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Long, Cai. "Identification of essential metabolites in metabolite networks." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43554.
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Metabolite essentiality is an important topic in systems biology and as such there has been increased focus on their prediction in metabolic networks. Specifically, two related questions have become the focus of this field: how do we decrease the amount of gene knock-out workloads and is it possible to predict essential metabolites in different growth conditions? Two different approaches to these questions: interaction-based method and constraints-based method, are conducted in this study to gain in depth understanding of metabolite essentiality in complex metabolic networks.
In the interaction-based approach, the correlations between metabolite essentiality and the metabolite network topology are studied. With the idea of predicting essential metabolites, the topological properties of the metabolite network are studied for the Mycobacterium tuberculosis model. It is found that there is strong correlation between metabolite essentiality and the degree and the number of shortest paths through the metabolite. Welch’s two sample T-test is performed to help identify the statistical significance of the differences between groups of essential metabolites and non-essential metabolites.
In the constraint-based approach, essential metabolites are identified in-silico. Flux Balance Analysis (known as FBA), is implemented with the most advanced in-silico model of Chlamydomonas Reinhardtii, which contains light usage information in 3 different growth environments: autotrophic, mixotrophic, and heterotrophic. Essential metabolites are predicted by metabolite knock out analysis, which is to set the flux of a certain metabolite to zero, and categorized into 3 types through Flux Sum Analysis. The basal flux-sum for metabolites is found to follow a exponential distribution, it is also found that essential metabolites tend to have larger basal flux-sum.
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Bi, Honggang. "Urinary metabolites of anabolic steroids : identification and synthesis." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=70304.
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The metabolism of several anabolic 17$ alpha$-methyl steroids, namely formebolone, mestanolone, methandienone, methyltestosterone, oxandrolone, oxymetholone and stanozolol, were investigated. The identification of metabolites was based on GC/MS analysis of different derivatives, and chemical synthesis of corresponding reference steroids. Common metabolic reactions have been studied and structure-metabolism relationships were discussed. New biotransformation routes, such as oxidative pathways of 2-formyl and 2-hydroxymethylene steroids, have been unveiled. The mechanism of 17-epimerization of these 17$ beta$-hydroxy-17$ alpha$-methyl steroids in vivo has also been clarified.A simple and convenient method for the preparation of 17$ beta$-tertiary sulfate derivatives of these steroids has been developed. A stereoselective approach was used for the partial synthesis of reference steroids in order to confirm the stereochemical features of an acidic metabolite of oxymetholone.
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Gough, A. J. E. "The isolation and identification of metabolites of interest from heteroptera." Thesis, Bucks New University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371221.
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Williams, David Ellis. "Novel secondary metabolites from selected cold water marine invertebrates." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/29175.
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A study of the secondary metabolism of two nudibranchs and one soft coral has led to the isolation of eighteen new and two known secondary metabolites. The structures of all compounds were determined by a combination of the interpretation of spectral data, chemical degradations and interconversions, and single crystal x-ray diffraction analysis.
The British Columbian dorid nudibranch Diaulula sandiegensis yielded two new steroidal metabolites, diaulusterols A (41) and B (42). The 25-(3-hydroxybutanoate) residue of diaulusterol A (41) and the 2α,3α-diol array of both 41 and 42 are not commonly encountered in naturally occurring steroids. Both metabolites exhibited considerable antibacterial and antifungal activity. Steroid 41 exhibited fish antifeedant activity. The relative concentration of 4.1 and 42 in the skin extracts of D. sandiegensis appears to be related to the animals' seasonal abundance.
Extracts of the British Columbian soft coral Gersemia rubiformis yielded a series of ten diterpenes possessing cembrane (170-175), pseudopterane (167-169) and gersolane (176) carbon skeletons. The structure of an eleventh diterpene remains unresolved. In addition, the structure of a degraded diterpene possessing a 13-membered ring (177) is tentatively proposed. G. rubiformis represents the first example of a soft coral to yield pseudopterane diterpenes. The organism is the first to contain cembrane, pseudopterane and gersolane metabolites, a fact which has biogenetic implications. Two new sesquiterpenes were also isolated. Tochuinyl acetate (165) and dihydrotochuinyl acetate (166) represent the first examples of cuparane sesquiterpenes to be isolated from a soft coral. A biogenesis is proposed. Metabolite 166 exhibited fish antifeedant activity.
Investigations of Gersemia rubiformis collected in Newfoundland waters revealed that the secondary metabolism differed from west coast specimens. The isolation of the new unstable sesquiterpene (+)-β-cubebene-3-acetate (178) resulted.
Skin extracts of the dendronotoid nudibranch Toquina tetraquetra were examined in an attempt to correlate its feeding dependency and lack of predation to the presence of allomones. Metabolites 165, 166, 170, 179 and the new butanoate diterpene 180 could be traced to the coelenterates which make up the animal's diet. Tochuinyl acetate (165), dihydrotochuinyl acetate (166) and rubifolide (170) were previously found in extracts of Gersemia rubiformis. Ptilosarcenone (179) has been reported as one of the major metabolites of the sea pen Ptilosarcus
gurneyi²¹³. The exact origin of a sixth metabolite, pukalide (63), remains unknown. It is proposed that Tochuina tetraquetra selectively sequesters dietary metabolites for defensive purposes.Science, Faculty of
Chemistry, Department of
Graduate
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Rashash, Diana M. C. "Identification and characterization of odorous metabolites produced by selected freshwater algae." Diss., This resource online, 1994. http://scholar.lib.vt.edu/theses/available/etd-06062008-164628/.
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Lundahl, Anna. "In vivo Pharmacokinetic Interactions of Finasteride and Identification of Novel Metabolites." Doctoral thesis, Uppsala universitet, Institutionen för farmaci, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-129362.
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The general aim of this thesis was to improve the understanding of the in vivo pharmacokinetics and, in particular, the metabolism of finasteride, a 5α-reductase inhibitor used in the treatment of enlarged prostate glands and male pattern baldness. CYP3A4 has been identified as the major enzyme involved in the sequential metabolism of finasteride to ω-OH finasteride (M1) and ω-COOH finasteride (M3). The consequences of induced and inhibited metabolism on the pharmacokinetics of finasteride and its metabolites were investigated in humans and pigs. Both studies included bile collection. The collected human and pig samples were used for the metabolite identification. As expected, induced metabolism led to reduced plasma exposure of finasteride and inhibited metabolism had the opposite effect. The interactions were investigated in detail and included examination of the biliary pharmacokinetics of finasteride and its metabolites. In pigs, the study included monitoring of the hepatic extraction over time, deconvolution and the development of a semi-physiological model for comparison of the effects on the gut wall and liver metabolism. For M3, the concentration ratios of bile to plasma and the renal clearance indicated that carrier-mediated processes are involved in the biliary and urinary excretion. This was not, however, the case for finasteride. The metabolite, M1, could not be quantified either in humans or pigs. Instead, two other OH metabolites, M1 isomers, were identified in humans. These metabolites were found to undergo glucuronide conjugation. In humans, one glucuronide was identified intact and in pigs, both glucuronides were identified intact in bile and in urine. In addition, a glucuronide of M3 was identified in human bile. In conclusion, advances have been made in the understanding of the pharmacokinetics of finasteride, in particular in relation to the metabolism. Hopefully, the findings of this comprehensive investigation can be applied to other drugs and novel chemical entities.
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Martin, Scott. "Identification and mechanistic study of novel drug metabolites by LC-MS." Thesis, Sheffield Hallam University, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713505.
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Understanding the metabolic fate of drug candidates in vitro and in vivo is a key component of drug development. Structural characterisation of drug candidate metabolites is important early in drug discovery to identify unwanted metabolic liabilities such as reactive, active, toxic or human specific metabolites. Reactive metabolite (RM) liability is a major concern for pharmaceutical companies during drug design, with most of the industry running primary RM trapping screens. This thesis investigates Fenclozic acid, homopiperazine reactivity, homomorphyline reactive aldehyde and methanol adduct formation, where RMs were not detected through routine glutathione/cyanide trapping assays. Fenclozic acid was withdrawn from clinical development due to hepatotoxicity; the mechanism of hepatotoxicity was never determined. In vitro covalent binding studies indicated phase I bioactivation in human liver microsomes, however no RMs were identified from in vitro experiments. As a part of this PhD thesis the metabolism of Fenclozic acid was investigated in bile duct cannulated rats using modern analytical techniques. Several new metabolites including glutathione related adducts formed through an epoxide RM were identified. A series of homopiperazine compounds were found to react with endogenous formaldehyde during rat in vivo studies. This thesis describes a detailed investigation into the identification and mechanism of formation of the resulting product, a bridged homopiperazine formed through a reactive Schiff base intermediate. A cysteineglycine conjugate observed for a series of homomorpholine compounds trapped by glutathione in human liver microsomes has been investigated in this thesis. NMR, detailed MS and methoxyamine trapping confirmed formation of a thiazolidine glycine product via a reactive aldehyde and subsequent glutathione rearrangement. A compound series in an early drug discovery programme formed unusual methanol adducts post incubation in human liver microsomes. The work undertaken in this thesis revealed the generation of a reactive aldehyde metabolite that did not form an adduct with glutathione, but reacted with the methanol mobile phase to form a pair of hemiacetal diastereoisomers. These examples would not have been detected using routine glutathione RM screening assays, this thesis highlights limitations of a screening approach, and where detailed metabolite identification studies employing modern LC-MS techniques are critical in understanding RM formation.
