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Genômica de listeria monocytogenes e transcriptômica do microrganismo na presença de óleo essencial extraído de baccharis psiadioides / Genomics of Listeria monocytogenes and transcriptomics of the microorganism in the presence of essential oil extracted from Baccharis psiadioidesPieta, Luiza January 2017 (has links)
Listeria monocytogenes é um bastonete Gram-positivo, anaeróbio facultativo, psicrotrófico, patogênico a humanos e transmitido por alimentos. É causador da listeriose, doença severa que acomete grupos de risco específicos, tais como idosos, imunocomprometidos, gestantes, crianças e recém-nascidos. Neste trabalho foi investigada a expressão diferencial de L. monocytogenes na presença de óleo essencial extraído de Baccharis psiadioides, planta da família Asteraceae popularmente chamada de “alecrim-do-campo”, “vassoura” ou “erva formiga”, utilizada pela população como planta medicinal. Além disso, os genomas de dois diferentes sorotipos de L. monocytogenes, frequentemente associados a surtos de listeriose, foram sequenciados através de plataforma MiSeq Illumina, sequências estas depositadas no GenBank, e comparados com genomas de referência. Anteriormente à execução das análises genômica e transcriptômica, foi determinada a composição do óleo essencial extraído de B. psiadioides utilizado nos experimentos, através de cromatografia gasosa com espectrômetro de massa (GC – MS), a qual demonstrou uma maior quantidade de β-pineno na fração composta majoritariamente por monoterpenos, composto este frequentemente encontrado em plantas medicinais aromáticas e apontado como um dos responsáveis pelo potencial antimicrobiano das mesmas. Os demais resultados obtidos no presente trabalho indicam que o óleo essencial testado apresenta potencial ação bacteriostática na concentração estudada, sendo que genes relacionados à virulência do microrganismo foram menos transcritos na sua presença, ao contrário do que foi observado para genes de resposta ao estresse, que apresentaram maiores níveis de transcrição nesta condição. A comparação genômica entre os genomas bacterianos sequenciados neste trabalho e as cepas referência sugere um maior número de proteínas expressas em L. monocytogenes do sorotipo 4b relacionadas à defesa e metabolismo do microrganismo, indicando mecanismos que podem estar envolvidos com a capacidade deste sorotipo estar mais envolvido nos casos humanos de listeriose. / LLiisstteerriiaa mmoonnooccyyttooggeenneess is a Gram-positive rod-shaped microorganism, facultative anaerobic, psychrotrophic, pathogenic to humans and transmitted by food. It causes listeriosis, a severe disease that affects specific risk groups such as elderly, immunocompromised, pregnant women, children and newborns. In this study, differential expression of LL.. mmoonnooccyyttooggeenneess in the presence of essential oil extracted from BBaacccchhaarriiss ppssiiaaddiiooiiddeess, a plant from AAsstteerraacceeaaee family popularly named as "alecrim-do-campo", "vassoura" or "erva formiga" used by population as a medicinal plant, was investigated. In addition, the genomes of two different LL.. mmoonnooccyyttooggeenneess serotypes, often associated with listeriosis outbreaks, were sequenced through the MiSeq Illumina platform. These sequences were deposited in GenBank and compared with reference genomes. Prior to the execution of genomic and transcriptomic analyzes, composition of the essential oil extracted from BB.. ppssiiaaddiiooiiddeess used in the experiments was determined by gas chromatography with mass spectrometer (GC-MS), which demonstrated a higher amount of β-pinene in the fraction composed mainly by monoterpenes. This compound is often found in aromatic medicinal plants and also pointed as one of those responsible for their antimicrobial potential. The other results obtained in the present study indicate that the essential oil tested has a potential bacteriostatic activity at the concentration studied, and genes related to the virulence of the microorganism were less transcribed in its presence, contrary to what was observed for stress response genes, which presented higher transcription levels on that condition. Comparative genomics between the bacterial genomes sequenced in this work and the reference strains suggests a higher number of proteins expressed in LL.. mmoonnooccyyttooggeenneess serotype 4b related to the defense and metabolism of the microorganism, indicating mechanisms that may be involved with the greater ability of this serotype to cause human listeriosis.
