques ou environnementaux pourraient expliquer en partie cette variation interindividuelle. Cependant, la salive pourrait aussi jouer un rôle dans cette perception. En effet, elle contient des molécules capables d’interagir avec le gras comme la lipase et les lipocalines. C’est aussi un fluide complexe qui contient une large diversité de protéines et de métabolites. De plus, sa régulation est complexe et peut varier à la suite d’une stimulation. En effet, des études récentes ont montré que le protéome salivaire est modifié à la suite d’une stimulation par des molécules correspondant aux saveurs primaires. Dans ce contexte, l’objectif de ce travail était de déterminer dans un premier temps s’il existe des liens entre la composition salivaire et la sensibilité gustative à un acide gras libre: l’acide oléique. Le deuxième objectif était d’étudier les modifications de la composition salivaire à la suite d’une stimulation gustative par l’acide oléique. Pour ceci deux approches ont été utilisées: des approches ciblées (activité enzymatique, capacité antioxydante etc) et des approches non-ciblées (protéomique et métabolomique). Deux groupes de treize sujets (hyper et hyposensible au goût de l’acide oléique) ont été sélectionnés à partir d’un panel de 73 participants. Leur salive a été collectée au repos et après stimulation par l’acide oléique. Les résultats montrent que la composition de la salive au repos est liée à la sensibilité à l’acide oléique. En effet, des marqueurs liés à la perception du goût ont été identifiés au sein du groupe des hypersensibles (anhydrase carbonique, cystatines et zinc alpha 2 glycoprotéine), alors que des marqueurs pouvant indiquer une activité bactérienne élevée (acides organiques) ont été identifiés au sein du groupe des hyposensibles. Par ailleurs, la composition de la salive collectée après stimulation par l’acide oléique est également modifiée et ces modifications sont différentes pour les sujets hyper et hyposensibles à cette stimulation
Human fat perception has recently triggered particular interest as it was shown that it does not only involve aroma and texture perception but also taste perception. The latter was supported by the presence of free fatty acids (FFA) taste receptors on the tongue. Recent studies have shown that fat taste sensitivity is variable among individuals. This inter-individual variation could be linked to genetic or environmental factors. However, saliva could also play a role in this perception. The role of saliva in taste perception is increasingly recognized. Saliva contains molecules able of interacting with fat such as lipase and lipocalin. It is also a complex fluid which contains a large diversity of proteins and metabolites. Its regulation is also complex and its composition may vary after a sensory stimulation. Indeed, studies have shown that when giving primary taste stimulations, the whole salivary proteome is modified. Thus, the first aim of the present work was to use both targeted (enzymatic activity, antioxidant capacity etc) and untargeted approaches (proteomics and metabolomics) to identify links between taste sensitivity to a fatty acid, oleic acid, and the salivary composition. The second aim was to investigate whether the salivary composition is modified after an oral stimulation by oleic acid.Two groups of thirteen male subjects (highly and weakly sensitive to the taste of oleic acid) were selected from an initial panel of 73 healthy participants. Their whole saliva was collected in two ways; the first without stimulation in order to study the links between oral sensitivity to oleic acid and saliva composition and the second using a stimulation by the same fatty acid in order to study potential modifications of saliva composition depending on sensitivity. Results show that salivary composition is linked to oral fatty acid perception. Markers previously reported as associated to taste perception were determined in the highly sensitive group (carbonic anhydrase, Zinc Alpha 2 glycoprotein and cystatins) while markers (organic acids) indicating a higher bacterial load were identified in weakly sensitive group. Furthermore, results obtained after stimulation by oleic acid suggest that saliva composition is modified, which confirms its dynamic nature. As different modifications were observed for the highly and weakly sensitive group, our results suggest that saliva is not only modified after a stimulation but also depending on the sensitivity to that particular stimulation

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Whitehill, Justin G. A. "INVESTIGATIONS INTO MECHANISMS OF ASH RESISTANCE TO THE EMERALD ASH BORER." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306863052.

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Vvedenskaya, Olga. "An in vivo study into the metabolic reprogramming of hepatocellular carcinoma." Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/19268.

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Die vorliegende Arbeit untersucht die Rolle des Metabolismus in der Entstehung und Progression des Hepatozellulären Karzinoms (HZK). Der Schwerpunkt der Studie liegt auf Veränderungen zentraler Stoffwechselwege, unter anderem der Glykolyse, der Gluconeogenese, des Citratzyklus und anderer Prozesse des Zellstoffwechsels. Umfassende Multiomikanalysen, wie etwa Proteomik, Metabolomik und gezielte Genomsequenzierung wurden angewandt, um in vivo die Mechanismen der HZK Entstehung zu verstehen. Es wurden zwei Systeme untersucht: das ASV-B Mausmodell und klinische Patientenproben. Die Kohorte bestehend aus Biopsien und Resektaten von 95 Patienten umfasste 47 Fälle von HZK und 48 Fälle ohne HZK. Das Proteom des Mausmodells und der Patientenkohorte zeigen eine deutliche Herabregulierung wesentlicher Energie bereitstellender Kreisläufe im HZK: Glykogenstoffwechsel, de novo Synthese von Glukose, Glutaminaufnahme in den Citratzyklus, des weiteren sind 60% der Enzyme des Citratzyklus, und des Transports von Pyruvat in Mitochondrien im HZK herabreguliert. In dieser Arbeit wurde ein Isoformenwechsel auf mehreren Ebenen des zentralen Kohlenstoffmetabolismus gezeigt. Sowohl das Mausmodell, als auch die Gewebeproben von HZK-Patienten weisen Isoformenwechsel der Phosphoglyzeratmutasen und der Pyruvatkinasen auf. Die Hauptmerkmale finden sich sowohl in Modellmäusen, als auch in Patienten, und stellen so einen universalen metabolomischen Fingerabdruck des HZK dar. Darüber hinaus demonstriert diese Studie, dass die Proteomanalyse von bioptischen Material ein aussagekräftiges und ausreichendes molekular-diagnostisches Instrument für die Krebsforschung ist: die Proteomanalyse von Lebermaterial erlaubt die Unterscheidung von Tumorgewebe und tumorfreien Proben und die Dokumentation des Krankheitsverlaufs.
The present work evaluates the role of metabolism in development and progression of hepatocellular carcinoma (HCC). This study focuses on changes of central metabolic pathways, including glycolysis, gluconeogenesis, tricarboxylic acid (TCA) cycle and other processes involved in cellular metabolism and known to be dysregulated during cancer formation. Comprehensive multiomics analyses, such as proteomics, metabolomics and targeted genome sequencing, were applied in order to better understand HCC developmental mechanisms in vivo. Two main systems were studied: the ASV-B mouse model and clinical samples from human patients. The human cohort was composed of biopsy and surgery material from 95 patients: 47 HCC and 48 non-HCC. Proteomic data from both mice and humans show a clear downregulation of the main energy-producing pathways in HCC. Glycogen metabolism, de novo glucose synthesis, glutamine uptake to the TCA cycle, approximately 60% of enzymes of TCA cycle, and transport of pyruvate to mitochondria are downregulated in HCC. An isoform switch at various levels of central carbon metabolism was demonstrated in this work. Both mice and humans with HCC reveal isoform switches at the level of phosphoglycerate mutases and pyruvate kinases. The key features are found in both mouse and human, showing a universal metabolic HCC fingerprint. This study also demonstrates that proteomic analysis of the bioptate material is a strong and sufficient molecular diagnostic tool for research in cancer: the proteomic analysis of liver material allows the distinction of tumor samples from non-tumor samples and also to track the level of disease progression. Targeted genome sequencing revealed that no clear distinction between cancer and precancerous conditions could be made exclusively from the mutation analysis. Human metabolomic data remains inconclusive, possibly due to the different sources of tissue samples.

