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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Dissection of phloem transport in cucurbitaceae by metabolomic analysis

Zhang, Baichen January 2005 (has links)
This thesis aimed to investigate several fundamental and perplexing questions relating to the phloem loading and transport mechanisms of <i>Cucurbita maxima</i>, by combining metabolomic analysis with cell biological techniques. This putative symplastic loading species has long been used for experiments on phloem anatomy, phloem biochemistry, phloem transport physiology and phloem signalling. Symplastic loading species have been proposed to use a polymer trapping mechanism to accumulate RFO (raffinose family oligosaccharides) sugars to build up high osmotic pressure in minor veins which sustains a concentration gradient that drives mass flow. However, extensive evidence indicating a low sugar concentration in their phloem exudates is a long-known problem that conflicts with this hypothesis. Previous metabolomic analysis shows the concentration of many small molecules in phloem exudates is higher than that of leaf tissues, which indicates an active apoplastic loading step. Therefore, in the view of the phloem metabolome, a symplastic loading mechanism cannot explain how small molecules other than RFO sugars are loaded into phloem. <br><br> Most studies of phloem physiology using cucurbits have neglected the possible functions of vascular architecture in phloem transport. It is well known that there are two phloem systems in cucurbits with distinctly different anatomical features: central phloem and extrafascicular phloem. However, mistaken conclusions on sources of cucurbit phloem exudation from previous reports have hindered consideration of the idea that there may be important differences between these two phloem systems. <br><br> The major results are summarized as below:<br> 1) O-linked glycans in <i>C.maxima</i> were structurally identified as beta-1,3 linked glucose polymers, and the composition of glycans in cucurbits was found to be species-specific. Inter-species grafting experiments proved that these glycans are phloem mobile and transported uni-directionally from scion to stock.<br> 2) As indicated by stable isotopic labelling experiments, a considerable amount of carbon is incorporated into small metabolites in phloem exudates. However, the incorporation of carbon into RFO sugars is much faster than for other metabolites.<br> 3) Both CO2 labelling experiments and comparative metabolomic analysis of phloem exudates and leaf tissues indicated that metabolic processes other than RFO sugar metabolism play an important role in cucurbit phloem physiology.<br> 4) The underlying assumption that the central phloem of cucurbits continuously releases exudates after physical incision was proved wrong by rigorous experiments including direct observation by normal microscopy and combined multiple-microscopic methods. Errors in previous experimental confirmation of phloem exudation in cucurbits are critically discussed.<br> 5) Extrafascicular phloem was proved to be functional, as indicated by phloem-mobile carboxyfluorescein tracer studies. Commissural sieve tubes interconnect phloem bundles into a complete super-symplastic network.<br> 6) Extrafascicular phloem represents the main source of exudates following physical incision. The major transported metabolites by these extrafacicular phloem are non-sugar compounds including amino acids, O-glycans, amines.<br> 7) Central phloem contains almost exclusively RFO sugars, the estimated amount of which is up to 1 to 2 molar. The major RFO sugar present in central phloem is stachyose. <br> 8) Cucurbits utilize two structurally different phloem systems for transporting different group of metabolites (RFO sugars and non-RFO sugar compounds). This implies that cucurbits may use spatially separated loading mechanisms (apoplastic loading for extrafascicular phloem and symplastic loading for central phloem) for supply of nutrients to sinks. <br> 9) Along the transport systems, RFO sugars were mainly distributed within central phloem tissues. There were only small amounts of RFO sugars present in xylem tissues (millimolar range) and trace amounts of RFO sugars in cortex and pith. The composition of small molecules in external central phloem is very different from that in internal central phloem.