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Beta cell differentiation status in Type 2 DiabetesJeffery, N. January 2019 (has links)
Type 2 Diabetes (T2D) affects over 415 million people globally and is characterised by cellular stresses including: poor glucose homeostasis, dyslipidaemia, inflammation, hypoxia and ER stress. Studies in mice have shown that exposure to these stresses influences beta cell differentiation status as well as cell survival and may explain the extent of beta cell mass loss that is seen in the disease. To date, studies of altered beta cell differentiation have largely been confined to murine models. I used the EndoC-bH1 human beta cell line, along with human pancreatic tissue sections, to better characterise this mechanism in human disease. To elucidate these mechanisms, I firstly established a humanised version of cell culture techniques for the EndoC βH1 cell model and assessed the influence on cell function. Secondly, I evaluated the effects of the diabetic microenvironment on beta cell differentiation and gene expression patterns. Finally, I investigated whether a diabetomimetic microenvironment induced differences in microRNA regulation in the cells. I found that the humanised EndoC-βH1 culture techniques improved glucose sensitive insulin release in the cell model. EndoC-βH1 cells exposed to a Diabetic microenvironment showed some degree of transdifferentiation and this may be due to dysregulation of splicing factor expression. These effects may be compounded by altered microRNA regulation in response to these cell stresses. These data suggest that altered gene regulation caused by a diabetic microenvironment may alter gene regulation to produce a reversible delta-like phenotype in human beta cells.
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Bioinformatics tools for the systems biology of dysferlin deficiency / Outils de bioinformatique pour la biologie des systèmes de la déficience en dysferlineMalatras, Apostolos 13 December 2017 (has links)
Le but de mon projet est de créer et d’appliquer des outils pour l’analyse de la biologie des systèmes musculaires en utilisant différentes données OMICS. Ce projet s’intéresse plus particulièrement à la dysferlinopathie due la déficience d’une protéine appelée dysferline qui est exprimée principalement dans les muscles squelettiques et cardiaque. La perte du dysferline due à la mutation (autosomique-récessive) du gène DYSF entraîne une dystrophie musculaire progressive (LGMD2B, MM, DMAT). Nous avons déjà développé des outils bio-informatiques qui peuvent être utilisés pour l’analyse fonctionnelle de données OMICS, relative à la dyspherlinopathie. Ces derniers incluent le test dit «gene set enrichment analysis», test comparant les profils OMICS d’intérêts aux données OMICS musculaires préalablement publiées ; et l’analyse des réseaux impliquant les diffèrent(e)s protéines et transcrits entre eux/elles. Ainsi, nous avons analysé des centaines de données omiques publiées provenant d’archives publiques. Les outils informatiques que nous avons développés sont CellWhere et MyoMiner. CellWhere est un outil facile à utiliser, permettant de visualiser sur un graphe interactif à la fois les interactions protéine-protéine et la localisation subcellulaire des protéines. Myominer est une base de données spécialisée dans le tissu et les cellules musculaires, et qui fournit une analyse de co-expression, aussi bien dans les tissus sains que pathologiques. Ces outils seront utilisés dans l'analyse et l'interprétation de données transcriptomiques pour les dyspherlinopathies mais également les autres pathologies neuromusculaires. / The aim of this project was to build and apply tools for the analysis of muscle omics data, with a focus on Dysferlin deficiency. This protein is expressed mainly in skeletal and cardiac muscles, and its loss due to mutation (autosomal-recessive) of the DYSF gene, results in a progressive muscular dystrophy (Limb Girdle Muscular Dystrophy type 2B (LGMD2B), Miyoshi myopathy and distal myopathy with tibialis anterior onset (DMAT)). We have developed various tools and pipelines that can be applied towards a bioinformatics functional analysis of omics data in muscular dystrophies and neuromuscular disorders. These include: tests for enrichment of gene sets derived from previously published muscle microarray data and networking analysis of functional associations between altered transcripts/proteins. To accomplish this, we analyzed hundreds of published omics data from public repositories. The tools we developed are called CellWhere and MyoMiner. CellWhere is a user-friendly tool that combines protein-protein interactions and protein subcellular localizations on an interactive graphical display (https://cellwhere-myo.rhcloud.com). MyoMiner is a muscle cell- and tissue-specific database that provides co-expression analyses in both normal and pathological tissues. Many gene co-expression databases already exist and are used broadly by researchers, but MyoMiner is the first muscle-specific tool of its kind (https://myominer-myo.rhcloud.com). These tools will be used in the analysis and interpretation of transcriptomics data from dysferlinopathic muscle and other neuromuscular conditions and will be important to understand the molecular mechanisms underlying these pathologies.