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Harmel, Robert Klaus. "New Analytical Tools to Interrogate Inositol Pyrophosphate Signaling." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21392.

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Inositolpyrophosphate (PP-InsPs) sind eine wichtige Gruppe eukaryotischer Botenstoffe, die mit verschiedenen Prozessen wie Apoptose, Phosphathomeostase und Insulinsignalkaskaden verknüpft sind. Trotz ihrer Entdeckung vor mehr als 20 Jahren bleibt es eine Herausforderung, die Signalmechanismen dieser Moleküle zu verstehen. Ursachen dafür sind der limitierte Zugang zu synthetischen PP-InsPs und ein Mangel an allgemein zugänglichen analytischen Methoden. Daher wurden in dieser Arbeit chemische und analytische Verfahren entwickelt, um unser Verständnis von diesen Molekülen sowohl auf ein biochemischer als auch auf zelluläre Ebene zu verbessern. Um der Knappheit an synthetischen PP-InsPs entgegen zu wirken, wurde eine hocheffiziente chemoenzymatische Synthese entwickelt, bei der mehr als 100 mg aller wesentlichen PP-InsPs aus Säugern hergestellt werden konnten. Parallel wurde ein neues analytisches Werkzeug entwickelt, dass Konzentrationen von PP-InsPs in komplexen Proben quantifizieren konnte. Mittels Enzymkatalyse konnten 13C-markiertes myo-inositol und 13C-markierte PP-InsPs hergestellt werden und niedrige Konzentrationen mit nuklearer Magnetresonanzspektroskopie detektiert werden. In vitro waren diese Verbindungen sehr nützlich, um PP-InsP Kinasen von Pflanzen und Säugern zu charakterisieren. Endogene Konzentrationen von PP-InsPs konnten durch metabolisches Markieren mit 13C-markiertem myo-inositol in humanen Zelllinien quantifiziert werden. Letztendlich wurde mittels eines neuen entwickelten proteomischen Ansatzes endogene Proteinpyrophosphorilierung, eine von PP-InsP eingebaute posttranslationale Proteinmodifikation, in menschlichen Zelllinien zum ersten Mal nachgewiesen. Zusammenfassend haben die aufgelisteten chemischen und analytischen Werkzeuge ein hohes Potenzial unser Verständnis der Signalmechanismen hinter den diversen Phänotypen der PP-InsPs zu stärken und Forschungsarbeit in dieser Richtung zu beschleunigen.
Inositol pyrophosphates (PP-InsPs) are an important group of second messengers that intersect with a wide range of processes in eukaryotic cells including phosphate homeostasis, insulin signaling and apoptosis. Despite their discovery more than two decades ago, elucidating the underlying signaling mechanisms remains a significant challenge. Therefore, a new set of chemical and analytical methods was developed here to improve our understanding of these intriguing molecules on the biochemical and cellular level. To overcome the shortage of synthetic PP-InsPs, a highly efficient and scalable chemoenzymatic approach was designed and the major mammalian PP-InsPs could be obtained in hundreds of milligram quantities and in high purity. In parallel, a new analytical tool was developed to quantify levels of PP-InsPs in complex samples. Chemoenzymatic access to 13C-labeled myo-inositol and 13C-labeled PP-InsPs enabled the detection of low concentrations of PP-InsPs using nuclear magnetic resonance spectroscopy. In vitro, these compounds were of great use for the biochemical characterization of PP-InsPs kinases from mammals and plants. Endogenous pools of PP-InsPs from human cell lines were identified and quantified by metabolic labeling with 13C-labeled myo-inositol. Finally, a new proteomics workflow towards the detection of protein pyrophosphorylation, a posttranslational modification mediated by PP-InsPs, using mass spectrometry was optimized and endogenously modified mammalian proteins could be identified for the first time and with high confidence. Taken together, the chemical and analytical tools presented here have great potential to accelerate the understanding of PP-InsP signaling and metabolism. Access to large amounts of PP-InsPs together with a reliable quantification method and the detection of endogenous protein pyrophosphorylation sites will be essential to unravel the signaling mechanisms underlying the diverse phenotypes associated with these metabolites.

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Budzinski, Ilara Gabriela Frasson. "Avaliação do metabolismo primário da região cambial e casca de Eucalyptus grandis." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-02012013-172036/.

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O gênero Eucalyptus é uma das principais espécies arbóreas comercialmente plantadas em todo o mundo. Apresenta inúmeras características favoráveis para a produção e comercialização de sua madeira, tais como o rápido crescimento, rotação de ciclo curto e produção de biomassa renovável, com potencial para geração de biocombustível. Apesar de sua grande importância econômica, pouco é conhecido sobre os processos moleculares que envolvem a formação da madeira e casca, principalmente no que diz respeito ao metabolismo primário. Ademais, não se têm muitas informações sobre mudanças moleculares que ocorrem durante a formação desses tecidos em resposta a variações sazonais. Sabe-se que a região cambial das árvores apresenta maior atividade metabólica durante o verão, quando comparada ao inverno. Deste modo, visando compreender mudanças dinâmicas ao nível transcricional, protéico e metabólico, principalmente em relação ao metabolismo primário, o presente trabalho teve por objetivo comparar os tecidos da região cambial e casca, coletados em diferentes períodos climáticos (verão e inverno). A expressão do padrão transcricional foi analisada por PCR em tempo real, seguido de normalização e análise estatística, usando os programas NormFinder e Rest, respectivamente. O perfil protéico foi obtido por eletroforese bidimensional (2D-PAGE), seguido de análise por espectrometria de massas associada a cromatografia líquida (LC-MS/MS) e posterior identificação das proteínas pelo programa Mascot Daemon. Já o perfil metabólico foi obtido por espectrometria de massas associada à cromatografia gasosa (GC/MS), com posterior análise dos picos identificados pelo programa MatLab. Em seguida, as análises estatísticas foram geradas pelo programa SIMCA P+ e \"R\". Para os transcritos, foi possível observar tanto para a região cambial quanto para a casca padrão diferencial na expressão dos genes analisados, principalmente aqueles atuantes na glicólise, durante os dois períodos sazonais. Quanto ao perfil protéico, foram identificadas um total de 77 e 75 proteínas estatisticamente significativas na região cambial e casca, respectivamente. Proteínas pertencentes ao metabolismo primário foram identificadas em ambos os tecidos. Metabólitos relacionados ao metabolismo de açúcares também foram encontrados.
Eucalyptus genus is the most widely planted hardwood crop in the world because of its superior growth, broad adaptability and multipurpose wood properties. In today\'s \"new carbon economy\", eucalypts are receiving attention as fast-growing, short-rotation, renewable biomass crop for energy production. In spite of its economical importance, little information is available about the molecular changes that occur in primary metabolism in the wood and bark forming tissues. Furthermore, there is less information about molecular changes that occur during wood and bark formation in response to seasonal variation. It\'s known that Eucalyptus cambial region presents higher metabolic activity in summer than in winter. Thus, in order to observe the dynamic changes in transcript, protein and metabolite levels, mainly related to primary metabolism, we compared cambial tissue and bark collected in two different seasons (summer high temp + high rainfall) and winter (lower temp and little rainfall). Transcript expression patterns were analyzed by Real-Time PCR, normalization chosen by NormFinder and statistical analysis carried out using REST. The protein profile was obtained by bidimensional electrophoresis (2D-PAGE) followed by liquid chromatography associated with mass spectrometry (LC-MS/MS) and analyzed by Mascot Daemon. Metabolite profile was obtained by GC-TOF/MS, peaks were analyzed in MatLab and statistical analyses were done using SIMCA and \"R\". The results obtained with transcripts indicate differential gene expression in the cambial region and bark during summer and winter. A total of 77 and 75 proteins in cambium and bark, respectively, presented statistically significant alterations and were identified and classified into functional categories. We identified many proteins from primary metabolism. Metabolites from carbohydrate metabolism were also identified.