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Jones, Donald J. L. "The identification and characterisation of quercetin metabolites in humans and rats." Thesis, University of Leicester, 2001. http://hdl.handle.net/2381/30754.
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Quercetin is a dietary flavonoid, known to possess a range of biological properties of in in vitro systems. These properties may be of potential therapeutic benefit against certain diseases. Considerable work has focused on its antioxidant capability, which has been shown to be more powerful than that of vitamin E. Quercetin also has antineoplastic activity, and at 10M it inhibits the proliferation of malignant cell lines derived from breast, ovarian and gastrointestinal tumours and leukaemias. Therefore, a phase I clinical trial on cancer patients was conducted at the Queen Elizabeth Hospital in Birmingham, UK, in which quercetin was administered intravenously. One patient received quercetin p.o. In parallel to this clinical trial, blood and urine, derived from rats, were analysed in order to study the metabolism of quercetin, and the elucidate its bioavailability in vivo. The results show that metabolism by F344 rats, which received quercetin i.v., reflected adequately the metabolism seen in humans. Furthermore, analysis of both human and rat samples demonstrated that quercetin is rapidly metabolised via phase II biotransformation pathways to methyl, sulphate and glucuronide conjugates, thus eliminating quercetin efficiently from the body. The study shows that quercetin undergoes extensive first pass hepatic metabolism which in addition to the poor adsorption, means that quercetin has low bioavailability. Consequently, quercetin might be of limited therapeutic value, especially when taken orally. Whether or not the constant intake of quercetin via the diet may be beneficial to buttress the antioxidant defence system within the body remains to be elucidated. The existence of GSH-conjugates of quercetin was investigated as a possible explanation for the nephrotoxicity exhibited in rats and the clinical trial. GSH-conjugates of quercetin were not found in vivo, although they could be synthesised in vitro. Quercetin and isorhamnetin, although not quercetin sulphate nor glycosidic quercetin, exhibited potent COX-2 inhibition as demonstrated by reduced levels of PGE2 in HCA-7 cells.
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Holman, Stephen William. "Rapid and definitive identification of pharmaceutical drug metabolites using mass spectrometry." Thesis, University of Southampton, 2010. https://eprints.soton.ac.uk/72950/.
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Low-energy collision-induced dissociation-tandem mass spectrometry (CIDMS/ MS) is a well-established approach for identifying pharmaceutical drug metabolites. The technique fulfils many necessary requirements for this task, such as a low limit of detection, simple interfacing to chromatographic techniques, capability of fast analyses and automation, and high sensitivity, selectivity and accuracy. However, one of the main limitations of low-energy CID-MS/MS is that unambiguous assignment of the site of metabolism is often not possible, particularly for oxidised metabolites. Further, data interpretation can be time-consuming, thus producing a bottleneck to high-throughput analyses. The aim of the presented study was to identify structurally dependent dissociation pathways using low-energy CID-MS/MS that could facilitate rapid and definitive assignment of the sites of metabolism of new chemical entities. Chapter 4 details a specific loss of 50 m/z units in a model S-oxide that arises due to an ortho-effect. This loss could be used to definitively assign the site of oxidation and discriminate between multiple sulfur atoms in a parent compound. The 50 m/z unit loss was also shown to be a two-step process involving sequential radical losses; a rare observation for even-electron precursor ions under low-energy CID conditions. Chapter 5 discusses the experimental investigation of two unexpected rearrangements during the dissociation of a model S-oxide that could prevent correct assignment of the site of metabolism. Chapter 6 presents a rapid and definitive approach to the characterisation of dialkyl tertiary amine-N-oxides. The work also elucidated generic dissociation behaviour under low-energy CID conditions. Finally, chapter 7 considers an observation of site-specific intra-ionic hydrogen/deuterium exchange in the gas phase. Seven sets of compounds were analysed to investigate the substructures that facilitate the exchange. The work demonstrates a method by which a deuterium label can be inserted into the carbon skeleton of a small molecule without having to synthetically produce the compound, which could be useful in performing timely and cost-effective structural elucidation studies. In summary, the presented study provides two potentially useful approaches for the rapid and definitive identification of oxidised metabolites, as well as increasing the body of knowledge relating to ion-chemistry under low-energy CID conditions.
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Hoang, Tiffany Truc. "Speciation and identification of low molecular weight organoselenium metabolites in human urine." Diss., Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/30671.
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Aitken, Carolyn M. "Identification of non-hydrocarbon metabolites of deep subsurface anaerobic petroleum hydrocarbon biodegradation." Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437844.
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SETTE, CARLA BORGES. "PHENANTHRENE AND ALKYL HOMOLOGOUS METABOLITES FORMATION AND IDENTIFICATION IN CRAB UCIDES CORDATUS." PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2010. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=16977@1.
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CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOO principal objetivo deste estudo foi avaliar a formação de metabólitos de fenantrenos, parental e alquilados, em urina de caranguejos da espécie Ucides cordatus. Foram realizados dois bioensaios em laboratório com exposição dos organismos ao fenantreno (2 ug.kg-1 e 3 ug.kg(-1))e um bioensaio com exposição aos homólogos alquilados de fenantreno 1-metilfenantreno e 2,6,9-trimetilfenantreno (0,0125 ug.kg-1 e 0,025 ug.kg(-1), respectivamente). O tempo do experimento médio foi de 96 h e nos tempos 0, 24, 48, 72 e 96 h foram coletadas amostras (1) urina para avaliação da resposta biológica através da formação de metabólitos, (2) hemolinfa para realização do ensaio do micronúcleo para verificar a formação de mutações celulares e (3) hepatopâncreas para determinação de HPA totais, principalmente os compostos de interesse no presente trabalho. Foram desenvolvidas metodologias para medidas de metabólitos em urina de caranguejo utilizando como ferramentas a cromatografia líquida de alta eficiência com detector de fluorescência (CLAE/F) e cromatografia líquida de alta eficiência com detecção por espectrometria de massas (CL/EM) com APCI. Foi identificada a formação dos metabólitos hidroxifenantreno (OHF) e, outros metabólitos de fenantreno di e tri hidroxilados em amostras de urina injetadas com fenantreno e trimetilfenantreno. Ambas as metodologias apresentaram boa sensibilidade (LQ = 0,001 e 0,01 ug.mL(-1), respectivamente) e boa linearidade (r2 =0.9998 e 0,9997, respectivamente). Não foi observada presença de mutações nas células presentes nas lâminas de hemolinfa em nenhum dos grupos dos bioensaios. As análises de HPA realizadas nas amostras de hepatopâncreas mostraram aumento na concentração de fenantreno naqueles grupos nos quais foi injetado o composto fenantreno, e não foi observada alteração nas concentrações dos metilados de fenantreno naqueles grupos injetados com 1-metilfenantreno e 2,6,9-trimetilfenantreno. Os métodos se mostraram eficiente para uma avaliação preliminar da formação de metabólitos de fenantreno e 2,6,9-trimetilfenantreno. Porém as baixas concentrações de 1-metilfenantreno e 2,6,9-trimetilfenantreno as quais os caranguejos foram expostos, não permitiram uma melhor avaliação dos dados.