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Génomique comparative et fonctionnelle de familles de gènes liés au métabolisme secondaire de la vigne (Vitis vinifera) et de ses proches parents / Comparative and functional genomics of gene families linked to secondary metabolism in grapevine (Vitis vinifera) and its relativesArista, Gautier 31 January 2017 (has links)
La vigne (Vitis vinifera) possède un métabolisme secondaire particulièrement riche donnant naissance à une large palette de molécules dont certaines sont impliquées dans les défenses contre les pathogènes et d'autres dans la grande diversité d’arômes qui fait la renommée des vins. L’analyse de la séquence de référence du génome de la vigne a permis de mettre en évidence une remarquable expansion de certaines familles de gènes liés au métabolisme secondaire par rapport aux autres plantes. Dans ce travail, j'ai étudié les familles gènes codant pour les cytochromes P450, dont certains sont impliqués dans la production d’arômes, les gènes codant pour les stilbènes synthases (STS), les endo-β-1,3-glucanases et les gènes de résistance de type NBS impliqués dans les défenses de la vigne. Ma thèse vise à proposer des hypothèses expliquant l’organisation structurale de ces familles de gènes et ainsi à mieux comprendre pourquoi certaines familles présentent une amplification dans le génome de la vigne. Des approches bioinformatiques ont été utilisées afin d’étudier ces différentes familles de gènes. Les gènes cytochromes P450 et gènes R de type NBS ont tout d'abord été annotés de manière manuelle dans le génome de référence de la vigne. L’expression des gènes endo-β-1,3- glucanases, STS et cytochromes P450 a été analysée en utilisant une approche transcriptomique à grande échelle. Pour ce faire, un outil a été développé durant cette thèse pour estimer le niveau d’expression des gènes à partir de données RNA-Seq disponibles dans les banques de données publiques. Parallèlement, des données de reséquençage d’ADN de 56 cépages et espèces de vigne ont été analysées, afin de déterminer les variations structurales de type CNV au sein des familles de gènes à domaine NBS et de gènes STS. Ces différents travaux ont permis de montrer que l’amplification des familles de gènes étudiées n’est pas spécifique du génome de référence mais est retrouvée dans l'ensemble du genre Vitis, mais également de mettre en évidence des variations structurales au sein des différents génomes étudiés. L'analyse de la famille STS a montré que ces gènes sont organisés en blocs de duplication, et que les gènes plus conservés sont aussi les plus exprimés. Nous avons également montré que les gènes à domaine NBS sont organisés en cluster, dont certains sont particulièrement soumis à variation. Ces travaux contribuent à une meilleure connaissance de facteurs de défense efficaces et durables ainsi que des gènes impliqués dans la synthèse d’arômes dans la vigne. Ces connaissances pourront bénéficier aux programmes de création variétale mis en œuvre à l’INRA de Colmar. / Grapevine (Vitis vinifera) has a particularly rich secondary metabolism, giving rise to a wide range of molecules, some of which are involved in defences against pathogens and others in the great diversity of aromas that make wines famous. Analysis of grapevine reference genome has shown a remarkable expansion of certain families of genes linked to secondary metabolism in comparison with the other plants. In this work, I have analysed gene families coding for cytochromes P450, some of them being involved in the production of aromas, genes coding for stilbene synthases (STS), endo-β-1,3-glucanases and NBS type resistance genes involved in grapevine defences. My thesis intends to propose hypothesis to explain the structural organisation of these families and therefore better understand why some of these families are amplified in the grapevine genome. Bioinformatic approaches have been used to study these different genes families. The cytochromes P450 and R genes of NBS type were manually annotated to improve the knowledge of these families of genes. The expression of endo-β-1,3-glucanases, STS and cytochromes P450 genes has been quantified using a large-scale transcriptomic approach. To this purpose, a tool has been developed during this thesis to estimate the level of genes expression from RNA- Seq data available in public