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38

Correia, Barbara dos Santos. "Linking omics and ecophysiology in Eucalytus: unravelling stress tolerance in a forest species." Doctoral thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/18399.

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Doutoramento em Biologia
Eucalyptus plantations are among the most productive forest stands worldwide. In Portugal and Spain, they are widely used for pulp production and as an energy crop. However, the region’s Mediterranean climate, with increasingly severe summer drought, negatively affects eucalypt growth and increases mortality. The aim of this doctoral thesis was to unravel drought tolerance in Eucalyptus globulus by investigating and interconnecting information on the processes mediating water deficit and rehydration, from gene and molecular regulation to physiological responses and plant performance, using two different genotypes and different stress trials. The thesis disclosed herein is presented in a series of research papers (chapters 2, 3, 4 and 5), preceded by a general introduction (chapter 1) and closed with concluding remarks (chapter 6). Chapter 2 describes a greenhouse trial and a slowly imposed water deficit, and is divided into three subchapters. Two genotypes (AL-18 and AL-10) were submitted to a 3-week water stress period at two different intensities (18% and 25% of field capacity), followed by 1 week of rewatering. Recovery was assessed 1 day and 1 week after rehydration. Several phytohormones were monitored in leaves, xylem sap and roots, 2 h, 4 h, 24 h, and 168 h after rewatering. Water deficit reduced height, biomass, water potential, and gas exchange. Contrarily, the levels of pigments, chlorophyll fluorescence parameters and MDA increased. ABA and ABA-GE levels increased, and JA content decreased in leaves and increased in xylem sap. During recovery, most of the physiological and biochemical responses of stressed plants were reversed. Comparative proteome (using difference gel electrophoresis) and metabolome (using gas chromatography–mass spectrometry) analyses enabled the separation and isolation of 2031 peptide spots, 217 of which were identified, and the detection of 121 polar metabolites. The analysis of the resilient clone AL-18, which presented a response network very distinct from the responsive clone AL-10, reinforced the role of specific photosynthetic and defence-related proteins as key players in mediating drought tolerance and revealed new players: glutamine synthetase, malate dehydrogenase and isoflavone reductase-like protein. Chapter 3 regards a climate chamber trial and a sudden water shortage, and is divided in two subchapters. The relative expression of 12 transcripts was analysed by quantitative PCR in two clones with different degrees of tolerance (AL-18 and AL-13) 7 and 11 days after water withholding and rehydration (2 h and 3 days after rewatering). Sudden water shortage was more detrimental to the plants than when slowly imposed, with heavier outcomes in clone AL-13, including plant death. Potential molecular indicators linked to enhanced water stress tolerance in Eucalyptus globulus were identified: rubisco activase (RCA), ferredoxin-NADP(H) oxireductase (FNR), mitochondrial malate dehydrogenase (mMDH), peroxisomal catalase (CAT) and isoflavone reductase (IFR). Afterwards, several biochemical markers of oxidative stress and DNA methylation patterns were quantified in the leaves of AL-18. The alterations detected using global and specific indicators reflected the parallel induction of redox and complex DNA methylation changes occurring during stress imposition and relief. Chapter 4 reports a field trial: the previously identified set of indicators for selection of water stress tolerance was tested in field-grown AL-18 and AL-13. Some of the plants were irrigated (IR), and others were left under environmental conditions of reduced rainfall (NI) during six and a half weeks prior to rewatering. Clone AL-18 showed few fluctuations in the conditions tested, and the alterations found in clone AL-13 highlighted an induction of photosynthetic and photorespiration metabolism after artificial rehydration. The results corroborated that responses to field conditions cannot be extrapolated from a stress applied individually in the context of developing selection markers. Chapter 5 describes a climate chamber trial that tested the isolated and combined effect of drought and heat. Physiological, biochemical and metabolomic alterations were monitored in AL-18 after a 5-day of consistent drought and/or 4 h at 40ºC. Testing drought-stressed plants subject to a heat shock revealed a decrease in gas exchange, Ψpd and JA, no alterations in electrolyte leakage, MDA, starch and pigments and increased glutathione pool in relation to control. The induction of cinnamate was a novel response triggered only by the combined stress. These results highlighted that the combination of drought and heat provides significant protection from more detrimental effects of drought-stressed eucalypts, confirming that combined stresses alter plant metabolism in a novel manner that cannot be extrapolated by the sum of the different stresses applied individually. This thesis describes a number of biological responses that enable E. globulus to thrive under conditions of water deficit and provides useful information of pathways to be explored in order to find suitable markers of abiotic stress tolerance in this species. Despite that, a bigger challenge remains and consists of the need to focus our studies in more realistic, field-like experiments, at least in the context of finding suitable selection markers in the climate change era.
As plantações de eucalipto estão entre as mais produtivas do mundo inteiro. Em Portugal e Espanha, são amplamente utilizadas na produção de polpa e como fonte de energia. No entanto, o clima mediterrânico da região, com secas de verão cada vez mais severas, afeta negativamente o crescimento do eucalipto e aumenta a sua mortalidade. Esta tese de doutoramento tem como objetivo desvendar a tolerância à seca da espécie Eucalyptus globulus, investigando e interligando informação dos processos que medeiam o défice hídrico e a reidratação, desde a regulação genética e molecular até às respostas fisiológicas e desempenho da planta, utilizando dois genótipos distintos e diferentes ensaios experimentais. Esta tese está estruturada sob a forma de estudos científicos (capítulos 2, 3, 4 e 5), precedidos por uma introdução geral (capítulo 1), e termina com as notas finais (capítulo 6). O capítulo 2 descreve um ensaio de estufa e um défice hídrico imposto lentamente, e está dividido em 3 subcapítulos. Dois genótipos (AL-18 e AL-10) foram sujeitos a um período de stress hídrico de 3 semanas com duas intensidades diferentes (18% e 25% da capacidade de campo), seguido de uma semana de reidratação. A recuperação foi avaliada um dia e uma semana depois da reidratação. Várias fitohormonas foram monitorizadas nas folhas, seiva xilémica e raízes, 2 h, 4 h, 24 h e 168 h depois da reidratação. A falta de água reduziu a altura, a biomassa, o potencial hídrico e as trocas gasosas. Pelo contrário, os níveis de pigmentos, parâmetros da fluorescência da clorofila e MDA aumentaram. Os níveis de ABA e de ABA-GE aumentaram, enquanto o JA diminuiu nas folhas e aumentou na seiva xilémica. Durante a recuperação, a maioria das alterações fisiológicas e bioquímicas provocadas pelo stress reverteram. Análises comparativas do proteoma (analisado por eletroforese em gel diferencial) e do metaboloma (analisado por cromatografia gasosa com espetrometria de massa) permitiram a separação de 2031 pontos peptídicos, dos quais 217 foram identificados, e a deteção de 121 metabolitos polares. A análise do clone resiliente AL-18, que apresentou uma rede de resposta bem distinta do clone responsivo AL-10, reforçou o papel de proteínas específicas da fotossíntese e relacionadas com a defesa como intermediários chave na tolerância à seca e revelou novos intermediários: glutamina sintetase, malato desidrogenase e isoflavona redutase. O capítulo 3 diz respeito a um ensaio em câmara climática e a uma rápida escassez de água, e está dividido em 2 subcapítulos. A expressão relativa de 12 transcritos foi analisada por PCR quantitativo em dois clones com diferentes graus de tolerância (AL-18 e AL-13) depois de 7 e 11 dias sem qualquer rega e posterior reidratação. A rápida escassez de água foi mais prejudicial para as plantas do que o défice hídrico imposto lentamente, com maior visibilidade no clone AL-13 que revelou morte de algumas plantas. Indicadores moleculares potencialmente ligados a uma tolerância aumentada foram identificados: rubisco ativase (RCA), ferredoxina-NADP(H) oxidorredutase (FNR), malato desidrogenase mitocondrial (mMDH) catalase peroxissomal (CAT) e isoflavona redutase (IFR). De seguida, vários marcadores bioquímicos de stress oxidativo e padrões de metilação do DNA foram quantificados nas folhas do clone AL-18. As alterações detetadas utilizando indicadores globais e específicos refletiram a indução de complexas modificações redox e de metilação do DNA, que ocorrem paralelamente durante a imposição e interrupção do stress. O capítulo 4 reporta um ensaio de campo: o conjunto de indicadores de seleção de tolerância hídrica identificado anteriormente foi testado em AL-18 e AL-13 plantados no campo. Algumas das plantas foram regadas artificialmente (IR) e outras foram deixadas nas condições ambientais de precipitação reduzida (NI) durante seis semanas e meia antes de voltar a regar. O clone AL-18 mostrou pouca variação nas condições testadas, e as alterações encontradas no clone AL-13 realçaram a indução do metabolismo fotossintético e fotorespiratório após a reidratação artificial. Estes resultados mostraram que as respostas das plantas no campo não podem ser extrapoladas a partir do estudo de um stress aplicado individualmente, particularmente no contexto de encontrar marcadores de seleção. O capítulo 5 descreve um ensaio em câmara climática que testou o efeito isolado e combinado de seca e calor. Alterações fisiológicas, bioquímicas e metabolómicas foram monitorizadas no clone AL-18 após 5 dias de seca consistente e/ou 4 h a 40ºC. Testar plantas em stress hídrico sujeitas a um choque térmico revelou uma diminuição das trocas gasosas, do potencial hídrico e do JA, nenhum efeito a nível da perda de eletrólitos, MDA, amido e pigmentos e um aumento na glutationa, em comparação com condições controlo. O stress combinado induziu também a produção do cinamato, uma resposta nova. Estes resultados realçam que a combinação de seca e calor fornece uma proteção significante contra os efeitos mais prejudiciais da seca isolada em eucalipto, confirmando que o stress combinado altera o metabolismo das plantas de uma forma nova que não pode ser extrapolada pela soma dos diferentes stresses aplicados individualmente. Esta tese descreve um conjunto de respostas biológicas que permitem ao eucalipto manter-se em condições de défice hídrico e revela informação útil de várias vias metabólicas a serem exploradas de modo a encontrar marcadores de tolerância ao stress abiótico apropriados. Apesar disso, um desafio maior permanece. Consiste na necessidade de focarmos os nossos estudos em experiências mais realistas, que mimetizem as condições de campo, pelo menos no contexto de encontrarmos marcadores de seleção ajustados a uma era de alterações climáticas.