<br> 10) Aggregated P-proteins were manually dissected from central phloem and analysed by both SDS-PAGE and mass spectrometry. Partial sequences of peptides were obtained by QTOF <i>de novo</i> sequencing from trypsin digests of three SDS-PAGE bands. None of these partial sequences shows significant homology to known cucurbit phloem proteins or other plant proteins. This proves that these central phloem proteins are a completely new group of proteins different from those in extrafascicular phloem. The extensively analysed P-proteins reported in literature to date are therefore now shown to arise from extrafascicular phloem and not central phloem, and therefore do not appear to be involved in the occlusion processes in central phloem. / Phloem transportiert ein ausgedehntes Spektrum an Molekülen zwischen Pflanzenorganen, um Wachstum und Entwicklung zu koordinieren. Folglich ist eine umfassende und unvoreingenommene Metabolom-Analyse notwendig, um unser Verständnis über den Transport von Stoffwechselprodukten sowie über Phloemtransport zu vertiefen. Phloemexsudate von Kürbispflanzen werden unter Verwendung der Metabolom-Analyse analysiert. Bei diesen Pflanzen wird angenommen, dass sie symplastische Beladungswege verwenden, um Photoassmilate als Ausgangsschritt des Phloemtransportes zu konzentrieren. Zwei neue Familien Callose-verwandter Substanzen, 1,3-Overknüpfte Glycane, sowie eine Reihe anderer kleinerer Metabolite werden in den Phloemexsudaten detektiert. Metabolom-Daten und physiologische Experimente widersprechen früher berichtetem Verständnis des Phloemexsudationsprozesses in Kürbispflanzen. Folglich bestätigt sich der Phloemexsudationsprozeß durch Kombination unterschiedlicher mikroskopischer Techniken. Kürbispflanzen besitzen zwei Phloemsysteme mit eindeutigen anatomischen Eigenschaften. Es zeigt sich, daß Phloemexsudate in Kürbissen hauptsächlich vom extrafaszikulären Phloem, nicht vom zentralen Phloem, stammen. In den letzten Jahrzehnten wurde gewöhnlich mißverstanden, daß Phloemexsudate vom zentralen Phloem stammen. Die eindeutigen metabolischen Profile der unterschiedlichen Phloemsysteme, die durch Metabolom-Analysen in der räumlichen Auflösung beobachtet werden, bestätigen die unterschiedlichen physiologischen Funktionen der zwei unterschiedlichen Phloemsysteme: das zentrale Phloem transportiert hauptsächlich Zucker, während das extrafaszikuläre Phloem ein ausgedehntes Spektrum von Metaboliten transportiert. Es kann auch ein unterschiedliches metabolisches Profil kleiner Moleküle zwischen internem und externem zentralem Phloem beobachtet werden. Von Strukturproteinen des zentralen Phloems wurden auch Proben genommen und mittels Massenspektrometrie analysiert. Diese Proteine erweisen sich als neuartige Proteine, die sich zu denen im extrafaszikulären Phloem unterscheiden. Dies bestätigt ferner den Funktionsunterschied der unterschiedlichen Phloemsysteme in Kürbispflanzen. Basierend auf diesen neuartigen Entdeckungen des Phloem-Metaboloms und dem vorhergehenden Wissen über den Phloemtransport in Kürbispflanzen, wird ein neues Modell vorgeschlagen, um den Mechanismus des Phloemtransports in der symplastischen Beladung zu verstehen.<br>
2

Studies on the global screening of functional food ingredients in tomato using LC-MS and metabolomic analysis / LC-MS及びメタボローム解析を利用したトマトに含まれる機能性成分の網羅的探索に関する研究 / # ja-Kana

Mori, Shinsuke 25 September 2018 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21375号 / 農博第2299号 / 新制||農||1067(附属図書館) / 学位論文||H30||N5148(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 橋本 渉, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM
3

Understanding the metabolic mechanisms stimulated by plant-associated bacteria to enhance cold tolerance in tomato plants

Licciardello, Giorgio 28 October 2024 (has links)
Climate change is expected to increase the frequency of mild winters and warm springs, which can induce premature plant development. This premature development results in a high risk of exposure of young plant tissues to cold stress leading to severe reductions in plant growth and agricultural production. Plants are associated with complex bacterial communities that can activate acclimation processes and positively affect plant performance at low temperatures. Beneficial effects of plant colonization by cold-tolerant bacteria include the modulation of cold-related genes and the reduction in cellular damage under cold stress, but scarce information is available on mechanisms stimulated by bacterial endophytes in tomato plants against cold stress. The aims of this work were i) to analyze the taxonomy and potential functions of plant-associated microbial communities in cold regions, ii) to understand metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress, and iii) to identify possible genomic traits of cold-tolerant endophytic bacteria responsible for plant growth promotion and cold stress mitigation. The first chapter