databases. In the meantime, DNA resequencing data from 56 cultivars and grapevine species have been analysed to identify structural variations of CNV types within the genes with a NBS domain and the STS genes. These works showed that the amplification of the gene families of interest was not specific to the reference genome but occurred at the scale of the Vitis genus, but also to highlighted structural variations in different genomes. Regarding the STS genes, blocks of duplication and more conserved and expressed genes were identified. For the genes with NBS domain, a clustered organisation has been highlighted with some clusters varying more than others in the studied genotypes. These works contribute to a better knowledge of gene families for efficient and durable defence against pathogens and optimal aromas synthesis in grapevine. This knowledge will benefit to breeding programs currently in progress at INRA Colmar.
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Defining the transcriptional and epigenetic signature of mouse embryonic stem cells with compromised developmental potencySchacker, Maria Anna January 2019 (has links)
Mouse embryonic stem (ES) cells have played a crucial role in studying developmental processes and gene function in vivo. They are extremely useful in the generation of transgenic animals as they can be genetically manipulated and subsequently microinjected into blastocyst stage embryos, where they combine with the inner cell mass and contribute to the developing embryo. Some of the resulting pups are chimaeric, consisting of a mixture of cells derived from the host blastocyst and the injected ES cells. We have identified several ES cell clones arising from gene targeting experiments with an impaired capacity to generate viable chimaeras. When injected into blastocysts, these clones cause embryonic death during mid to late gestation, suggesting that the cells are able to contribute to the embryo but interfere with normal embryonic development. The aim of this work was to identify the underlying changes in the transcriptome, epigenome or cell surface markers that have occurred in these compromised ES cells and to further define the developmental phenotype of the chimaeric embryos. Different stages during development were analysed and whereas there was little difference in embryonic death at gestational day e13.5, there was a significant decrease in embryos surviving to gestational day e17.5. Additionally, severe haemorrhaging was observed in all the dead embryos and small foci of haemorrhaging could also be seen in a number of embryos that were still alive. This was also observed at e13.5, albeit to a less severe extent. Using RNA sequencing to discover differences in the transcriptome between control ES cells and the compromised ES cells, five genes were identified that were downregulated in the compromised cells. Four of these, Gtl2, Rtl1as, Rian and Mirg are all located in the imprinted Dlk1-Dio3 region on chromosome 12 and are normally expressed from the maternal genome. This pattern was also validated in tissues from e17.5 chimaeric embryos. The expression of this locus is to a large extent regulated by a differentially methylated region located approximately 13kb upstream of the Gtl2 promoter, the IG-DMR. Whereas this is usually only methylated on the paternal copy, in the compromised ES cells both the paternal and the maternal copy were fully methylated, likely causing the silencing of Gtl2, Rtl1as, Rian and Mirg. Using the DNA methyltransferase inhibitor 5-azacytidine, expression of Gtl2 could be rescued. Injection of those 5-azacytidine treated cells into blastocysts did partially rescue the embryonic lethal phenotype. Additionally, cell surface markers were analysed in a phenotypic screen using phage display. NGS analysis of the phage outputs indicates that there may be additional differences in cell surface markers between the control and compromised ES cell clones, but their specific details remain to be identified. Overall, we have identified the maternally expressed genes of the Dlk1-Dio3 region as markers that can distinguish between ES cells with normal or compromised developmental potency and propose to include these genes in the pre-blastocyst injection screening routine for experiments involving the production of chimaeras or genetically modified mouse strains.