The main goal of this study was to evaluate the formation of phenanthrenes metabolites, parental and alkylated, in crabs Ucides cordatus urine. Laboratory experiments were carried out exposing organisms to phenanthrene (2 ug.kg-1 and 3 ug.kg-1), to the phenanthrene homologues alkylated, 1-methylphenanthrene and 2,6,9-trimethylphenanthrene (0,0125 ug.kg-1 and 0,025 ug.kg-1, respectively), to a Arabe oil and to a PAH contaminated sediment. The experiment maximum time was 96 h and at 0, 24, 48, 72 and 96 h were collected samples of: (1) urine to perform tests in order to evaluate the biological response about the formation of phenanthrene metabolites, (2) hemolymph to perform the micronucleus assay in order to verify cell mutations formation and (3) hepatopancreas to the determination of total PAH, especially this work’s interest compounds. Methodologies were developed to metabolites measurements in crab’s urine using kind of tools as the high performance liquid chromatography with fluorescence detection (HPLC/F) and high performance liquid chromatography with mass spectrometry detection (LC/MS). Were identified the formation of hydroxyphenanthrene (OHF) and other metabolites of phenanthrene, di- and tri-hydroxylated in urine samples injected with phenanthrene and trimethylphenanthrene. Both methods showed good sensitivity (LOQ = 0,001 and 0,01 ug.mL(-1), respectively) and good linearity (r2 = 0,9998 and 0,9997, respectively). There was no presence of mutated cells in the hemolymph slides in either group of bioassays. Analysis of PAH in hepatopancreas samples showed an increase in phenanthrene concentration in those groups of which the phenanthrene was the compound injected, and there was no concentrations change in methylphenanthrene on those groups injected with 1-methylphenanthrene and 2,6,9 - trimethylphenanthrene. The methods were showed to be effective in a preliminary assessment of phenanthrene and 2,6,9-trimethylphenanthrene metabolites formation. But the 1-methylphenanthrene and 2,6,9-trimethylphenanthrene low concentrations which the crabs were exposed, did not allow having a better evaluation of the data to this groups.
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Coras, Roxana Georgiana. "Identification of Metabolites as Biomarkers and Mediators of Inflammation in Inflammatory Arthritis." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/673867.
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L’artritis inflamatòria representa una gran càrrega social i econòmica tot i els recents avenços terapèutics. Malgrat un millor coneixement dels mecanismes patogénics, l’elecció de l’tractament encara es realitza a manera de prova, el que condueix a una manca de control de l’activitat de la malaltia en aproximadament el 30% dels pacients i una alta taxa d’efectes secundaris. La identificació de mediadors de malaltia i predictors de resposta a el tractament és necesaria per permetre el tractament adequat i aconseguir la remissió clínica o al menys una baixa activitat de la malaltia. És poc prob-able que un sol biomarcador proporcioni informació suficient per explicar aquestes malalties heterogènies. La metabolòmica és una eina que es pot utilitzar per al descobriment de biomarcadors, ja que pot identificar perfils d’una gran quantitat de metabòlits en diferents tipus de mostres. Els metabòlits no són només el resultat final dels processos químics que tenen lloc en la cèl·lula, sinó que també juguen un paper crític en una varietat de processos cel·lulars, com les modificacions postranslacionals i la regulació de les cèl·lules immunes. Amb la hipòtesi que els metabòlits circulants reflecteixen processos sinovials, aquest projecte va tenir com a objectiu estudiar els metabòlits circulants en relació amb l’activitat de la enfermedad y la resposta als fàrmacs antireumàtics modificadors de la malaltia. Descrivim diferents perfils metabolòmics en pacients amb artritis inflamatòria comparats amb controls. També identifiquem metabòlits que es correlacionen amb l’activitat de la malaltia i que poden ser mediadors de la malaltia, així com metabòlits associats amb la resposta a el tractament.La artritis inflamatoria representa una gran carga social y económica a pesar de los recientes avances terapéuticos. A pesar de un mejor conocimiento de los mecanismos patogénicos, la elección del tratamiento todavía se realiza a modo de prueba, lo que conduce a una falta de control de la actividad de la enfermedad en aproximadamente el 30 % de los pacientes y una alta tasa de efectos secundarios. La identificación de mediadores de enfermedad y predictores de respuesta al tratamiento es necesaria para permitir el tratamiento adecuado y lograr la remisión clínica o al menos una baja actividad de la enfermedad. Es poco probable que un solo biomarcador proporcione información suficiente para explicar estas enfermedades heterogéneas. La metabolómica es una herramienta que se puede utilizar para el descubrimiento de biomarcadores, ya que puede identificar perfiles de una gran cantidad de metabolitos en diferentes tipos de muestras. Los metabolitos no son solo el resultado final de los procesos químicos que ocurren en la célula, sino que también juegan un papel crítico en una variedad de procesos celulares, como las modificaciones postranslacionales y la regulación de las células inmunes. Con la hipótesis de que los metabolitos circulantes reflejan procesos sinoviales, este proyecto tuvo como objetivo estudiar los metabolitos circulantes en relación con la actividad de la enfermedad y la respuesta a los fármacos antirreumáticos modificadores de la enfermedad. Describimos diferentes perfiles metabolómicos en pacientes con artritis inflamatoria comparados con controles. También identificamos metabolitos que se correlacionan con la actividad de la enfermedad y que pueden ser mediadores de la enfermedad, asi como metabolitos asociados con la respuesta al tratamiento.
Inflammatory arthritis represents a great social and economic burden despite recent therapeutic advances. Although we have a better understanding of the pathogenic mechanisms, treatment election is still made on a trial basis, which leads to lack of control of disease activity in approximately 30% of patients and a high rate of side effects. The identification of disease mediators and predictors of response to treatment are needed to allow the adequate treatment and achieve clinical remission or at least low disease activity. A single biomarker is unlikely to provide sufficient information to explain these heterogeneous diseases. Metabolomics is a tool that can be used for biomarker discovery as it can identify profiles of a large number of metabolites in different types of samples. Metabolites are not just the end result of chemical processes that occur in the cell, but also play critical role in a variety of cellular processes, such as post-translational modifications and immune cell regulation. With the hypothesis that circulating metabolites reflect synovial processes, this project aimed to study circulating metabolites in relation to disease activity and response to disease modifying anti- rheumatic drugs. We described different metabolomic profiles in patients with inflammatory arthritis compared to controls. We also identified metabolites that correlate with disease activity and that may be mediators of disease, as well as metabolites that are associated with response to treatment.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina
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Hofvander, Lotta. "Polychlorinated biphenyls and their metabolites in human blood : Method development, identification and quantification." Doctoral thesis, Stockholm : Dept. of environmental chemistry, Stockholm university, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-805.
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Deo, Anand K. "Hepatic microsomal bile acid biotransformation : identification of metabolites and cytochrome p450 enzymes involved." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/15222.
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Bile acids are end-products of cholesterol metabolism and essential for absorption of
dietary lipids in the body. Impaired bile flow leads to hepatic bile acid accumulation and liver damage. Hepatic microsomal oxidation offers a potential mechanism for efficient elimination of bile acids. The present study investigated the cytochrome P450 (P450)-mediated hepatic microsomal biotransformation profiles of lithocholic acid, cholic acid and chenodeoxycholic
acid using a liquid chromatography-mass spectrometry (LCIMS) based assay.
Incubation of lithocholic acid with rat hepatic microsomes resulted in the formation of a major 6β-hydroxylated metabolite, murideoxycholic acid, followed by isolithocholic acid and 3-ketocholanoic acid. Ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid were
identified as minor metabolites. Studies using P450-specific antibodies, chemical inducers, and rat recombinant enzymes showed that formation of murideoxycholic acid and 3-ketocholanoic acid were mediated by CYP3A2 and CYP2C 11. Similar metabolite profiles were obtained by incubation of lithocholic acid with mouse hepatic microsomes generating murideoxycholic acid
as the major metabolite. Studies using P450 inducers and chemical inhibitors suggested the involvement of murine CYP3A in murideoxycholic acid and 3-ketocholanoic acid formation, and CYP1A, CYP2B and CYP3A enzymes in ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid formation by mouse liver microsomes. Biotransformation of lithocholic
acid by human hepatic microsomes generated 3-ketocholanoic acid as the major metabolite, and hyodeoxycholic acid, ursodeoxycholic acid, 6-ketolithocholic acid and murideoxycholic acid, as minor metabolites. Studies with chemical inhibitors and human recombinant enzymes
demonstrated that CYP3A4 catalyzed the formation of all five metabolites.
The biotransformation of cholic acid and chenodeoxycholic acid by human hepatic
microsomes revealed the formation of a single cholic acid metabolite, 3-dehydrocholic acid. Chenodeoxycholic acid biotransformation generated 7α-hydroxy-3 -oxo-5β-cholan-24-oic acid as the major metabolite followed by у-muricholic acid, 7-ketolithocholic acid and cholic acid,
respectively. CYP3A4 was found to be the major enzyme involved in the biotransformation of cholic acid and chenodeoxycholic acid in human liver microsomes. A comparison of metabolite profiles demonstrated the dominant role of human CYP3A4 in the oxidation of bile acids at the C-3 position. In contrast, 6β-hydroxylation catalyzed by multiple P450 (CYP1A, CYP2B, CYP2C and CYP3A) enzymes was the preferred biotransformation pathway in rodent liver microsomes.