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39

Nehme, Ali. "Ciblage Tissu-Spécifique des Cascades Enzymatiques de l’Angiotensinogène dans l’Athérome Humain." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10267/document.

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L'Athérosclérose est la principale cause de décès et d'invalidité dans le monde. L'implication du système rénine-angiotensine-aldostérone (RAAS) dans le développement de la maladie est expérimentalement et cliniquement bien documentée. Toutefois, en raison de la complexité du système, ces études ne donnent pas de vision claire sur l'association entre le système et la maladie. À cet égard, nous avons étudié l'organisation fonctionnelle d'un ensemble de 37 gènes codant pour les composants classiques et nouvellement découverts du RAAS, y compris les substrats, les enzymes et les récepteurs. Cet ensemble a été appelé RAAS étendu (extRAAS). En utilisant une analyse statistique des données du transcriptome de l'athérome carotidien humain, nous avons révélé des caractéristiques spéciales de l'expression de l'extRAAS associées au remodelage athéromateux. Une caractéristique importante de ce modèle est la coordination de 2 groupes de gènes qui sont connus pour favoriser la formation de l'athérome. Le premier groupe est constitué de gènes codant pour les peptidases de l'angiotensine, y compris ACE, CTSG, CTSD et RNPEP. Le deuxième groupe est constitué des gènes codant pour les récepteurs AGTR1, MR, GR et LNPEP
Atherosclerosis remains and continues to be the leading cause of death and disability in the world. The implication of Renin-angiotensin-aldosterone system (RAAS) in the development of the disease is well experimentally and clinically documented. However, due to the complexity of the system, these studies remain dispersed and give no clear global view of the association between the system and the disease. In this regard, we studied the functional organization of a set of 37 genes encoding classical and newly discovered RAAS participants, including substrate, enzymes and receptors. This set was called extended RAAS (extRAAS). Using statistical analysis of human carotid atheroma transcriptome involving gene clustering, we revealed special features of extRAAS expression associated with atheromatous remodeling. An important feature of this pattern was the coordination of 2 clusters of genes that are known to favor atheroma formation. The first cluster constitutes genes that encode for angiotensin peptidases, including ACE, CTSG, CTSD and RNPEP. Whereas the second encode for receptors (AGTR1, MR, GR and LNPEP). We hypothesized that the local pattern of extRAAS gene expression plays a key role in the development of atherosclerosis by orienting the metabolism of active peptides

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40

Ebah, Leonard. "Extraction and analysis of interstitial fluid, and characterisation of the interstitial compartment in kidney disease." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/extraction-and-analysis-of-interstitial-fluid-and-characterisation-of-the-interstitial-compartment-in-kidney-disease(8e9ce4a2-8ec4-41ee-ac22-bc142ca9e0b0).html.