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Computational analysis and method development for high throughput transcriptomics and transcriptional regulatory inference in plantsGuo, Wenbin January 2018 (has links)
RNA sequencing (RNA-seq) technologies facilitate the characterisation of genes and transcripts in different cell types as well as their expression analysis across various conditions. Due to its ability to provide in-depth insights into transcription and post-transcription mechanisms, RNA-seq has been extensively used in functional genetics and transcriptomics, system biology and developmental biology in animals, plants, diseases, etc. The aim of this project is to use mathematical and computational models to integrate big genomic and transcriptomic data from high-throughput technologies in plant biology and develop new methods to identify which genes or transcripts have significant expression variation across experimental conditions of interest, then to interpret the regulatory causalities of these expression changes by distinguishing the effects from the transcription and alternative splicing. We performed a high resolution ultra-deep RNA-seq time-course experiment to study Arabidopsis in response to cold treatment where plants were grown at 20<sup>o</sup>C and then the temperature was reduced to 4<sup>o</sup>C. We have developed a high quality <i>Arabidopsis thaliana</i> Reference Transcript Dataset (AtRTD2) transcriptome for accurate transcript and gene quantification. This high quality time-series dataset was used as the benchmark for novel method development and downstream expression analysis. The main outcomes of this project include three parts. i) A pipeline for differential expression (DE) and differential alternative splicing (DAS) analysis at both gene and transcript levels. Firstly, we implemented data pre-processing to reduce the noise/low expression, batch effects and technical biases of read counts. Then we used the limma-voom pipeline to compare the expression at corresponding time-points of 4<sup>o</sup>C to the time-points of 20<sup>o</sup>C. We identified 8,949 genes with altered expression of which 2,442 showed significant DAS and 1,647 were only regulated by AS. Compared with current publications, 3,039 of these genes were novel cold-responsive genes. In addition, we identified 4,008 differential transcript usage (DTU) transcripts of which the expression changes were significantly different to their cognate DAS genes. ii) A TSIS R package for time-series transcript isoform switch (IS) analysis was developed. IS refers to the time-points when a pair of transcript isoforms from the same gene reverse their relative expression abundances. By using a five metric scheme to evaluate robustly the qualities of each switch point, we identified 892 significant ISs between the high abundance transcripts in the DAS genes and about 57% of these switches occurred very rapidly between 0-6h following transfer to 4<sup>o</sup>C. iii) A RLowPC R package for co-expression network construction was generated. The RLowPC method uses a two-step approach to select the high-confidence edges first by reducing the search space by only picking the top ranked genes from an initial partial correlation analysis, and then computes the partial correlations in the confined search space by only removing the linear dependencies from the shared neighbours, largely ignoring the genes showing lower association. In future work, we will construct dynamic transcriptional and AS regulatory networks to interpret the causalities of DE and DAS. We will study the coupling and de-coupling of expression rhythmicity to the Arabidopsis circadian clock in response to cold. We will develop new methods to improve the statistical power of expression comparative analysis, such as by taking into account the missing values of expression and by distinguishing the technical and biological variabilities.