includes an introduction on cold stress and acclimation processes in plants, and the second chapter defines the aims of the project. In the third chapter, the taxonomic and functional characterization of plant-associated microbial communities of alpine, Arctic, and Antarctic regions was reviewed, highlighting the main environmental factors affecting their taxonomic structure. e. The key findings of this chapter are the functional roles of microbial communities in plant growth and survival in cold environments, and the suggestion of potential biotechnological applications of ubiquitous and endemic cold-tolerant microorganisms. In the fourth chapter, metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress were studied by metabolomic analyses, and compounds possibly associated with cold stress mitigation were found. 14 Tomato seeds were inoculated with two bacterial endophytes isolated from Antarctic Colobanthus quitensis plants (Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10) or with Paraburkholderia phytofirmans PsJN, while mock-inoculated seeds were used as control. The metabolic composition of tomato plants was analyzed immediately after cold stress exposure (4°C for seven days) or after two and four days of recovery at 25°C. Under cold stress, the content of malondialdehyde, phenylalanine, ferulic acid, and p-coumaric acid was lower in bacterium-inoculated compared to mock-inoculated plants, indicating a reduction of lipid peroxidation and the stimulation of phenolic compound metabolism. The content of two phenolic compounds, five putative phenylalanine-derived dipeptides, and three further phenylalanine-derived compounds was higher in bacterium-inoculated compared to mock-inoculated samples under cold stress. Thus, the presented work suggests that psychrotolerant endophytic bacteria can reprogram polyphenol metabolism and stimulate the accumulation of secondary metabolites, like 4-hydroxybenzoic and salicylic acid, which are involved in cold stress mitigation, and phenylalanine-derived dipeptides possibly involved in plant stress responses. In the fifth chapter, functional and genomic traits of Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10 were studied. In the framework of the present study, Ewingella sp., Pseudomonas sp., and the bacterial consortium showed plant growth-promoting activity on tomato seedlings at low temperatures. Ammonia was produced by both bacterial isolates and their consortium, while indole-3-acetic acid and proteases were produced by Ewingella sp. and Pseudomonas sp., respectively. Ewingella sp. and Pseudomonas sp. genomes (51.57% and 60.63% guanine-cytosine, 4,148 and 5,983 predicted genes, respectively) encompassed genes related to amino acid metabolism, plant hormone metabolism (auxin, cytokinins, ethylene, and salicylic acid), nitrogen metabolism, lytic activities (amylases, cellulases, and proteases). Traits related to plant growth promotion included genes for iron transport, phosphate metabolism, potassium transport, siderophore metabolism and 15 transport, and zinc transport. Moreover, Ewingella sp. and Pseudomonas sp. encompassed genes related to cold tolerance, such as cold shock and heat shock-related proteins, lipid desaturases, and genes related to polyamine metabolism, proline metabolism, proline and glycine betaine transport, reactive oxygen species detoxification, and trehalose metabolism. Thus, in this chapter, it was discovered that Antarctic cold-tolerant endophytes include multiple genomic and functional traits to survive under cold conditions and some of them can contribute to promote the host plant growth at low temperatures. These findings indicate that plant-associated bacteria of cold regions have a great biotechnological potential to mitigate cold stress in crop plants. In particular, Antarctic bacterial endophytes encompass genomic traits responsible for plant growth promotion and protection against cold stress, and they can mitigate cold stress in tomato plants by a complex reprogramming of plant metabolism. Although further metabolomic and functional studies are required to verify compound annotations and to better clarify the role of phenylalanine-derived compounds and phenylalanine-derived dipeptides in cold stress mitigation, these results provided a better understanding of metabolic changes stimulated by psychrotolerant endophytic bacteria in cold-stressed tomato plants. Thus, the validation of cold stress mitigation activated by psychrotolerant endophytic bacteria under field conditions will pave the way for the further development of endophytic bacterial inoculants as sustainable products to protect crops against cold stress.