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Kidney failure results in fluid and toxin accumulation within body fluid compartments, contributing to the excess mortality seen in this condition. Such uremic toxins have been measured in plasma, with levels assumed to reflect extraplasmatic concentrations such as in interstitial fluid (ISF). ISF is separated from plasma by nanometre-order microvascular pores; toxins may not circulate “freely” between the two compartments. This work set out to characterise the ISF in uremic subjects, with the hypothesis that there may be differences with plasma. Any such difference may be clinically relevant, owing to the much larger size of the ISF compartment, its proximity to cell metabolic processes, and its expansion in renal impairment.We used a modified microdialysis technique to successfully sample subcorneal ISF of some the uremic toxins (urea, creatinine, urate, phosphate). Reverse iontophoresis (RI) was also used as a non-invasive technique to sample epidermal ISF of urea. Hollow microneedles were developed and their ability to extract ISF tested in CKD patients and controls. The mechanical properties (pressure, volume, permeability) and biochemical composition (proteomic and metabolomic profiles) of the interstitial compartment were also investigated.Microdialysis and RI performed very well as interstitial uremic toxin sampling techniques. Small differences were seen in steady states between ISF and plasma urea, creatinine, phosphate and urate, with slightly lower ISF levels. Dialysis seemed to enhance this difference, with a lag in the clearance of ISF toxins seen in some patients, most remarkable with phosphate. Metabolomic analysis identified several uremic toxins in ISF, whilst proteomics found some significant differences between the two compartments, with toxins like beta-2 microglobulin occurring in ISF only. Microneedle arrays successfully extracted ISF in 68.8% of patients with oedema. Successful extraction of ISF with microneedles occurred mainly in oedematous patients, who were found to have raised interstitial pressures (ISP) and volumes. ISP correlated significantly with body fluid volumes and seemed time-dependent, lower in more chronic oedema. ISP and volumes also correlated with the oedema depitting time (after thumb pressure), a potential novel parameter that probably relates to tissue hydraulic conductivity and hence volume status and fluid mobility within the interstitium.This study demonstrates that interstitial fluid may need to be considered as a separate active compartment in patients with renal dysfunction, with a different “uremic" composition and unique pathophysiological characteristics that cannot be explained by blood compartment based measurements alone. There is a need for more studies, to further characterise this compartment and elucidate its importance.

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41

Güell, Cargol Marc. "Big complexity in a minimal bacterium." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7235.

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With only 689 genes Mycoplasma pneumoniae (M. pneumoniae) is among the simplest known organisms. Because of this simplicity, mycoplasma represents an attractive organism for systems-wide analyses. Such approaches aiming at the whole quantitative understanding of an entire organism are expected to illustrate the basic principles of life. Strand-specific tiling arrays complemented by transcriptome sequencing, were combined with more than 252 spotted arrays to study M. pneumoniae transcriptional organization. An important presence of alternative transcripts (42%) within operons and a high frequency of antisense RNA (89) were detected. Metabolism was also studied in detail. A manually curated metabolic network allowed the definition of a minimal medium with 19 essential nutrients. This has been complemented with measurements of biomass indicators, metabolites and fluxes. Integration with transcriptional profiling has provided keys in the metabolic regulation. Protein organization and interactions have been addressed systematically by Tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The biochemical analysis revealed 178 protein complexes which have been complemented by structural models, single-article electron microscopy and electron tomography. By integrating the datasets from these different approaches, we show that this small bacterium harbors an unexpected complexity with features such as the frequent occurrence of alternative transcripts and antisense RNA, a small but tightly controlled metabolic network and a high level of proteome organization.
Amb només 689 gens Mycoplasma pneumoniae es troba entre els organismes més simples que es coneixen. Degut a aquesta simplicitat, mycoplasma representa un organisme atractiu per dur a terme estudis a nivell genòmic. S'espera d'aquests treballs que pretenen descriure de manera quantitativa l'organisme sencer que ajudin a entendre els principis bàsics de la vida. Per tal l'estudiar amb profunditat del transcriptoma, s'ha fet ús d'una combinació de dades de "tiling arrays" amb especificitat de cadena, ultraseqüenciació i més de 252 microarrays. Després d'analitzar els resultats s'ha detectat una alta presència de transcrits alternatius (42%) dintre operons i una alt contingut de ARN de tipus "antisense" (89). També s'ha realitzat un estudi detallat del metabolisme. S'ha revisat i completat manualment el mapa metabòlic de M. pneumoniae, fet que ha permès el disseny d'un medi mínim amb l'ús de 19 ingredients essencials. El mapa s'ha completat amb diferents mesures d'indicadors de biomassa, metabòlits i fluxos. També s'ha estudiat la regulació de metabolisme mitjançant microarrays. Per altra banda, s'han mesurat sistemàticament les interaccions proteïna-proteïna mitjançant "Tandem affinity purification-mass spectrometry (TAP-MS)". Aquest anàlisis ha detectat 178 complexes diferents, els quals han estat complementats amb models estructurals, microscòpia electrònica i tomografia electrònica. Mitjançant la integració d'aquestes col·leccions de dades, es pot mostrar que aquest petit bacteri amaga un inesperada complexitat amb característiques com la freqüència de transcrits alternatius i ARN "antisense", una xarxa metabòlica petita però fortament controlada i una alta organització del proteoma.

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42

Lamouche, Florian. "Analyse comparative des mécanismes de différenciation des bactéroïdes au cours des symbioses Bradyrhizobium Aeschynomene." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS036/document.

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En cas de carence azotée, les légumineuses sont capables de mettre en place une symbiose avec des bactéries du sol fixatrices d’azote appelées rhizobia. Cette symbiose a lieu dans un organe appelé nodosité où les bactéries sont endocytées et appelées bactéroïdes. Certains clades de légumineuses imposent un processus de différenciation à leurs bactéroïdes qui agrandissent considérablement et deviennent polyploïdes, menant à des morphotypes bactériens allongés ou sphériques. Au cours de cette thèse, j’ai étudié la différenciation des bactéroïdes de Bradyrhizobium spp. en association avec Aeschynomene spp.. Les bactéroïdes de ces plantes présentent des degrés de différenciation distincts qui dépendent de l’espèce hôte. Mes données suggèrent que les bactéroïdes les plus différenciés sont aussi les plus efficaces. J’ai cherché à savoir quels facteurs procaryotes pourraient être impliqués dans les adaptations des bactéroïdes au processus de différenciation et à leurs divers hôtes, le tout en lien avec cette différence d’efficacité symbiotique au travers d’approches globales sans a priori de type -omiques. Les conditions considérées sont des bactéroïdes de différents morphotypes et des cultures libres de référence. Les fonctions activées en conditions symbiotiques ont été identifiées, ainsi que les gènes spécifiques d’un hôte donné. Des analyses fonctionnelles des gènes d’intérêt ont également été menées. Les mutants bactériens n’ont toutefois pas présenté de phénotype symbiotique drastique, montrant ainsi l’existence de réseaux de gènes complexes menant à la résilience des génomes de rhizobia
In case of nitrogen starvation, legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. This interaction takes place in nodules where the symbionts are internalized and become bacteroids. Some legume clades also impose a differentiation process onto the bacteroids which become enlarged and polyploid, leading to elongated or spherical morphotypes. During my PhD work, I have studied bacteroid differentiation of Bradyrhizobium species in association with Aeschynomene spp.. These bacteroids display distinct differentiation levels depending on the plant host, and my analyses suggest that the most differentiated ones are also the most efficient. I investigated the bacterial factors potentially involved in the adaptations to differentiation and host-specificity, and related to the higher efficiency of the most differentiated bacteroids using global-omics approaches without a priori. The analyzed conditions were bacteroids of distinct morphotypes and free-living reference cultures. Activated functions under symbiotic conditions were identified, as well as host-specific ones. Functional analyses were performed on genes of interest. However, the bacterial mutants did not display drastic symbiotic phenotypes, showing the existence of complex gene networks leading to high resilience of rhizobial genomes

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Moraes, Fabricio Edgar de. "Bioinformática aplicada à biologia sistêmica para a identificação dos fatores regulatórios do acúmulo de sacarose no colmo da cana-de-açúcar." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-01112016-112458/.