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Comparative Studies of Fungal Dimorphism in DikaryaTeeratas Kijpornyongpan (7887371) 20 November 2019 (has links)
<p>Fungi display
diverse growth forms. Some grow as unicellular yeasts, some grow as
multicellular hyphae, while others switch between these two growth forms, i.e.,
the dimorphic fungi. Dimorphism is found in many pathogenic fungi, and it is
thought to be a strategy to maximize their fitness during different stages of
life cycles. The corn smut fungus <i>Ustilago maydis</i> serves as a renowned
model organism for studying fungal dimorphism and its role in pathogenesis. However, knowledge
only from the model species may not be expanded to other species unless
multispecies studies have been demonstrated. In this dissertation, I performed
comparative analyses to examine if knowledge from <i>U. maydis</i> is
translational to other dimorphic fungi. First, a physiological study was
conducted to find what can serve as a common signal for dimorphic transition of
several Ustilaginomycotina species. I found that the lipid serves as a potential
common cue for yeast-to-hyphal transition in most dimorphic species, while alternate
types of energy-source carbohydrate do not affect fungal dimorphism. In
addition, pectin and high temperature can also trigger filamentous growth in
some Ustilaginomycotina species. Second, I performed comparative
transcriptomics to determine if a mechanism for yeast-to-hyphal dimorphic
transition is conserved across multiple dimorphic species. Three species of
Ustilaginomycotina (<i>U. maydis</i>, <i>Tilletiopsis washingtonensis </i>and <i>Meira
miltonrushii</i>) plus one species from Ascomycota (<i>Ophiostoma novo-ulmi</i>)
were included in the analyses. I found that the similarity of transcriptomic
alteration is not dependent on phylogenetic relatedness. Genes in amino acid transport
and metabolism, energy production and conversion and cytoskeleton are commonly
altered during the dimorphic transition of all studied species. Moreover, I
discovered several core genes which can play a conserved role in transducing
signals for the dimorphic transition. Finally, I performed comparative analyses
of 190 fungal genomes to determine genomic properties that are associated with
types of fungal growth form. I found that small genome size is a characteristic
for yeast-like fungi. Few indicator genes, such as genes encoding proteins in
the NADPH oxidase complex and cytoskeletons, which are predominantly lost in
yeast-like fungi in both Ascomycota and Basidiomycota. However, many other
genes are associated with types of growth form in a lineage-specific manner. Findings
from this dissertation will serve as fundamentals for future research in fungal
cell biology, especially in fungal dimorphism. Additionally, results from this
study suggest cautions when extrapolating results from model species onto non-model
species.</p>
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Cisco Science: Using Omics To Answer A Range Of Key QuestionsLachance, Hannah 01 January 2019 (has links)
Coregonines, including cisco (Coregonus artedi), kiyi (Coregonus kiyi), and bloater (Coregonus hoyi), are a focus for prey fish conservation and restoration efforts throughout the Laurentian Great Lakes. However, fundamental questions about coregonine ecology and genetics remain. For example, we know little about how the early life stages of coregonines respond to environmental change at either the genotypic or phenotypic level. We also have limited knowledge about how to identify different species at the larval stage and the genetic relationships among species, which makes the different species difficult to study at the larval stage. To increase the probability for success in restoration efforts, current and future research need to integrate traditional and novel approaches to better understand what leads to current and future coregonine successes. We used DNA and RNA omics tools, genomics and transcriptomics to boost our comprehension of current coregonine populations and to help understand how C. artedi may respond to environmental change. During the winter of 2017, we conducted a pilot experiment to evaluate how C. artedi eggs may respond to increased light exposure resulting from current and expected reductions in annual ice and snow cover due to global warming. We used transcriptomics to assess differences in gene expression between a continuous light and continuous dark treatment. Our results indicate that light is an environmental factor that could lead to earlier hatch dates, smaller yolk sacs, changes in mortality and differential gene expression in metabolic related and other functionally important genes. In 2018, we sampled larval coregonines in the Apostle Islands of Lake Superior each week from hatch in May until late July. We used genomic sequencing to genetically identify 197 larvae to species: C. artedi, C. hoyi, and C. kiyi. The larval demographic characteristics of each species was assessed and revealed that length ranges, growth rates, yolk sac condition, and effective population size varied among species. Larvae of all three species were found throughout the entirety of the Apostle Islands and the genetic diversity within each species appears high. The results from our pilot experiment and field observations help advance our understanding of the important early life stages of coregonines and how changes in light exposure or growth rates could affect their success or failure in a changing climate.