4

Μεταβολομική ανάλυση κυττάρων HeLa μετά από υπερέκφραση της πρωτεΐνης DGCR14, ενός παράγοντα που σχετίζεται με το σωματίδιο συναρμογής (spliceosome)

Καυκιά, Ελένη 02 March 2015 (has links)
Στην μετα-γονιδιωματική εποχή, την εποχή της συστημικής βιολογίας, η κατανόηση της πολυπλοκότητας της κυτταρικής φυσιολογίας απαιτεί την ανάλυση της δυναμικής των δικτύων βιομοριακών αλληλεπιδράσεων σε όλα τα μοριακά επίπεδα κυτταρικής λειτουργίας. Με τη σειρά της, η λειτουργική γονιδιωματική, ένας θεμελιώδης λίθος της συστημικής βιολογίας, στοχεύει στον πολυδιάστατο χαρακτηρισμό ενός γονιδίου, συνδυάζοντας δεδομένα από τις τεχνολογίες υψηλής απόδοσης. Είναι αυτή ακριβώς η ενοποίηση όλων των μοριακών προτύπων για ένα διαταραγμένο βιολογικό σύστημα που μπορεί να δώσει πληροφορίες αναφορικά με την λειτουργία ενός αγνώστου γονιδίου. Στο πλαίσιο αυτό, η παρούσα Διπλωματική Εργασία αποτελεί μέρος της ολιστικής λειτουργικής ανάλυσης δύο αλληλεπιδρώντων, αγνώστου βιολογικού ρόλου, πρωτεϊνών, της DGCR14 και της FRA10AC1, οι οποίες έχουν απομονωθεί ως συστατικά του σωματιδίου συναρμογής και έχουν συσχετιστεί με νευρολογικές ασθένειες. Η παρούσα εργασία επικεντρώνεται στην μεταβολομική μελέτη των μοριακών επιπτώσεων της υπερέκφρασης της DGCR14 σε ένα ανθρώπινο κυτταρικό μοντέλο, τα κύτταρα HeLa, με την χρήση της αέριας χρωματογραφίας - φασματομετρία μάζας. Ωστόσο, για να επιτευχθεί αυτό, θέματα σχετικά με τις δυνατότητες ποσοτικοποίησης των πολυβηματικών ομικών αναλύσεων έπρεπε να επιλυθούν. Μια σημαντική παράμετρος αφορά στην γρήγορη αδρανοποίηση των ενζυματικών διεργασιών έτσι ώστε οι αποκτηθέντες μετρήσεις να αντικατοπτρίζουν την πραγματική κυτταρική φυσιολογία. Για τον σκοπό αυτό, ο πειραματικός σχεδιασμός πρέπει να τροποποιείται κατάλληλα έτσι ώστε οποιεσδήποτε απαιτούμενες προ-αναλυτικές διαδικασίες χειρισμού των κυττάρων να έχουν ελάχιστη επίδραση στην φυσιολογία τους. Μελετήσαμε συνεπώς την επίδραση τεσσάρων πρωτοκόλλων συλλογής προσκολλημένων κυττάρων και δύο διαφορετικών διαλυμάτων έκπλυσης στο μεταβολικό πρότυπο κυττάρων HeLa. Τα μεταβολομικά δεδομένα αξιολογήθηκαν στο πλαίσιο της καρκινικής μεταβολικής φυσιολογίας και το πρωτόκολλο με την ελάχιστη δυνατή επίδραση στην κυτταρική φυσιολογία καθορίστηκε. Μεταξύ των αποτελεσμάτων αυτής της μελέτης, πολύτιμες πληροφορίες σχετικά με την μεταβολική φυσιολογία των αθανατοποιημένων κυτταρικών σειρών προέκυψαν, οι οποίες ενίσχυσαν σημαντικά την υπάρχουσα γνώση γύρω από τον καρκινικό μεταβολισμό, σε σταθερές ή μεταβαλλόμενες περιβαλλοντικές συνθήκες. Επακόλουθα, η βελτιστοποίηση της διαδικασίας συλλογής είχε ως αποτέλεσμα την δημιουργία ενός αντιπροσωπευτικού μεταβολικού προτύπου κυττάρων HeLa πάνω στο οποίο πραγματοποιήθηκε η αξιολόγηση της υπερέκφρασης της πρωτεΐνης DGCR14 χωρίς να επισκιάζεται από πειραματικές αποκλίσεις εισαγόμενες από την διαδικασία χειρισμού των κυττάρων. Αναφορικά με τα κύτταρα που υπερεκφράζουν την DGCR14, η μεταβολομική ανάλυση εντόπισε μια αλλαγή φυσιολογίας συνδεόμενη με συγκεκριμένα μεταβολικά μονοπάτια τα οποία υποδηλώνουν έντονο μεταβολικό στρες. Για να διερευνήσουμε την συσχέτιση της υπερέκφρασης της DGCR14 με τον παραπάνω μεταβολικό φαινότυπο, χρησιμοποιήσαμε το ανακατασκευασμένο δίκτυο πρωτεϊνικών αλληλεπιδράσεων του σωματιδίου συναρμογής στον άνθρωπο και το δίκτυο πρωτεϊνικών αλληλεπιδράσεων στον άνθρωπο από την μετα-βάση δεδομένων PICKLE, προκειμένου να αντλήσουμε επιπλέον πληροφορίες για τον ρόλο της DGCR14 βάσει της θέσης της σε σχέση με άλλους κόμβους και