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Vlachopoulos, Evangelos G. "Phenolic metabolites as a chemotaxonomic aid for the identification of cyst forming nematodes." Thesis, University of Newcastle Upon Tyne, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235749.
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Chen, Tzu-Yu. "Pilot study: identification of anthocyanin metabolites in the mice fed purple-fleshed sweetpotato." Thesis, Kansas State University, 2012. http://hdl.handle.net/2097/13764.
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Master of ScienceDepartment of Human Nutrition
Weiqun Wang
Anthocyanins may prevent chronic diseases such as cancer and cardiovascular disease, however, the anthocyanin metabolites are not well elucidated. We previously selected a purple-fleshed sweet potato clone P40 that contained anthocyanins at up to 7.5 g/kg dry matter, most of which are cyanidin and peonidin derivatives. The main objective of this study is to identify anthocyanin metabolites in the mice fed 20-30% of purple sweet potato P40 (287 mg and 430 mg peonidin-3-glucoside equivalent /kg body weight) diet for 6 weeks. Plasma, liver, and feces were analyzed for anthocyanin metabolites using HPLC/MS and MALDI-TOF-MS. Fifteen hours after consumption of P40 diet, we identified 4 anthocyanin metabolites cyanidin 3,5- diglucoside; cyanidin 3-sophoroside-5-glucoside; cyanidin3-p-hydroxybenzoylsophroside-5-glucoside; and peonidin 3-p-hydroxybenzoylsophroside-5-glucoside in fecal samples. No anthocyanin metabolites were detected in plasma or liver extracts by HPLC/MS or MALDI-TOF-MS. The results indicate that anthocyanin metabolites in fecal samples might provide health benefits for colonic mucosal cells. However, the lack metabolites in both plasma and liver samples suggest a continuous intake of the anthocyanins may be required for systemic benefits due to their quick degradation and low bioavailability.
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Cece, Esra Nurten 1984. "Metabolite identification in drug discovery : from data to information and from information to knowledge." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/403648.
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Drug metabolism studies provide the opportunity to enhance metabolic properties of new drugs. The overall aims of the drug metabolism studies are to (1.) optimize pharmacokinetic properties of the drug candidates, (2.) characterize the polymorphic enzyme contribution to clearance, and (3.) support the selection of safe drugs with respect to bioactivation potentials.
The ultimate goal in drug metabolism assays in early drug discovery is to translate analytical data to build final knowledge. By contribution of this translation, metabolism scientists can rationalize how structures of new drug compounds could be changed and how metabolic pathways could be better understood. Analytical techniques, such as High Resolution Mass Spectrometry (HRMS), have progressed and now it is possible to generate large datasets through High Throughput Screening (HTS) assays in drug metabolism laboratories. However, the transformation of these data into information and information into knowledge is insufficient. In-depth data inspection is necessary to support the generation of high quality results which are consistent across experiments. For this purpose, innovative software solutions can be utilized to process analytical data. In this respect, by applying standardized and fully automated data evaluation tools, it is possible to (1.) enable comprehensive data analysis, (2.) accelerate structure-based information handling, (3.) eliminate human error and finally (4.) improve chemical features of lead molecules in terms of biotransformation properties.
This thesis research aimed to use a novel automated workflow within HRMS to identify the drug metabolites and their structures. Final results confirmed that this new workflow can be used to translate HRMS data into information, which is required for building useful and ultimate knowledge in drug metabolism.Los estudios de metabolismo de fármacos ofrecen la oportunidad de mejorar las propiedades metabólicas de nuevos fármacos. Los objetivos generales de los estudios de metabolismo de fármacos son: (1.) optimizar las propiedades farmacocinéticas de los fármacos candidatos, (2.) caracterizar la contribución de las enzimas polimórficas a la elimicación y (3.) apoyar la selección de fármacos seguros con respecto a los potenciales bioactivación. El objetivo final en los ensayos de metabolismo de fármacos es traducir los datos analíticos para construir conocimiento final. Mediante la aportación de esta traducción, los científicos que trabajan en metabolismo pueden racionalizar cómo las estructuras de los nuevos compuestos de fármacos podrían ser cambiadas y cómo las vías metabólicas podrían ser mejor entendidaa. Las técnicas analíticas, como la espectrometría de masas de alta resolución (HRMS), han progresado y ahora es posible generar grandes volúmenes de datos a través de ensayos masivos (HTS) en laboratorios de metabolismo de fármacos. Sin embargo, la transformación de estos datos a información y la información a conocimiento es insuficiente. Es necesario un estudio en profundidad de los datos para ayudar a la generación de resultados de alta calidad que sean consistentes con los experimentos. Para este propósito, las soluciones innovadoras de software pueden ser utilizados con el objetivo de procesar los datos analíticos. A este respecto, mediante la aplicación de datos estandarizados y totalmente automatizados herramientas de evaluación, es posible (1.) permitir el análisis de datos completos, (2.) acelerar el manejo de información basada en la estructura, (3.) eliminar el error humano y finalmente (4.) mejorar las características químicas de las moléculas en términos de sus propiedades metabólicas. La investigación de esta tesis tuvo el objetivo que utilizar una novedoso y automatizado “sistema de trabajo” dentro HRMS para identificar los metabolitos de compuestos así como sus estructuras. Los resultados finales se confirmaron que se pueden utilizar herramientas de software para convertir los datos en información de HRMS, necesario para la construcción del conocimiento útil en el metabolismo de fármacos
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Roberts, Joanne B. "Determination and identification of drug and chemical metabolites in waste water by LCMS/MS." Thesis, Glasgow Caledonian University, 2017. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.726806.
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Wang, Cheng. "Structure Elucidation and Identification of Known and Unknown Metabolites by Nuclear Magnetic Resonance Spectroscopy." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574290782016797.
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Esberg, Boysen Marianne. "Molecular identification and quantification of the Penicillium roqueforti group /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1999. http://epsilon.slu.se/avh/1999/91-576-5469-7.pdf.
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Zheng, Ming. "Isolation, identification and synthesis of hydromorphone metabolites, analysis and antinociceptive activities in comparison to morphine." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0021/NQ46456.pdf.
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Gallant, Vicki Ann. "Liquid chromatography tandem mass spectrometry identification of apple polyphenol metabolites in human urine and plasma." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/14072/.
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Apples are a major dietary source of polyphenols in the Western diet and contain procyanidins, hydroxycinnamic acids, flavanols, dihydrochalcones and flavonols. Despite their abundance and familiarity very little research into their metabolism has been performed; research is required to elucidate the metabolic products of these polyphenols and characterise their absorption and excretion pathways. A human intervention study was designed specifically to investigate the absorption, metabolism, excretion and biokinetcs of apple polyphenols. Male volunteers (n = 9) consumed a supermarket apple juice substituted with water as the control phase, and the same apple juice substituted with a high polyphenol cider apple extract as the test phase. Blood samples were taken over 0-24 h and urine samples were collected at 0-4 h, 4-8 h and 8-24 h. A rapid, validated and novel single LC/ES/IMS/MS method was developed and validated for the analysis of a wide range of polyphenols and their metabolites in these urine and plasma samples (after sample preparation). Apple polyphenolrelated metabolites were identified using LC/MS/MS and MS2; nine urinary metabolites and seven plasma metabolites were identified, mostly for the first time after apple consumption. Data on the excretion, bioavailability and biokinetics of these metabolites, including products of the colonic micro flora, were obtained. In urine, the major apple-related polyphenolic metabolites identified were dihydroxyphenyl valerolactone sulfate and 5- (3', 4'- dihydroxyphenyl) -y- valerolactone glucuronide; both colonic bacterial metabolites which appear at their maximum concentrations 4-8 h post apple ingestion. Minor metabolites included (-) epicatechin sulfate and glucuronide conjugates. In plasma, 3, 4-dihydroxyphenylacetic acid, 5- (3',4'-dihydroxyphenyl) -y- valerolactone glucuronide and dihydroxyphenyl valero lactone sulfate predominate; Tmax values of 5-6 h were observed. Minor plasma metabolites included phloretin (Cmax291 ± 175 nM) and p-coumaric acid (Cmax 634 ± 225 nM). In conclusion, the project has identified apple-related polyphenol metabolites in human urine and plasma; many for the first time after apple consumption. Important biokinetic parameters have also been reported for these metabolites.