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High thoughput study of biofilm and virulence in Listeria monocytogenes using innovative approaches / Étude à haut débit du biofilm et de la virulence de Listeria monocytogenes en utilisant des approches innovantesLee, Bo-Hyung 28 May 2019 (has links)
Listeria monocytogenes est un pathogène d'origine alimentaire à multiples facettes caractérisé par sa capacité d'adaptation dans des conditions défavorables et par sa prolifération dans une vaste gamme d'environnements, du sol aux cellules hôtes des mammifères. L'hétérogénéité génétique de L. monocytogenes se reflète dans sa structure clonale diversifiée, ce qui corrèle, dans une certaine mesure, avec des traits phénotypiques tels que la virulence ou la résistance au stress. La thèse portait sur deux phénotypes les plus éminents, la formation d'un biofilm et le potentiel de virulence, sous différents angles et à l'aide des technologies les plus récentes. Tout au long des études, des grands panels d'isolats ont été utilisés pour représenter la diversité intraspécifique. Stimulants défavorables tels que le choc froid et la privation d'éléments nutritifs induits par l'étape d'adhésion bactérienne. L'ajout de NaCl aux cultures de croissance a stimulé la production de biofilm et, de manière surprenante, il a considérablement intensifié la maturation du biofilm de cellules privées de nutriments. Un degré élevé de variation de la productivité relative du biofilm a été observé parmi les sérotypes, les génotypes, de même que les isolats selon les conditions de culture. Cependant, un certain génotype (complexe clonal 26) a révélé de manière caractéristique une production de biofilm plus élevée à froid (10°C), suggérant une association du génotype avec le phénotype du biofilm. Pan-GWAS a identifié un certain nombre de gènes parmi lesquels ceux impliqués dans des fonctions telles que la ‘transformation/compétence’, les ‘gènes liés aux phages’ et le ‘métabolisme du phosphate’ devront faire l'objet d'études plus approfondies sur leur rôle dans la formation du biofilm. L'analyse du séquençage de l'ARN a révélé une grande hétérogénéité intraspécifique dans les profils de transcriptome basal qui mettaient en évidence le rôle du réseau de régulation, y compris certains facteurs transcriptionnels avec des rôles clés dans la virulence tels que σB, PrfA, et CodY. La plasticité transcriptomique entre les lignées I et II ainsi que les génotypes hyper et hypovirulents ont confirmé les caractéristiques évolutives et épidémiologiques de L. monocytogenes. De plus, la voie métabolique centrale a été impliquée dans l'infection dans le système modèle de Galleria mellonella. En conclusion, la thèse a exploré la diversité intraspécifique de L. monocytogenes et a donné lieu à de nombreux résultats phénotypiques, génomiques et transcriptomiques. Grâce à l'approche intégrative des omiques en listeriologie, le présent travail contribuera à dévoiler la physiologie et la pathogenèse de la bactérie. / Conditions and proliferation in a wide range of environments from soil to mammalian host cells. The genetic heterogeneity in L. monocytogenes is reflected on its diversified clonal structure which correlates, to some extent, with phenotypic traits such as virulence or stress resistance. The thesis investigated two most prominent phenotypes, biofilm formation and virulence potential, from various perspectives using state-of-the art technologies. Throughout the studies, large panels of isolates were used to represent the intraspecific diversity. Unfavourable stimuli such as cold shock and nutrient deprivation induced bacterial adhesion step. Addition of NaCl to growth cultures stimulated biofilm production and, surprisingly, it significantly intensified biofilm maturation of nutrient-deprived cells. High degree of variation in relative biofilm productivity was observed among serotypes, genotypes, as well as isolates across culture conditions, however, certain genotype (clonal complex 26) revealed distinctively higher biofilm production under cold temperature (10°C) suggesting an association of genotype with biofilm phenotype. Pan-GWAS identified a number of genes among