υπερ-κόμβους. Μια πιθανή λειτουργική συσχέτιση της DGCR14 με αυτοφαγικούς και λυσοσωμικούς μηχανισμούς βρέθηκε, η οποία θα αξιολογηθεί και μελλοντικά μέσω της ανάλυσης, ξεχωριστά και συνδυαστικά, των μοριακών συνεπειών της υπερ- και υπο-έκφρασης σε όλα τα μοριακά επίπεδα κυτταρικής λειτουργίας. / In the post-genomic, systems biology era, developing a systems level understanding of a physiological process requires the analysis of biomolecular network dynamics at all molecular levels of cellular function. Likewise, functional genomics, an essential foundation of systems biology research, aims to define and analyze gene function at a global level by integrating data obtained from multiple high-throughput technologies. It is the integration of all the molecular profiles for a systematically perturbed system that can provide insight about the function of unknown genes. Along these lines, the present study is part of the systematic functional analysis of two interacting, but yet of unknown biological role, spliceosomal proteins, DGCR14 and FRA10AC1, that have both been implicated in neurological diseases. The present work focuses on studying the molecular consequences of DGCR14 overexpression in a human cell model, HeLa cells, at the metabolic level using Gas Chromatography-(ion trap) Mass Spectrometry. However, to succeed in this, issues regarding the quantification capabilities of the multistep omic analysis procedures needed to be resolved. A major concern refers to the fast quenching of any enzymatic processes, so that the acquired measurements indeed reflect the cellular physiology in vivo. To this end, the experimental design should be appropriately adjusted so that any required sample handling actions before quenching have a minimal effect on cellular physiology. Thus, we investigated the effect of four cell collection protocols and two different washing solutions on the intracellular metabolic profile measurements of a HeLa cell culture. The measurements were interpreted in the context of the known cancer cell metabolic physiology and the protocol with the minimum possible effect on cellular physiology was specified. Among the results of this study, valuable information about the metabolic physiology of the immortal cell line arise, which improved our knowledge about cancer metabolism under steady or varying environmental conditions. Subsequently, the optimization of the collection procedure enabled us to establish a representative metabolic profile of HeLa cells against which the overexpression of DGCR14 was evaluated without being obscured by the effect of the sample handling. Regarding the overexpressing cells, the metabolomic analysis detected a trend of physiological change connected to specific metabolic pathways indicating strong metabolic stress. To understand how DGCR14 overexpression generates this particular metabolic phenotype, we used the human spliceosomal complex protein-protein interaction (PPI) network and the integrated human PPI meta-database PICKLE to extract additional information about DGCR14 role based on its location with respect to other nodes and hubs. A possible functional correlation of DGCR14 to autophagic and lysosomal mechanisms was established, that will be further evaluated in the future through the analysis, separately and in combination, of the consequences of DGCR14 over- and under-expression at all molecular levels of cellular function.

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