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A cana-de-açúcar (Saccharum spp.) é uma das principais gramíneas cultivadas do mundo e o Brasil é seu maior produtor, ela se tornou uma importante cultura devido às altas taxas de assimilação de carbono permitindo a síntese e acumulação de grandes quantidades de sacarose em seus entrenós. Com isso, faz-se necessário uma melhor compreensão dos mecanismos moleculares que regulam o acúmulo de sacarose nesta planta. Tais mecanismos têm sido estudados em vários níveis, tais como, identificação e localização de genes, identificação de lócus controladores de características quantitativas, transcriptoma, proteôma, caracterização e identificação de metabólitos. Com todos esses estudos é evidente a necessidade de uma abordagem holística para o entendimento global da planta durante o acúmulo de sacarose. Assim este trabalho teve por objetivo integrar dados de metabolômica e proteômica de tecidos da cana-de-açúcar da variedade SP80-3280 durante o desenvolvimento e o acúmulo de sacarose, utilizando a bioinformática para unir esses resultados por meio da análise de correlação canônica regularizada em uma abordagem de biologia sistêmica. Os resultados obtidos indicam diferenças no perfil metabólico e proteico da cana-açúcar durante o desenvolvimento e acúmulo de sacarose. Foram propostas classes de metabólitos que podem estar relacionados com o acúmulo de sacarose na cana-de-açúcar tais como glicerolipídeos, glicerofosfolipídeos, cumarinas e derivados, esteroides e derivados de esteroides e acil graxos. Também foram propostas proteínas que podem estar relacionadas com o acúmulo de sacarose, onde as histonas foram as que mais se destacaram. Nas redes biológicas de correlações também foram observadas correlações entre possíveis metabólitos e proteínas que podem estar correlacionadas com o acúmulo de sacarose na cana-de-açúcar
Sugarcane (Saccharum spp.) is one of the most important cultivated grasses of the world and Brazil is the largest producer, it has become an important crop due to high carbon assimilation rates allowing the synthesis and accumulation of large amounts of sucrose in their internodes. Thus, it is necessary a high understanding of the molecular mechanisms involved in the regulation of sucrose accumulation in this plant. These mechanisms have been studied at various levels, such as gene identification and localization, identification of quantitative trait locus controlling, transcriptome, proteome, characterization and metabolites identification. With all these studies is evident the necessity for a holistic approach to global understanding of the plant during the sucrose accumulation. Thus, this work aims to integrate metabolomics and proteomics data from tissues of sugarcane variety SP80-3280, during plant development and sucrose accumulation, using bioinformatics to link these results by regularized canonical correlation analysis in a systems biology approach. The results indicate differences in the metabolic and protein profile of sugarcane during development and sucrose accumulation. Metabolites classes have been proposed that may be related to sugarcane sucrose accumulation as glycerolipids, glycerophospholipids, coumarins and derivatives, steroids and steroid derivatives and fatty acyl. In addition, some proteins have been proposed that may be related to sucrose accumulation, where the most highlighted were the histones. In the biological correlations networks, have been also observed correlations between possible metabolites and proteins that can be correlated with the accumulation of sucrose in sugarcane

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Juárez, Ortega Paloma. "Production of recombinant Immunoglobulin A in plants for passive immunotherapy." Doctoral thesis, Universitat Politècnica de València, 2014. http://hdl.handle.net/10251/37015.

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Mucosal passive immunization is the transfer of active antibodies from one organism to the mucosal surfaces of another organism for preventing or treating infectious diseases. Mucosal passive immunization has a great potential for the prevention and treatment of enteric infections like Rotavirus, which causes more than 114 million episodes of diarrhoea annually with a death toll of more than 450.000 per year. However, the high cost of recombinant antibodies with the current manufacturing systems based on mammalian cells hampers the production of the high antibody quantities required for passive immunization strategies. Alternative expression platforms such as plants could provide higher scalability and reduced costs. Moreover, the use of edible plant organs, which are Generally¿Regarded¿As¿ Safe (GRAS), could reduce manufacturing costs even further by easing the requirements for antibody purification. We analyze here the feasibility of utilizing fruits as inexpensive biofactories of human antibodies that can be orally delivered as crude extracts or partially purified formulations in mucosal passive immunization strategies. In the first section of this thesis, the construction of tomato plants producing a model human Immunoglobulin A (IgA) against rotavirus in their fruits is described. As a result, an elite homozygous line was obtained whose fruits produced on average 41 ¿g of IgA per gram of fresh weigh, equivalent to 0.69 mg IgA per gram of dry tomato powder. Minimally processed products derived from IgA¿expressing tomatoes were shown to strongly inhibit virus infection in an in vitro neutralization assay. Moreover, in order to make IgA¿expressing tomatoes easily distinguishable from wild¿type tomatoes, they were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. The resulting transgenically¿labelled purple tomatoes contained not only high levels of recombinant neutralizing human IgA but also increased amounts of anthocyanins. In the second section of the thesis the composition of IgA¿expressing tomatoes was analyzed in search of possible unintended effects that could compromise the GRAS status of the final product. To this end, transgenic IgA¿tomatoes were compared with wild type tomatoes and also commercial tomato varieties using proteomic and metabolomic approaches. 2D¿DIGE gels coupled with LC¿MSMS for protein identification showed that all the uptrend differential proteins detected corresponded only to immunoglobulin chains or antibody fragments. On the other hand, non¿targeted metabolite data obtained by UPLC¿MS
Juárez Ortega, P. (2014). Production of recombinant Immunoglobulin A in plants for passive immunotherapy [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37015
TESIS

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Ros, i. Simó Clara 1984. "Drug consumption and stressful experiences in adolescent mice: behavioural, neorotoxic and neurochemical responses." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/111169.

