京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21813号 / 農博第2326号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5185(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21875号 / 人博第904号 / 新制||人||215(附属図書館) / 2018||人博||904(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 瀬戸口 浩彰, 教授 加藤 眞, 教授 市岡 孝朗, 准教授 西川 完途 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22281号 / 理博第4595号 / 新制||理||1659(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 沼田 英治, 准教授 森 哲, 教授 曽田 貞滋 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Salinity is a major abiotic stress in plants that causes significant reductions in crop yield. The need for improvement of food production has driven research to understand factors underlying plant responses to salt and mechanisms of salt tolerance. The aim of improving tolerance in traditional crops has been initiated but most crops can only tolerate a limited amount of salt in their systems to survive and produce biomass. Studies of naturally occurring high salt-tolerant plants (halophytes) are now being promoted for economic interests such as food, fodder or ecological reasons. Suaeda fruticosa, a member of the family Chenopodiaceae, belongs to a potential model halophyte genus for studying salt tolerance. However, published reports on the identification of genes, expression patterns and mechanisms of salinity tolerance in succulent halophytes are very limited. Next generation RNA-sequencing techniques are now available to help characterize genes involved in salinity response, along with expression patterns and functions of responsive genes. In this study, we have optimized the assembly of the transcriptome of S. fruticosa. We have annotated the genes based on their gene ontology characteristics and analyzed differential expression to identify genes that are up- and down-regulated in the presence of salt and have grouped the genes based on their putative functions. We also have provided evidence for groups of transcription factors that are involved in salt tolerance of this species and have identified those that may affect the regulation of salt tolerance. This work elucidates the characterization of genes involved in salinity tolerance to increase our understanding of the regulation of salt in a succulent halophyte.

Le combat contre les infections bactériennes reste un enjeu majeur de santé publique notamment avec la dissémination des Entérobactéries productrices de carbapénèmases, capables d’hydrolyser l’ensemble des β-lactamines. On assiste à l’émergence de certains clones épidémiques, se distinguant par leur distribution mondiale, leur forte transmissibilité et leur capacité à persister chez les patients. L’exemple le plus parlant est le cas du clone de Klebsiella pneumoniae appartenant au « sequence type » (ST) 258 et responsable de la diffusion mondiale de la carbapénèmase KPC (Klebsiella Pneumoniae Carbapenemase) portée majoritairement par des plasmides de la famille InFIIk. Les raisons du succès de ce clone et de l’association KPC/IncFIIk/ST258 ne sont pas encore totalement élucidées. Par ailleurs, il n’existe pas de corrélation entre le niveau d’expression d’une carbapénèmase, la sensibilité in vitro de la souche vis à vis des carbapénèmes et l’efficacité clinique d’un traitement par ces molécules. Les phénomènes d’héterorésistance aux carbapénèmes sont fréquents chez les souches produisant KPC, mais l’impact clinique est inconnu. Les mécanismes de régulation de l’expression des carbapénèmases ne sont pas élucidés.Les objectifs de cette thèse résident dans l’analyse des facteurs génétiques associés à la diffusion et la persistance de clones multi-résistants ainsi que l’analyse de l’expression des β-lactamases associées.La première partie de ce travail porte sur l’analyse de l’évolution in vivo d’une souche de K. pneumoniae ST258 produisant KPC ayant persisté chez un patient pendant près de 5 ans. L’analyse comparative des génomes provenant de 17 isolats a permis de mettre en évidence une diversification génétique importante ainsi que la sélection de mutations modifiant la virulence de la souche et sa sensibilité aux antibiotiques. Afin de caractériser les raisons du succès de certains plasmides portant KPC, une analyse transcriptomique d’une souche de Escherichia coli TOP10 transformée par un plasmide multi-réplicon IncFIIk-IncFIB exprimant KPC-2, a été réalisée en présence ou non d’imipénème. Les gènes les plus exprimés dans ces conditions sont les gènes de résistance aux antibiotiques et certains gènes essentiels à la réplication du plasmide. La présence d’imipénème affecte peu la transcription des gènes plasmidiques mais induit un stress oxydatif important dans l’ensemble de la souche. Par ailleurs, l’analyse de l’expression du gène blaKPC-2 dans différentes espèces par 5’-RACE a permis de révéler que ce gène de résistance est sous la dépendance de plusieurs promoteurs, dont la force diffère selon le fond génétique. Cette caractéristique pourrait expliquer le succès de certains isoformes du transposon Tn4401 permettant une meilleure expression du gène blaKPC-2, dans certaines espèces. Les outils développés dans cette thèse ont également été appliqués à l’analyse d’un clone d’Enterobacter kobei ST125 dont la céphalosporinase naturelle ACT-28 possède une activité d’hydrolyse accrue vis à vis de l’imipénème. Enfin, l’analyse du génome de la première souche Shewanella sp. produisant une BLSE de type CTX-M-15 a permis de révéler la présence d’un nouveau variant oxacillinase chromosomique avec activité carbapénèmase, appelé OXA-535. OXA-535 est proche d’OXA-436, un autre variant carbapénèmase porté par un plasmide ayant déjà disséminé chez les Entérobactéries. L’analyse de l’environnement génétique des gènes blaOXA-535 et blaOXA-436 confirme le rôle du genre Shewanella comme progéniteur des carbapénèmases de classe D. Ce travail contribue à une meilleure compréhension de la diffusion de certains clones multi-résistants et des mécanismes contrôlant l’expression des gènes de résistance aux β-lactamines. / Multidrug resistant bacteria and in particular carbapenemase-producing Enterobacteriaceae remain a major health public challenge. Some successful clones are emerging globally, due to their high transmissibility and their ability to colonize and persist in patients over time. Genomic analyses revealed that the dissemination of KPC carbapenemase is closely related to the spread of Klebsiella pneumoniae of the sequence-type (ST) 258 and to few successful plasmids linked to IncFIIk family. However, the reasons of the association between K. pneumoniae ST258, IncFIIk plasmids and KPC that led to the rapid spread of this clone are currently unknown.Furthermore, there is no correlation between expression level of a carbapenemase-encoding gene, in vitro susceptibility to carbapenems and efficiency of a carbapenem-based treatment. Most of the time, KPC-producing K. pneumoniae exhibit a heteroresistant phenotype with carbapenems, but its clinical impact remains unknown. The mechanisms underlying the regulation of carbapenemases expression remain to be explored.The objectives of the thesis are to obtain deeper insights into genomic plasticity of carbapenemase–producing clones, and into the expression of their β-lactamases.The first part of this work was dedicated to the in vivo evolution of a single strain of KPC-producing K. pneumoniae ST258 that colonized a patient for almost 5 years. Genomic comparison of 17 isolates revealed a remarkable diversification with occurrence of several mutations with impact on bacterial virulence and susceptibility to antibiotics.Several studies extensively described the genetic structures of blaKPC-carrying plasmids, but information regarding gene expression at the whole plasmid level are lacking. Accordingly, we performed RNA-seq on Escherichia coli TOP10 transformed with an IncFIIk-IncFI blaKPC-2-carrying plasmid, with or without imipenem exposure. In both conditions, plasmid-encoded genes related to antimicrobial resistance and involved in plasmid replication were the most expressed. Imipenem exposure led to a more general response with overexpression of E. coli numerous chromosome-encoded genes involved in oxidative stress response. In addition, analysis of blaKPC-2 gene expression in several species using 5’RACE revealed the presence of several promoters whose strength depends on the bacterial genetic background. This could promote higher expression of blaKPC-2 gene and explain the association of some isoforms of Tn4401 in different species. The tools developed in the frame of this work were also applied to study a single Enterobacter kobei ST125 clone whose natural cephalosporinase (ACT-28) has increased hydrolytic activity towards imipenem. Finally, genomic analysis of the first ESBL-producing Shewanella sp. was performed. It revealed the presence of blaCTX-M-15 and blaSHV-2 genes carried on an IncA/C plasmid and a new chromosomally-encoded oxacillinase variant of OXA-48 with carbapenemase activity, called OXA-535. OXA-535 was found to be closely related to OXA-436, another carbapenemase which has recently spread in Enterobacteriaceae. Analysis of the genetic environment of both blaOXA-48-like genes confirmed the role of Shewanella spp. as progenitors of class D carbapenemases.Overall, this work contributes to a better comprehension of the diffusion of multi-drug resistant clones and of the mechanisms implicated in β-lactamase expression.

Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted

Avocado is an economically important crop with ~70% oil in its fruit tissue, which is an essential component of human diet. The steady increase in global demand for avocado production (9%/year) has drawn attention to the importance of understanding the genetic regulation of triacylglycerol (TAG) accumulation. Using RNA-seq approach, mesocarp-specific regulation and biosynthesis of TAG in developing avocado fruit was analyzed. During the period of TAG accumulation in the mesocarp, an increased expression was noted for genes mostly associated with hexose metabolism in plastids, including pyruvate kinase, relative to cytosol, which is likely associated with the need for higher pyruvate flux directed toward plastid fatty acid synthesis. A corresponding increase in expression for plastidial fatty acid synthesis genes was also noted but not for TAG assembly genes. Additionally, WRINKLED1 (WRI1), a regulatory element typically associated with seed oil biosynthesis, was also highly expressed in oil-rich mesocarp of avocado, along with two other isoforms of WRI. Transcriptomics also revealed that multiple acyltransferases that participate in rate-limiting step in TAG synthesis might be active concomitantly in mesocarp to achieve higher levels of TAG accumulation. Similar observations were previously made with transcriptome analysis of oil-rich seed and non-seed tissues. Together these data suggest a ubiquitous role for WRI1 and that a major point of regulation of oil biosynthesis in oilrich mesocarp tissue most likely occurs at the level of source and not sink. Overall, this study provides a foundation for functional genomics required to direct metabolic engineering efforts to enhance avocado oil yield.

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Unlike most other fruits that accumulate oil in seed tissues, avocado stores large amounts of oil in the form of triacylglycerol (TAG) in its mesocarp tissue. The regulation of TAG biosynthetic pathways in such nonseed tissues is poorly understood. RNA-Seq was used to identify the transcriptional networks underlying TAG biosynthesis in developing mesocarp of avocado. Deep transcriptional profiling studies revealed that several transcripts were differentially represented between the early and late developmental stages of mesocarp. The temporal expression pattern of transcripts associated with fatty acid biosynthesis in plastid coincided with increasing oil content. Furthermore, except for the transcripts that likely encode enzymes involved in the terminal step in TAG synthesis, others involved in TAG assembly in the endoplasmic reticulum were poorly expressed. This pattern of higher expression for genes involved in fatty acid synthesis but not TAG assembly was similar to previously observed pattern in other seed and nonseed tissues. Comparative analysis of transcript levels for about 1500 transcription factors, across different species and oil-storing tissues, also revealed that WRINKLED1 may be highly conserved across species but transcriptional regulation of oil biosynthesis, includes distinct tissue-specific features. In conclusion, while the biochemical pathways for oil biosynthesis in nonseed tissue are highly similar to that of seed tissues, certain distinctive modes of regulation of fatty acid biosynthesis and TAG assembly likely occur in oil-rich nonseed tissues.