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Schwiebs, Anja [Verfasser]. "Identification, characterization and physiological significance of metabolites of the B-type natriuretic peptide / Anja Schwiebs." Gießen : Universitätsbibliothek, 2013. http://d-nb.info/1065064829/34.
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Rezende, de Castro Moretti Fernanda. "Identification of candidate resistance metabolites to Leifsonia xyli subsp. xyli in sugarcane through metabolomic profiling." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512478087354334.
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Moro, Sabrina [Verfasser], and Angela [Akademischer Betreuer] Mally. "Identification of target proteins of furan reactive metabolites in rat liver / Sabrina Moro. Betreuer: Angela Mally." Würzburg : Universitätsbibliothek der Universität Würzburg, 2011. http://d-nb.info/1013619838/34.
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Malmberg, Tina. "Identification and characterisation of hydroxylated PCB and PBDE metabolites in blood : congener specific synthesis and analysis /." Stockholm : Institutionen för miljökemi, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-245.
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Eloraby, Ghada. "Identification of protein targets of nevirapine reactive metabolites using click chemistry and mass spectrometry-based differential proteomics." Mémoire, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8995.
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Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials.
In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin.
The specific objectives derived from the general objective were as follow:
1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction.
2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity.
3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence.
4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response.
The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK.Résumé : Les effets indésirables (EI) sont les effets indésirables causés après l'administration d'une dose unique ou une administration prolongée du médicament ou le résultat de la combinaison de deux médicaments ou plus. La Réaction idiosyncratique (IDR) est une réaction indésirable qui ne se produit pas dans la plupart des patients traités avec un médicament et qui ne comporte pas l'effet thérapeutique du médicament. IDR sont imprévisibles et peuvent mettre la vie du malade en danger. Cette réaction dépend des caractéristiques chimiques du médicaments et/ou de la réponse immunitaire individuelle du patient. IDR est un problème majeur pour le développement de médicaments car ils ne sont généralement pas détectés au cours des essais cliniques. Dans cette étude, nous nous sommes concentrés sur la Réaction idiosyncratique de névirapine (NVP) qui est un inhibiteur de transcriptase inverse non nucléosidique utilisé pour le traitement du virus d'immunodéficience humaine (VIH). L'utilisation de NVP est limitée par une incidence relativement élevée d'éruption cutanée. NVP provoque également une éruption cutanée chez les rats femelles de souche Brown Norway. Notre étude vise à mieux comprendre les IDRs induites par l'administration de NVP chez l'animal. La présente étude vise à vérifier l'hypothèse que les problèmes cutanés associés à la prise de NVP soient attribuables à la modification post-traductionnelle de protéines détactable par spectrométrie de masse. Les principaux objectifs de ce projet étaient : 1) Déterminer si la Nevirapine alcynes (NVP-ALK), un analogue de la NVP peut développer la même éruption cutanée que la NVP. La NVP-ALK a été couplé avec de la biotine en utilisant la réaction chimique (click chemistry). 2) Détecter les modifications post-traductionelles des proteines par Western blot et des techniques de spectrométrie de masse, pour comprendre le mécanisme de la toxicité induite par la NVP. 3) Identifier les protéines modifiées en utilisant le moteur de recherche MASCOT pour l'identification des protéines, en comparant le les spectres de masse obtenus avec les spectres théoriques pour trouver une séquence correspondante de peptide. 4) Tester si la NVP et ses métabolites peuvent provoquer des effets nocifs, comme l'induction d'un stress oxydatif dans les cellules (par la mesure de la glutathionylation des protéines). Les résultats ont montré que la névirapine est métabolisé en métabolite réactif ce qui provoque une modification de la kératine. Ainsi nos résultats suggèrent que la kératine est la cible des métabolites de la NVP-ALK.
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Mazlan, Noor Wini Binti. "Identification of bioactive secondary metabolites from mangrove plant Avicennia lanata and its endophytic fungi by using metabolomics." Thesis, University of Strathclyde, 2015. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=26167.
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Wanjie, Sylvia W. "Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc4474/.
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The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.
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Takahashi, Haruya. "Studies on the identification and function of metabolites involved in peroxisome proliferator-activated receptor (PPAR) α activation." Kyoto University, 2014. http://hdl.handle.net/2433/188765.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第18327号
農博第2052号
新制||農||1022(附属図書館)
学位論文||H26||N4834(農学部図書室)
31185
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 河田 照雄, 教授 金本 龍平, 教授 入江 一浩
学位規則第4条第1項該当
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Schatz, Nina Jane [Verfasser], and Kulling S. [Akademischer Betreuer] E. "Ozonation of Chloridazon Metabolites: Identification of Oxidation Products and Reaction Pathways / Nina Jane Schatz. Betreuer: S. E. Kulling." Karlsruhe : KIT-Bibliothek, 2012. http://d-nb.info/1030315981/34.
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Welbeck, Edward William. "Separation, identification and standardisation of plant metabolites in traditional Chinese Medicines by chromatographic spectroscopic and multivariate analysis processess." Thesis, London Metropolitan University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517140.
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Pope, Simon Alexander Samuel. "The analysis and identification of urinary metabolites of vitamin E in man using mass spectrometry and chemical synthesis." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247078.
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Vitamin E (a-tocopherol) is the major lipid soluble antioxidant in vivo and is important for maintaining the integrity of cell membranes. Oxidative stress, defined as an imbalance between oxidants and antioxidants, has been implicated in the aetiology of numerous diseases. There is, therefore, interest in establishing methods to measure oxidative stress. It has been suggested that metabolites of vitamin E such as atocopheronolactone (a-TL), with an oxidised chroman ring, may be an indicator of in vivo oxidative stress and that the carboxyethyl-hydroxychromans (CEHCs), with a shortened phytyl side chain, may provide a measure of adequate or excess vitamin E status. However, doubts have been raised about the authenticity of a-TL since a-CEHC has been shown to be artefactually oxidised to a-TL in many of the procedures described. In the course of the current study a relatively simple method using gas chromatographymass spectrometry (GC-MS) was developed which allowed the reproducible measurement of a wide range of deconjugated vitamin E metabolites in urine. This method was used to study the urinary metabolites produced by normal subjects before and after supplementation with vitamin E. The CEHCs were confirmed as the major urinary metabolites of vitamin E, a-TL was detected and a novel group of metabolites, the carboxymethylbutyl-hydroxychromans (CMBHCs), was also tentatively identified. A range of conjugated (sulphated and glucuronidated) and free metabolites of vitamin E were synthesised chemically and used to a) confirm the identity of (x-CMBHC, b) provide standards for GC-MS and tandem mass spectrometry, c) elucidate the mechanism of artefactual oxidation and to develop new methods for the precise measurement of endogenously produced a-TL and d) investigate the type of conjugation of the various metabolites of vitamin E in human urine.
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Benatrehina, Paule Annecie. "Identification and Isolation of Secondary Metabolites from Podocarpus neriifolius Using Bioactivity-Guided and 1D-NMR-Based Dereplication Approaches." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu153193675651081.
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Tan, Choon Yong. "Identification and Dereplication of Bioactive Secondary metabolites of Penicillium aurantiacobrunneum, a Fungal Associate of the Lichen Niebla homalea." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586533114478772.
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Li, Zhenchi. "Applications of 16S rRNA metagenomics and metabolomics in correlation of toxicity of puffer fishes with gut microbiota and identification of potential precursors in tetrodotoxin biosynthesis." HKBU Institutional Repository, 2020. https://repository.hkbu.edu.hk/etd_oa/775.