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Adolescence is a critical developmental period in which the brain emerges from an immature state to adulthood. This process of brain development is associated to greater cognitive capacity but also to altered emotional behaviour, such as anxiety and depressive symptoms; as well as increased sensation-seeking and risk taking behaviour. The proper development of brain and behaviour into adulthood can be negatively affected by external factors such as drug abuse and environmental conditions. This work consists firstly on, studying the impact of binge ethanol, 3, 4-Methylenedioxymethamphetamine (MDMA) and its combination in adolescent mice. Secondly, study the consequences of early-life stressful experiences (social isolation) into adulthood. Main results obtained from the first objective are that the combination of binge ethanol and MDMA induces emotional-like alterations. These alterations can be prevented by antidepressant treatment. In addition, MDMA induces memory impairments that may be associated to oxidative damage to specific proteins in the hippocampus. Neuroinflammation is also present after MDMA treatment, but not after binge ethanol treatment, in mice striatum. Metabolomic studies indicate that brain metabolism is altered after binge ethanol, MDMA or its combination. Even though these are only preliminary results, these alterations might be due to an imbalance in tryptophan metabolism. Regarding the second objective, our findings indicate that social isolation during adolescence induces an altered response to novel and stressful situations. These alterations are probably due to altered HPA axis activity.
L'adolescència és un període crític en el desenvolupament de l’individu en el qual el cervell va d’un estat immadur a l’edat adulta. Aquest procés va acompanyat d’una elevada capacitat cognitiva però també de freqüents alteracions de tipus emocional, com l’ansietat o els símptomes depressius, així com la cerca de sensacions de risc. Un bon desenvolupament del cervell i del comportament es pot veure negativament afectat per factors externs com són l’abús de drogues i les condicions ambientals desfavorables. Aquest projecte consisteix en primer lloc, a estudiar l'impacte de l’alcohol en excés, la 3, 4-Metilendioximetamfetamina (MDMA) i la seva combinació en ratolins adolescents. En segon lloc, estudiar les conseqüències en l’edat adulta d’experiències estressants durant l’adolescència. Els principals resultats obtinguts referents al primer objectiu són que la combinació d'alcohol en excés i MDMA provoca alteracions de tipus emocional. Aquestes alteracions poden ser previngudes pel tractament amb antidepressius. A més, la MDMA indueix un deteriorament de la memòria que pot estar associada amb el dany oxidatiu a proteïnes específiques de l'hipocamp. També s’ha observat una resposta neuroinflamatòria en el cos estriat dels ratolins després del tractament amb MDMA, però no després del tractament amb etanol en excés. Finalment, estudis de metabolòmica indiquen que el metabolisme cerebral es veu alterat després de l’alcohol en excés, la MDMA o la seva combinació. Tot i que només són resultats preliminars, aquestes alteracions poden ser conseqüència d'un desequilibri en el metabolisme del triptòfan. Referent al segon objectiu, els nostres resultats indiquen que l'aïllament social durant l’adolescència indueix una resposta alterada a situacions novelles i estressants. Aquestes respostes anormals són probablement conseqüència d’alteracions en l’activitat de l’eix HPA.

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Zang, Tuo. "Quantitative characterization of paediatric burn blister fluid." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/122968/1/Tuo_Zang_Thesis.pdf.

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Burn injury is a highly traumatic event for any infant or child. The degree of burn severity often determine the treatment operations and the extent of later scar formation, which may require long-term surgical remediation or skin grafting. This investigation quantitatively characterises the biochemical composition of burn blister fluid from paediatric patients using advanced analytical techniques. The correlation of the abundance of proteins and metabolic molecules were explored by statistical and bioinformatics methods. Thus, this study is able to provide a timely and objective measurement that may reflect the burn wound microenvironment and assist clinical diagnosis.

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Morin-Sardin, Stéphanie. "Etudes physiologiques et moléculaires de l'adaptation des Mucor aux matrices fromagères." Thesis, Brest, 2016. http://www.theses.fr/2016BRES0065/document.

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Dans le contexte fromager, les champignons filamenteux du genre Mucor ont un rôle ambivalent. En fonction du fromage considéré, ils peuvent être assimilés à des microorganismes d’altération responsables de défauts de fabrication ou au contraire contribuer au développement des qualités organoleptiques des produits. Dans le cadre de ce travail, nous avons souhaité confirmer et objectiver la dichotomie classiquement faite en industrie fromagère entre espèces technologiques et espèces contaminantes, et investiguer les mécanismes d’adaptation potentiels mis en oeuvre chez les espèces technologiques. La morphologie et la croissance radiale de 7 souches représentatives d’espèces technologiques, contaminantes et non-fromagères (endophyte) de Mucor ont été étudiées sur différents milieux (synthétique, mimant le fromage et fromager) en fonction de facteurs clés du processus de production des fromages (température, aw, pH). Les valeurs cardinales de croissance ont été déterminées sur milieu synthétique, un modèle prédictif a été proposé et validé sur matrices fromagères pour le facteur température et la meilleure faculté de croissance des souches technologiques sur milieux fromagers par rapport au milieu synthétique a été démontrée. Une approche de protéomique comparée a permis de décrire les voies métaboliques mises en jeu par 4 de ces souches dans les deux types d’environnement, fromager et non-fromager, et 35 protéines spécifiquement surexprimées par la souche technologique M. lanceolatus UBOCC-A-109153 sur milieu mimant le fromage ont été identifiées. Les avantages compétitifs associés à ces potentiels marqueurs d’adaptation vont faire l’objet d’investigations complémentaires
In the cheese industry context, Mucor species exhibit an ambivalent behavior, as some species are essential technological organisms contributing to the required organoleptic characteristics of some cheeses while some others can be spoiling agents. The present study aimed at better understanding this ambivalence and investigating the putative adaptation mechanisms to cheese existing in Mucor technological species. Morphology and radial growth of 7 representative Mucor species: technological, contaminant and non-cheese related (plant endophyte) species were monitored on different media (synthetic, cheese-mimicking media and cheese) in function of key parameters for cheese manufacture (temperature, aw, pH). Cardinal values were determined on synthetic medium and as a result a predictive model was proposed and validated on cheese matrices for the temperature parameter. Interestingly, cheese technological species exhibited higher optimal growth rates on cheese related matrices than on synthetic media, while the opposite was observed for non-technological species. A comparative proteomic approach allowed unraveling the main metabolic pathways playing a role in growth of 4 of the 7 studied strains on both synthetic medium and cheese-mimicking medium. This proteomic study also highlighted the occurrence of 35 proteins specifically expressed by the technological strains M. lanceolatus UBOCC-A-109153 on the cheese-mimicking medium. Putative competitive and adaptative advantages of these hypothetical adaptation markers will be tested through additional investigations

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Boutant, Marc. "Etude comparative du métabolisme des lipides chez Streptomyces coelicolor en culture avec le glucose ou le glycérol comme source de carbone." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS507.