which those implicated in functions such as ‘transformation/competence’, ‘phage-related genes’, and ‘metabolism of phosphate’ will need further investigations for their roles in biofilm formation. RNA sequencing analysis revealed high intraspecific heterogeneity in basal transcriptome profiles that featured the role of regulatory network including certain transcriptional factors with key roles in virulence such as σB, PrfA, and CodY. The transcriptomic plasticity between lineage I and II as well as hyper- and hypovirulent genotypes supported the evolutionary and epidemiological characteristics of L. monocytogenes. Moreover, the central metabolic pathway was implicated in the infection in Galleria mellonella model system. Conclusively, the thesis explored intraspecific diversity in L. monocytogenes and resulted in ample phenotypic, genomic, and transcriptomic findings. With the integrative omics approach in listeriology, the present work will contribute to unveiling the physiology and pathogenesis of the bacterium.
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The significance of feedback de-excitationKülheim, Carsten January 2005 (has links)
<p>During photosynthesis sunlight is absorbed by photosynthetic pigments and converted into organic compounds, such as carbohydrates. Photosynthesis needs to be highly regulated, since both too much and too little light are harmful to plant. If too little light is absorbed, a plant cannot store enough energy, which will have effects on growth and fitness of the plant. With too much light absorbed, a dangerous side reaction of photosynthesis, the production of reactive oxygen species can happen. These reactive oxygen species can damage the proteins in the chloroplast and the lipids of the chloroplast.</p><p>To avoid the production of reactive oxygen species, plants have evolved many mechanisms, which act on different time-scales and different levels of organization. As a first measure, when the absorbed light is exceeding the capacity for its utilization, is to switch the light-harvesting antenna from efficient light harvesting to energy dissipation. This process is called feedback de-excitation (FDE). The protein PsbS is essential for this process as well as a functioning xanthophylls cycle with the enzyme violaxanthin de-epoxidase (VDE).</p><p>I have investigated the effects of plants with changes in their ability to dissipate excess excitation energy in the model plants species Arabidopsis thaliana. Three genotypes with either increased or decreased capacity for FDE were used during my experiments. The first genotype over-expresses the PsbS gene, having approximately two-fold increased amounts of PsbS and FDE. The second is a PsbS deletion mutant with no PsbS protein and no FDE. The third genotype cannot perform the conversion of violaxanthin to zeaxanthin, because the enzyme VDE is missing. This mutant has some FDE left. </p><p><i>Arabidopsis thaliana</i> is an annual plant, which flowers only once in its lifetime. Therefore, when counting the seeds produced an estimation of fitness can be made from the amount of seeds produced. This was done during my experiments and shown that FDE is a trait and that plants with increased FDE have a higher fitness and vice versa. </p><p>This was also the case for a collection of plants lacking a single protein from the light harvesting antenna. All of these genotypes had a fitness reduction, proving that their function is not redundant. </p><p>In an attempt to explain why the fitness is reduced in plants with altered FDE, photosynthetic measurements, as well as a determination of the transcriptome and the metabolome was performed. Plants lacking FDE had higher levels of photoinhibition, leading both to lower rates of photosynthesis and to higher repair cost. This could in part explain the reduction in fitness. These plants also had major changes in their transcriptome and their metabolome. Primary metabolism was most effected, for example carbohydrate and amino acid metabolism. But there were also changes in secondary metabolism such as an up regulation of the biosynthesis of anthocyanins.</p>
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