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Tetrodotoxin (TTX) is a lethal neurotoxin isolated mainly from the organs of wild puffer fishes. Although the neurotoxicity mechanisms of TTX are well known, the TTX origin and the biosynthetic mechanisms inside its hosts remain unresolved. In recent decades, the numerous reports of TTX-producing bacteria strongly suggested its bacterial origin. However, this origin is currently being challenged by the low and inconsistent TTX productions in vitro by the previously reported TTX-producing bacteria. Culturable TTX-producing bacteria were frequently isolated and reported from the guts of TTX-bearing animals including puffer fishes, however, these bacteria were estimated to account for 0.1% of the total gut bacteria. Moreover, the identification and functions of the non-culturable gut bacteria participating in TTX biosynthesis have never been reported. I hypothesize that the puffer fish gut bacteria and the entire gut environment serve as a functional integrality responsible for TTX biosynthesis. In this study, 16S rRNA amplicon metagenomics pipeline was established to profile the entire gut bacterial structures of both toxic and non-toxic puffer fishes respectively. UniFrac based principal coordinate analysis showed that bacterial diversities were significantly different (P-value < 0.001) between the gut environments of toxic puffer fishes and the non-toxics. Vibrio and Cyanobacteria were identified as centralities of gut bacteria co-occurrence network in toxic puffer fishes, implying their key roles in TTX biosynthesis. The results of metagenome prediction and gene set enrichment indicated that arginine biosynthesis was significant enriched (P-value < 0.05) in the toxic group. To further investigate the roles of key bacteria and arginine biosynthesis in producing TTX, metabolomics pipeline was established along with 16S rRNA amplicon metagenomics to monitor the dynamics of metabolites and bacterial compositions in guts of toxic puffer fishes during their detoxification process. The average TTX concentrations in the liver after a 60-day culture (6.41 ± 3.00 µg/g) was found significantly lower (P-value < 0.01) than that of the same species from the wild (31.86 ± 22.20 µg/g). The relative abundance of Vibrio was found positively correlated with the liver TTX concentrations. With the increase of culture periods, the relative abundance of Vibrio and Cyanobacteria decreased. In addition, both the metabolites and functional genes in arginine biosynthesis metabolic pathway were found significantly down-regulated (P-value < 0.05). These results indicated that both Vibrio and Cyanobacteria bacterial symbionts participated in TTX biosynthesis using arginine as a potential precursor in the gut environment of toxic puffer fishes.
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Yuan, Baowen [Verfasser], and Barbara [Akademischer Betreuer] Burwinkel. "Identification of Plasma Metabolites Associated with Breast and Ovarian Cancer and Breast Cancer Prognosis / Baowen Yuan ; Betreuer: Barbara Burwinkel." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1214371698/34.
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Gómez, Castellà Cristina 1985. "Improving detection capabilities of doping agents by identification of new phase I and phase II metabolites by LC-MS/MS." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/132539.
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Metabolic studies of doping agents have been traditionally performed by using gas chromatography coupled to mass spectrometry (GC-MS). In the last years, liquid chromatography coupled to mass spectrometry (LC-MS) has demonstrated to offer new possibilities to perform metabolic studies. The objective of this thesis was to study the metabolism (phase I and phase II) of different doping agents by LC-MS/MS in order to improve the detection capabilities of the studied substances.
For mesocarb, a thermolabile compound, the parent drug 19 metabolites were detected in urine including mono-, di- and tri-hydroxylated metabolites excreted free or conjugated with glucuronic acid and sulphate. For toremifene, an anti-estrogenic drug with poor electron ionization properties, the parent drug and 20 metabolites were detected in urine. The structure of most of the metabolites was proposed.
Anabolic androgenic steroids (AAS) metabolites conjugated with sulphate were investigated to improve the retrospectivity of the detection of these compounds. A study of hydrolysis and MS/MS behaviour of sulphate metabolites of AAS was performed, and sulphate conjugated metabolites of boldenone, methyltestosterone and metandienone were studied. Boldenone sulphate and epiboldenone sulphate were identified as boldenone metabolites in humans. They can be used as markers of exogenous boldenone administration. For methyltestosterone, three new sulphate metabolites were detected and structures were proposed. One of them, 17β-methyl-5α-androstan-3α,17α-diol 3α-sulphate was detected in urine up to 21 days after methyltestosterone administration, improving three times the retrospectivity of the detection with respect to other previously reported long-term metabolites. Several new sulphate metabolites were detected after metandienone administration. One of them was characterized as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one conjugated with sulphate, and it was detected up to 26 days after administration, improving the retrospectivity of the detection with respect to other long-term metabolites described.
The usefulness of LC-MS/MS for the detection and characterization of metabolites of doping agents has been demonstrated, especially for the study of new phase II metabolites and for metabolic studies of compounds where GC-MS shows relevant limitations.Els estudis metabòlics de substàncies dopants han estat tradicionalment realitzats mitjançant l’ús de cromatografia de gasos acoblada a espectrometria de masses (GC-MS). En els últims anys, s’ha demostrat la utilitat de la cromatografia líquida acoblada a espectrometria de masses (LC-MS) per realitzar estudis de metabolisme. L’objectiu d’aquesta tesi va ser estudiar el metabolisme (fase I i fase II) de diferents substàncies dopants mitjançant LC-MS/MS per tal de millorar la capacitat de detecció dels compostos estudiats. Per a mesocarb, compost termolàbil, es van detectar en orina el compost inalterat i 19 metabòlits incloent metabòlits mono-, di- i tri-hidroxilats excretats lliures o conjugats amb àcid glucurònic i sulfat. Per a toremifè, un fàrmac anti-estrogènic amb espectre de masses d’impacte electrònic amb pocs ions diganòstic, es van detectar el compost inalterat i 20 metabòlits en orina. Es va proposar l’estructura de la major part de metabòlits detectats. Per tal de millorar la retrospectivitat de la detecció dels esteroides anabolitzants androgènics (AAS) es van estudiar els metabòlits conjugats amb sulfat. Es va realitzar un estudi de la hidròlisi i del comportament espectromètric dels metabòlits conjugats amb sulfat dels AAS. Es van estudiar els metabòlits conjugats amb sulfat de boldenona, metiltestosterona i metandienona. Es van identificar boldenona sulfat i epiboldenona sulfat com a metabòlits de boldenona en humans. Aquests metabòlits poden ser usats com a marcadors de l’administració exògena de boldenona. Per a metiltestosterona, es van detectar i proposar les estructures de tres nous metabòlits conjugats amb sulfat. Un d’ells, el 17β-metil-5α-androstà-3α,17α-diol 3α-sulfat, va ser detectat en orina fins a 21 dies després de l’administració de metiltestosterona. Es van detectar diversos metabòlits de metandienona conjugats amb sulfat no descrits prèviament. Un d’ells, identificat com a 18-nor-17β-hidroximetil-17α-metilandrost-1,4,13-trien-3-ona conjugat amb sulfat, va ser detectat fins 26 dies després de l’administració. Tant per a metiltestosterone com per a metandienone, els metabòlits conjugats amb sulfat permeten millorar la retrospectivitat de la detecció respecte a altres marcadors descrits anteriorment. S’ha demostrat la utilitat del LC-MS/MS per a la detecció i caracterització de metabòlits de substàncies dopants, especialment per a l’estudi de nous metabòlits de fase II i per a estudis de metabolisme de compostos que mostren limitacions en GC-MS.
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McClean, Stephen. "An investigation of modern analytical techniques for the identification and determination of selected drugs and pollutants, their degradation products and metabolites." Thesis, University of Ulster, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322413.
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Ma, Cunxian. "Tentative Identification of Hydroxylated 2,2',3,5',6-pentachlorobiphenyl Metabolites in Whole Poplar Plants by a Combination of Chromatographic and Spectrometry Techniques." Thesis, University of Iowa, 2014. https://ir.uiowa.edu/etd/4691.
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2,2',3,5',6-pentachlorobiphenyl (PCB95) is a chiral congener of the persistent organic pollutants (POPs) family of PCBs. It has been shown that chiral PCBs can be enantioselectively transformed into hydroxylated metabolites by cytochrome P450 in animals. Previous studies in our group suggested that PCB 95 can be enantioselectively translocated and metabolized in whole poplar plants. In this work, healthy whole poplar plants were hydroponically exposed to PCB95 for 30 days. Two unknown OH-PCB95 metabolites were detected in the roots by HPLC-MS. Different chromatographic and spectrometry techniques, including HPLC-MS, NMR and GC-MS, were tried to determine the structure of the more abundant metabolite of the two. It was identified to be 4'-OH-PCB95 (4'-95) by GC-MS method. The data show that PCB95 can be transformed into at least two hydroxylated metabolites by whole poplar plants, with one of them being 4'-95. Chiral analysis of 4'-95 by HPLC-MS showed slightly more abundance of the second eluting enantiomer E2-4'-95 in the roots, suggesting that the biotransformation of PCB95 to 4'-95 is enantioselective. Comparison with animal studies shows a distinct metabolite profile in whole poplar plants.
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Razmilic, Neira Valeria Isabel. "Metabolism analysis of streptomyces leeuwenhoekii C34 with a genome scale model and identification of Biosynthetic genes of specialized metabolites by genome mining." Tesis, Universidad de Chile, 2017. http://repositorio.uchile.cl/handle/2250/144111.