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Les Streptomyces sont des bactéries filamenteuses à Gram positif que l’on retrouve dans les couches superficielles du sol. La souche modèle S. coelicolor M145 est capable de produire des antibiotiques et accumuler de faibles quantités de triacylglycérol lorsqu’elle est mise en culture avec le glucose ou le glycérol comme source de carbone. La souche produit de l’actinorhodine seulement en culture avec le glycérol comme source de carbone, et nous assistons à une accumulation d’acides gras, notamment sous forme d’esters d’acide oléique avec l’une ou l’autre source de carbone. Ces accumulations d’acides gras ne durent que pendant une certaine période de temps, à l’issu de laquelle l’accumulation s’arrête, et on assiste à la production de métabolites secondaires qui peut s’accompagner de la consommation des acides gras précédemment accumulés. Ces observations suggèrent d’une part que la source de carbone est un effecteur différentiel dans la production de métabolites secondaire, et que d’autre part le métabolisme des lipides est lié au métabolisme secondaire. Ce travail de thèse va tenter d’établir les liens entre le métabolisme primaire et le métabolisme secondaire chez S.coelicolor via le métabolisme des lipides. Pour se faire, nous avons dans un premier temps défini un milieu synthètique, ainsi que des stratégies d’alimentation de type fed-batch en bioréacteur, pour permettre d’obtenir d’une part une phase de croissance exponentielle, et d’autre part une phase de croissance non exponentielle et imposée par une limitation en azote. Cette limitation nutritionnelle est classiquement utilisée à la fois lors des études de l’accumulation de lipides chez les microorganismes oléagineux tels que Yarrowia lipilytica ou Rhodotorula glutinis, et aussi dans le cas d’études de la production d’antibiotiques par les Streptomyces. L’étude du métabolome couplée à l’étude du protéome nous a permis d’établir la direction générale des flux globaux de carbone, d’énergie et de pouvoir réducteur lors des différentes phases de croissances. Ces travaux démontrent une hétérogénéité métabolique dans la population bactérienne totale, avec la présence de 2 à 3 sous-populations différentes pouvant coexister. Les évolutions temporelles du protéome et notamment des régulateurs transcritptomiques montrent des réactions tardives, très certainement dues à la présence d’effecteurs intracellulaires accumulés, ainsi qu’au dynamisme de la culture
Streptomyces are filamentous Gram positive bacteria found in the soil upper layers. The model strain S. coelicolor M145 can produce antiobiotics and accumulate low levels of triacylglycerol when cultivated with glucose or glycerol as carbon source. This strain produces actinorhodin only when glycerol is used as carbone source, but the fatty acids accumulation as esterified oleic acid occurs with both carbon sources. However, the fatty acids accumulation only last for a short period of time, and afterward, a production of secondary metabolites is observed and the previously accumulated fatty acids are consummed. Those results suggest that the carbon source act as an effector for the production of secondary metabolites and the lipids metabolism is some ways linked to the secondary metabolism. With this work, we will try to establish the links between the primary and secondary metabolism via the lipids metabolism with S. coelicolor. First, in order to obtain cultures with an exponential growth phase and a non-exponential growth phase under a nitrogen limitation, a synthetic media and fed-batch feeding strategies have been designed. The nitrogen limitation is usually used to study lipids accumulation with oleaginous microorganisms such as Yarrowia lipilytica or Rhodotorula glutinis, and also during studies of antibiotics production with Streptomyces. The metabolomics study paired with the proteomics study made possible to establish the global directions of carbon, energy and reduced power fluxes during all the obtained growth phases. This work also showed that the microbial population is heterogeneous and 2 to 3 subpopulations can coexist with both carbon sources. The proteomics temporal changes and in particular of transciptional regulators show some late reactions to the nitrogen limitation especially when glycerol is used, probably because of the intracellular accumulation of effector compounds over time, and because of growth kinetics

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Pasquier, Grégory. "Etude de l’impact de l’Esca sur la qualité des raisins par une approche protéomique." Thesis, Bordeaux 2, 2012. http://www.theses.fr/2012BOR21909/document.

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La vigne est sensible à de nombreuses maladies cryptogamiques qui vont altérer la qualité des raisins et des vins. Depuis plusieurs années, l’Esca est devenue un fléau pour la viticulture moderne, car elle est responsable de la mort des pieds de vigne lorsqu’elle se présente sous forme apoplectique. Sa forme chronique est liée à la présence d’un cortège de champignons pathogènes dans le tronc et les bras, qui vont provoquer le plus souvent des symptômes foliaires. L’altération de la photosynthèse que cela provoque, va avoir des conséquences sur la maturation du fruit. Cependant, peu de travaux de recherche ont été menés pour caractériser précisément l’impact de cette maladie sur le métabolisme et la composition chimique des baies de raisin. Notre étude avait pour but d’apprécier, grâce à une approche protéomique, les conséquences de cette maladie sur la qualité des raisins. Nos travaux ont permis de mettre en évidence que l’expression de symptômes foliaires d’Esca provoque une modification d’abondance des protéines liées aux mécanismes de défense et de stress oxydatif de la pellicule du raisin. Il est également observé un changement de capacité antioxydante par modulation des teneurs en peroxyde d’hydrogène, en glutathion ou en pyridoxine. Les concentrations en molécules d’intérêt œnologique comme les acides aminés, les amines biogènes ou les tannins sont également modifiées en présence de symptômes foliaires d’Esca
The vine is susceptible to many fungal diseases that will affect the quality of grapes and wines. For several years, Esca has become the bane of modern viticulture because it is responsible for the death of the vines when presented in the form apoplectic. Its chronic form is associated with the presence of a procession of pathogenic fungi in the trunk and arms that will most often cause leaf symptoms. The alteration of photosynthesis that this causes will affect fruit ripening. However, little research has been conducted to characterize precisely the impact of this disease on the metabolism and chemical composition of grape berries. Our study aimed to assess, using a proteomic approach the disease's impact on the quality of grapes and get specific markers. Our work allowed us to demonstrate that the expression of foliar symptoms of Esca causes a change in abundance of proteins related to defense mechanisms and oxidative stress in the grape skin. It is also observed a change in antioxidant capacity by modulating levels of hydrogen peroxide, glutathione or pyridoxine. The concentrations of molecules of oenological interest such as amino acids, biogenic amines or tannins are also modified in the presence of foliar symptoms of Esca

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Mansor, Rozaihan. "Proteomic and metabolomic studies on milk during bovine mastitis." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3207/.

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The principal objectives of the study presented in this thesis were to study the changes of milk proteomes, peptidomes and metabolomes during the course of bovine mastitis in comparison with normal milk samples and to discover new bovine mastitis biomarkers using various modern and up-to-date methodologies such as proteomics, peptidomics and metabolomics. Bovine mastitis caused by bacterial infection of the mammary gland of dairy cows is often associated with loss of milk production due to a reduction in milk composition and quality which in turns, lead to negative economic impact on dairy industry. Two important acute phase proteins (APPs) which serve as valuable biomarkers in bovine mastitis were investigated in every chapter using developed and validated enzyme linked immunosorbent assay (ELISA) for bovine milk haptoglobin and commercially available ELISA for bovine milk serum amyloid A3 (M-SAA3). These APPs were quantified alongside somatic cell counts (SCC) and California Mastitis Test (CMT) to confirm the disease status of each animal used in this study. Proteomic methodologies were applied including 1D gel electrophoresis, 2D gel electrophoresis, MALDI-TOF analysis and difference gel electrophoresis to investigate the changes of milk proteome in both subclinical and clinical mastitic milk samples in comparison with healthy milk samples. However these investigations did not reveal novel biomarkers for mastitis. Next, peptidomic methodologies were used to study the changes in milk peptidome and to detect the presence of any significant disease biomarkers in the presence of bovine mastitis by using CE-MS and LC-MS/MS. A total of 31 and 14 polypeptides can be used to discriminate control from infected groups and E. coli from S. aureus infected groups respectively. Lastly, metabolomic methodology was applied with an intention to study the changes in milk metabolome and ultimately to detect the presence of novel biomarkers in bovine mastitis. Di- and tri-peptides were found higher in S. aureus than in E. coli infected groups and based on metabolic pathways, arachidonic, arginine and galactose metabolites were seen increased in mastitic milk samples in comparison to healthy milk samples. Overall, the findings detailed in this thesis indicate that the use of advanced proteomic and metabolomic methodologies could deliver on their promise of the discovery of potential significant bovine mastitis biomarkers. Further studies are needed for validation of these proposed biomarkers and it was hoped that better prevention and treatment methods for bovine mastitis can be achieved in the future.

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Dissertations / Theses on the topic 'Proteomics and metabolomics'

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