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Doctor en Ciencias de la Ingeniería, Mención Ingeniería Química y BiotecnologíaStreptomyces leeuwenhoekii C34 es una nueva cepa que fue aislada desde la laguna Chaxa ubicada en el Desierto de Atacama, Chile. Esta cepa produce metabolitos especializados con actividad contra Staph. aureus resistente a meticilina (MRSA): chaxamicinas y chaxalactinas. La secuencia genómica de S. leeuwenhoekii C34 se obtuvo mediante las tecnologías de Illumina Miseq y PACbio RS II SMRT. El genoma se utilizó para identificar clústers de genes biosintéticos (BGCs) que codifican para metabolitos especializados a través de minería de genomas, y para desarrollar un modelo a escala genómica (GSM) para estudiar las rutas de biosíntesis de producción de metabolitos especializados. Se encontraron 34 BGCs en el genoma de S. leeuwenhoekii C34, más un BGC ubicado en el plásmido pSLE2. Se encontró tres BGCs para lazo-péptidos. Específicamente, se identificó el producto del BGC del lazo-péptido 3 en el sobrenadante de S. leeuwenhoekii C34 cultivado en medio TSB/YEME y se expresó exitosamente en el huésped heterólogo S. coelicolor M1152. Se confirmó que este lazo-péptido era el mismo que la chaxapeptina, recientemente descrita para S. leeuwenhoekii C58. Por otra parte, se identificó un BGC de 64 kb (locus 1083651 a 1147687) que codifica para un híbrido trans-AT PKS/NRPS. Es probable que el producto de este BGC sea un compuesto halogenado debido a la presencia de un gen, sle09470, que codifica para una enzima cloradora. Para estudiar este clúster de genes, se desarrollaron diferentes cepas derivadas de S. leeuwenhoekii. También, el BGC se clonó en huéspedes heterólogos: S. coelicolor M1152, M1154 and S. albus. A través de análisis de HPLC MS/MS y comparación de perfiles de metabolitos, se identificó un grupo de compuestos con patrón clorado, sin embargo se descartaron como posibles productos del BGC ya que además de encontrarse en las cepas de S. leeuwenhoekii también se encontraron en muestras de S. coelicolor M1152. Por otra parte, se detecto un metabolito con una señal de m/z 611.53 [M + H]+ solamente en las muestras de S. leeuwenhoekii M1614 ( chaxamycin BGC) y M1619 ( chaxamycin BGC; sle09560). Se requieren msá estudios para confirmar si los metabolitos expresados diferencialmente corresponden a un producto del híbrido transAT-PKS/NRPS BGC. Para construir el GSM de S. leeuwenhoekii C34 se desarrolló una interfaz basada en python, que permite: buscar genes de Streptomyces asociados a reacciones en la base de datos KEGG, realizar BLAST local contra S. leeuwenhoekii C34, comparar los dominios de proteínas, descargar información de los metabolitos, construir el GSM y realizar simulaciones usando COBRApy. Las rutas biosintéticas de chaxamicinas, chaxalactinas, desferrioxaminas, ectoina y el producto del híbrido transAT-PKS/NRPS BGC (híbrido PK-NP) se incluyeron en el modelo. El modelo, iVR1007, consiste de 1722 reacciones, 1463 metabolitos y 1007 genes, y se validó usando información experimental de crecimiento en diferentes fuentes de carbono, nitrógeno y fósforo, mostrando un 83.7 % de precisión. El modelo se usó para encontrar deleción y sobre-expresión de genes no intuitivas que predicen un aumento en la producción de precursores de chaxamicinas, chaxalactinas e híbrido PK-NP. Las modificaciones predichas podrán ser usadas para realizar ingeniería metabólica de S. leeuwenhoekii C34 para incrementar la producción de metabolitos especializados.
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Wimberley, James. "De novo Sequencing and Analysis of Salvia hispanica Transcriptome and Identification of Genes Involved in the Biosynthesis of Secondary Metabolites." Chapman University Digital Commons, 2019. https://digitalcommons.chapman.edu/cads_theses/5.
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Salvia hispanica L. (commonly known as chia) is gaining popularity worldwide and specially in US as a healthy oil and food supplement for human and animal consumption due to its favorable oil composition, and high protein, fiber, and antioxidant contents. Despite these benefits and its growing public demand, very limited gene sequence information is currently available in public databases. In this project, we generated 90 million high quality 150 bp paired-end sequences from the chia leaf and root tissues. The sequences were de novo assembled into 103,367 contigs with average length of 1,445 bp. The resulted assembly represented 92.2% transcriptome completeness. Around 69% of the assembled contigs were annotated against the uniprot database and represented a diverse array of functional and biological categories. A total of 14,267 contigs showed significant expression difference between the leaf and root tissues, with 6,151 and 8,116 contigs upregulated in the leaf and root, respectively. The sequence data generated in this project will provide valuable resources for future functional genomic research in chia. With the availability of transcriptome sequences, it would be possible to identify genes involved in the important metabolic pathways that give chia its unique nutritional and medicinal properties. Finally, the generated data will contribute to the genetic improvement efforts of chia to better serve the public demand.
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Tisler, Selina Kornelia [Verfasser], and Christian [Akademischer Betreuer] Zwiener. "Identification, occurrence and fate of transformation products and metabolites of fluoxetine and metformin in the aquatic environment / Selina Kornelia Tisler ; Betreuer: Christian Zwiener." Tübingen : Universitätsbibliothek Tübingen, 2019. http://d-nb.info/1205002499/34.
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Sundström, Ingela. "Liquid Chromatography-Mass Spectrometry as a Tool for Drug Metabolite Identification in Biological Fluids : With Application to Ketobemidone." Doctoral thesis, Uppsala University, Analytical Pharmaceutical Chemistry, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7909.
Full textAbstract:
Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone.
Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.
On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites.
Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.
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Bücherl, Daniel [Verfasser], and Jörg [Akademischer Betreuer] Heilmann. "Isolation of kaempferol glycosides from Ginkgo biloba leaves and synthesis, identification and quantification of their major in vivo metabolites / Daniel Bücherl. Betreuer: Jörg Heilmann." Regensburg : Universitätsbibliothek Regensburg, 2013. http://d-nb.info/1047236923/34.
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Zhou, Qiuqin [Verfasser], Helge B. [Gutachter] Bode, and Martin [Gutachter] Grininger. "Identification of selected secondary metabolites from Xenorhabdus and investigation on the biosynthesis of anthraquinones from Photorhabdus / Qiuqin Zhou. Gutachter: Helge B. Bode ; Martin Grininger." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2016. http://d-nb.info/1112601457/34.
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Bujor, Oana-Crina. "Extraction, identification and antioxidant activity of the phenolic secondary metabolites isolated from the leaves, stems and fruits of two shrubs of the Ericaceae family." Thesis, Avignon, 2016. http://www.theses.fr/2016AVIG0261/document.
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La myrtille et l’airelle rouge, deux arbrisseaux de la famille des Ericacées, sont consommées comme des aliments, boissons et suppléments alimentaires pour leur valeur nutritionnelle et leur richesse en polyphénols antioxydants. Dans les plantes, la qualité et la quantité de composés phénoliques sont influencées par les parties morphologiques de la plante à utiliser. En particulier, les composés phénoliques des végétaux exercent leur activité antioxydante dans la protection des lipides alimentaires et le compartiment gastrique a été proposé comme le site majeur pour le stress oxydatif lié au régime alimentaire. L’objectif général de cette thèse était d’étudier les variations saisonnières des composés phénoliques d’extraits de feuilles, branches et fruits de la myrtille et de l’airelle rouge ainsi que l’activité antioxydante de ces extraits. Pour cette étude, des extraits aqueux et hydroéthanoliques (fruits uniquement) des échantillons collectés en mai, juillet et septembre pendant les années 2013-2014 ont été obtenus par extraction assistée par microondes.Les analyses qualitatives et quantitatives par UPLC / MS des extraits de la myrtille ont montré la présence de dérivés de l’acide caféique et de l’acide p-coumarique et des glycosides de flavonols dans les feuilles tandis que des oligomères de flavanols étaient aussi présents dans les branches, et ce dans des quantités élevées. La thioacidolyse a révélé de faibles degrés de polymérisation (2-4) et l’(-)-épicatéchine comme unité principale des flavan-3-ols. Il existe une très bonne corrélation entre la Somme des Composés phénoliques par UPLC et la Teneur en Polyphenols Totaux ou l’activité antioxydante dans le test DPPH, excepté pour les feuilles du mois de mai. Ces dernières sont relativement riches en dérives de l’acide p-coumarique. Les effets de la saison apparaissent plus marqués pour les feuilles qui présentent une plus grande activité antioxydante et teneur en polyphénols en juillet et septembre. Ces paramètres sont optimaux en juillet pour les branches de myrtille. La période de de cueillette peut être définie en fonction des structures phénoliques désirées.Dans l’airelle rouge, la présence prédominante de monomères et oligomères de flavanols et de glycosides de quercétine a été identifiée dans toutes les parties morphologiques. Les proanthocyanidines contiennent la (+)-catéchine et la (-)-épicatéchine comme unités d'extension et terminale. De plus, la teneur en polyphénols totaux (méthode de Folin, UPLC) a montré une augmentation légère mais significative de mai à septembre pour les feuilles et les branches. Cette augmentation a été confirmée pour l'activité antioxydante dans le test DPPH pour les feuilles et les branches en 2014.L’activité antioxydante des extraits de myrtille et d’airelle rouge lors de l’inhibition de l’oxydation lipidique (accumulation de diènes conjugués) a été évaluée dans des conditions in vitro simulant la digestion. Tout d'abord, l'inhibition de l’oxydation lipidique a été conduite sur des émulsions huile de tournesol-dans-eau stabilisées par la sérum albumine bovine (BSA) ou des phospholipides d’œuf (PL), qui simulent l’état physique des lipides alimentaires lors de la digestion gastrique. L’oxydation a été initiée par la metmyoglobine, une forme de fer apportée par la viande rouge. Dans les deux modèles d’émulsions, les extraits aqueux des branches et des feuilles et l’extrait hydroethanolique de fruit de myrtille sont des inhibiteurs plus efficaces de l'oxydation lipidique durant la première phase de digestion (pH 5) que durant la seconde phase (pH 3). D’autre part, un extrait de feuilles de myrtille a été testé dans un modèle complet de digestion in vitro statique (étapes orale, gastrique et intestinale). L'oxydation lipidique, rapide lors de la l’étape gastrique (systèmes BSA et PL) et puis plus lente lors de l'étape intestinale (système PL), a été totalement inhibée par l'extrait de feuilles de myrtilleBilberry and lingonberry, two shrubs of the Ericaceae family, are consumed as food, beverage and dietary supplements due to their nutritional value and richness in antioxidant polyphenols. In plants, the quality and quantity of phenolic compounds are influenced by the parts of the plant to be used. In particular, plant phenolic compounds provide antioxidant activity in the protection of dietary lipids from oxidation and the gastric compartment has been proposed as a major site for diet-related oxidative stress. The aim of this thesis is to simultaneously assess the seasonal variations of phenolic compounds in leaves, stems, and fruits of bilberry and lingonberry extracts, as well as their antioxidant activity. For this study, aqueous and hydroethanolic (only fruits) extracts of bilberry and lingonberry samples collected in May, July and September during the years 2013-2014 were obtained under microwave-assisted extraction.In bilberry extracts, qualitative and quantitative analyses by UPLC/MS showed the presence of caffeoyl derivatives, p-coumaroyl derivatives, and flavonol glycosides in leaves whereas in stems, flavanol oligomers were additionally identified in significant amounts. Thioacidolysis revealed low degrees of polymerization (2-4) and (-)-epicatechin as the main flavan-3-ol unit. The sum of the phenolic compounds by UPLC was highly correlated with the Total Polyphenol Content and the antioxidant activity in the DPPH test for all the extracts except those of May leaves. The latter were relatively richer in p-coumaric acid derivatives. Seasonal effects were more marked for leaves which exhibited higher antioxidant activities and phenolic contents in July and September when these parameters were maximum in July for bilberry stems. The harvest period can be refined based on the desired phenolic structures. For lingonberry, the predominant presence of monomers and oligomers of flavanols and quercetin glycosides was found in all the morphological parts. Proanthocyanidins contain (+)-catechin and (-)-epicatechin as both extension and terminal units. The sum of the phenolic compounds by UPLC was less correlated with the Total Polyphenol Content and the antioxidant activity in the DPPH test than in bilberry. Furthermore, the total phenolic content (Folin method, UPLC) showed a slight but significant increase from May to September for both leaves and stems. This increase was confirmed for the antioxidant activity by the DPPH test for both leaves and stems in 2014.The antioxidant activity of bilberry and lingonberry extracts against lipid oxidation (formation of lipid-derived conjugated dienes) was evaluated under in vitro simulated digestion conditions. Firstly, the inhibition of lipid oxidation was performed using sunflower oil-in-water emulsions stabilized by bovine serum albumin (BSA) or egg yolk phospholipids (PL), both emulsifiers mimicking dietary components. Oxidation was initiated by metmyoglobin, a form of dietary iron from red meat. In both emulsion models, aqueous extracts from stems and leaves and the hydroethanolic fruit extract of bilberry proved to be more efficient inhibitors of lipid oxidation in the early phase of digestion (pH 5) than during the second phase (pH 3). Secondly, a bilberry leaf extract was tested in the inhibition of lipid oxidation in a complete static in vitro digestion model (oral, gastric and intestinal phases). The fast lipid oxidation in the gastric step (BSA and PL systems) and the slower lipid oxidation in the intestinal step (PL system) were totally inhibited by the bilberry leaf extract
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Förster, Yvonne, Johannes R. Schmidt, Dirk K. Wissenbach, Susanne E. M. Pfeiffer, Sven Baumann, Lorenz C. Hofbauer, Bergen Martin von, Stefan Kalkhof, and Stefan Rammelt. "Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-217570.
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Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis.
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Dallery, Jean-Félix. "Identification et régulation des gènes du métabolisme secondaire et caractérisation de métabolites chez le champignon phytopathogène Colletotrichum higginsianum." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS102.
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Les espèces du genre Colletotrichum sont majoritairement des agents pathogènes de nombreuses plantes et provoquent des dégâts importants aux cultures partout dans le monde. Colletotrichum higginsianum est un champignon phytopathogène hémibiotrophe capable d’infecter de nombreuses Brassicaceae dont la plante modèle Arabidopsis thaliana. L’intérêt du pathosystème C. higginsianum – A. thaliana réside dans la possibilité de manipuler génétiquement les deux partenaires. Par ailleurs, la disponibilité de très nombreuses lignées transgéniques et d’outils génétiques aide à disséquer les mécanismes de résistance et de sensibilité de la plante. Nous avons re-séquencé (technologie PacBio) le génome de C. higginsianum afin d’obtenir les séquences complètes des 12 chromosomes. L’analyse détaillée de cette seconde version du génome a permis de mettre en évidence l’un des plus grands arsenaux fongiques de gènes du MS et de résoudre les problèmes de fragmentation de la première version du génome. Afin d’identifier les métabolites secondaires que C. higginsianum produit au cours de l’infection de la plante, nous avons supprimé des répresseurs du MS qui modifient l’état de la chromatine (Kmt1, Kmt6, Hep1, CclA) et sur-exprimé des inducteurs du MS (LaeA, Sge1) décrits chez d’autres mycètes. Grâce à des analyses de transcriptomique (RNA-Seq), des gènes du MS différentiellement exprimés dans ces mutants ont pu être identifiés. Ensuite, par des analyses de chimie analytique nous avons pu identifier et caractériser deux familles de métabolites secondaires. Enfin, nous avons mis en évidence des activités inédites pour ces deux familles de composés. Ces résultats ouvrent de nouvelles perspectives pour aborder le déterminisme du pouvoir pathogène de C. higginsianumColletotrichum spp. are major plant pathogens of numerous crops worldwide. Colletotrichum higginsianum uses a hemibiotrophic lifestyle to infect many members of the Brassicaceae including the model plant Arabidopsis thaliana. The C. higginsianum – A. thaliana pathosystem facilitates dissection of the mechanisms of plant resistance and susceptibility and the traits underlying fungal pathogenicity. Both partners can be genetically manipulated and powerful genetic tools and transgenic lines are available on the plant side. With a view to obtain a more complete inventory of C. higginsianum secondary metabolism (SM) genes, we re-sequenced the genome de novo using PacBio technology, providing 12 complete chromosome sequences. The in-depth analysis of this second version of the genome revealed one of the largest SM repertoires described to date for any ascomycete and solved numerous fragmentation problems in the first genome assembly. In order to identify secondary metabolites produced by C. higginsianum during plant infection, we deleted negative SM regulators that modify chromatin status (Kmt1, Kmt6, Hep1, CclA), and over-expressed positive SM regulators (LaeA, Sge1), all of which were well-described in other fungi. Using transcriptomics (RNASeq), we identified SM genes differentially expressed in these mutants. Using metabolite profiling, we also identified and characterised two families of secondary metabolites. Finally we describe novel biological activities for these compound families. These results provide new insights into the molecular basis of pathogenicity in C. higginsianum