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1
Maynaud, Géraldine. « Adaptation aux métaux lourds de populations de rhizobia impliquées dans la phytostabilisation de déblais miniers : Identification des mécanismes d’adaptation au Zn et au Cd, et structuration des populations de rhizobia adaptées aux sites miniers ». Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20074/document.
Texte intégralRésumé :
La symbiose entre Anthyllis vulneraria et Mesorhizobium metallidurans, mise en évidence dans l'ancienne mine Zn/Pb des Avinières à St Laurent-le-Minier (Gard) permet une entrée d'azote dans les sols grâce à la fixation biologique qui favorise l'installation d'une végétation pérenne d'intérêt pour limiter la pollution métallique via l'érosion éolienne et hydrique. M. metallidurans ayant la particularité de résister à de fortes concentrations en Zn et Cd, la recherche des gènes d'adaptation aux métaux lourds a été mise en œuvre pour mieux connaitre cette bactérie impliquée dans des opérations de phytostabilisation, par des approches de génétique moléculaire et de transcriptomie (RNAseq). Les gènes codant pour des systèmes de séquestration, d'exclusion, de réduction et d'efflux des métaux lourds, dont cadA1, codant pour une PIB-ATPase exportant Cd/Zn ont été identifiés chez deux souches métallicoles associées à Anthyllis ; M. metallidurans STM 2683T (mine des Avinières) et Mesorhizobium sp. STM 4661 (mine d'Eylie). Une étude fonctionnelle de cadA1 a permis de caractériser son rôle dans la résistance aux métaux lourds chez M. metallidurans. Ce gène a ensuite été testé comme marqueur de résistance pour étudier la diversité et la répartition des symbiotes d'Anthyllis sur des sites miniers et non pollués. Pour cela ce travail a été complété par des analyses phénotypiques de tolérance au Zn et Cd et des analyses phylogénétiques basées sur des marqueurs taxonomiques et symbiotiques. La contrainte métallique exercée par les environnements miniers semble influencer la composition et la diversité bactérienne avec une plus forte proportion (i) de phénotype métallicole lié à la présence du gène cadA1 et (ii) de rhizobia appartenant à M. metallidurans ou à une espèce très proche. La plante hôte semble quant à elle influencer la diversité symbiotique des rhizobia, indépendamment de la contrainte métalliqueEfficient nitrogen-fixing symbiosis between Anthyllis vulneraria and Mesorhizobium metallidurans, identified in the highly Zn/Pb polluted mining site of Avinières (St Laurent-le-Minier, Gard county, France) has recently been described as a potential key bioremediation agent for stimulating the growth of a sustainable plant cover and thus limit heavy metal dispersion from contaminated sites. M. metallidurans strains were shown to be resistant to high Zn and Cd concentrations. The aim of our work was to identify and characterize genes involved in heavy metal adaptation in M. metallidurans by using genetic and transcriptomic approaches (RNAseq technology). Putative genes involved in heavy metal adaptation mechanisms such as exclusion, binding, reduction and efflux, like cadA1, encoding an efflux system PIB-type ATPase involved in Zn and Cd export, were identified in two Mesorhizobium strains associated with Anthyllis: M. metallidurans STM 2683T (Avinières mine) and Mesorhizobium sp. STM 4661 (Eylie mine). Functional studies allowed us to characterize the cadA1 efflux protein as involved in metal tolerance in M. metallidurans. Then, cadA1 was used as a metal-resistance marker to study the diversity and the distribution of Anthyllis symbionts from mine soils and unpolluted soils. This work was completed by Zn- and Cd-tolerance phenotype assays and phylogenetic analyses using taxonomical and symbiotic markers. Metals in mine environments seemed to influence the bacterial composition and the diversity with a high proportion of (i) metal-tolerant phenotypes consistent with the detection of the cadA1 gene and (ii) strains belonging to the M. metallidurans species or to a bacterial species close to it. The plant-hosts seemed to impact symbiotic diversity independently of the metal-tolerant property
2
Dubé, Delphine. « Différence dans la capacité de fibroblastes à être reprogrammés par le cytoplasme de l'ovocyte : étude d'une situation différentielle chez le bovin ». Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS252.
Texte intégralRésumé :
La reprogrammation, qui est la réversion d’un noyau d’un état somatique vers un état moins différencié, constitue un enjeu majeur pour la thérapie cellulaire. Cependant, les mécanismes initiaux qui président à la reprogrammation restent mal connus. Le transfert nucléaire (clonage) met à profit les propriétés de reprogrammation uniques du cytoplasme ovocytaire, et constitue une approche expérimentale intéressante pour analyser ces processus. Le but de cette thèse est d’étudier la différence de capacité de cellules fibroblastiques à être reprogrammées efficacement, en tirant partie d’une situation-modèle d'efficacité différentielle de reprogrammation après clonage chez le bovin. Ce modèle est constitué de deux lots de fibroblastes donneurs de noyaux, qui forment des embryons clonés dont la différence d’efficacité de développement à terme varie d’un facteur 8. L’analyse des cellules donneuses a montré une augmentation des anomalies de ploïdie dans les cellules à faible potentiel, et la similitude transcriptomique entre les cellules donneuses, alors que la comparaison des transcriptomes des embryonsclonés a montré des différences de reprogrammation de l’expression génique dès le stade suivant l’activation du génome embryonnaire. Des différences de méthylation de l’ADN entre cellules donneuses ont été observées dans les promoteurs de gènes candidats différentiellement reprogrammés, ainsi que dans une analyse plus globale par RRBS. Nous avons enfin étudié la distribution des cellules filles des deux premiers blastomères au stade blastocyste, la distribution « orthogonale » et l’aptitude au développement à terme des embryons de souris clonés étant liées (Liu et al., 2012). Nous avons montré l’existence de trois distributions dans les embryons fécondés mais n’avons pas observé de différence de proportions de celles-ci entre embryons bovins clonés. En conclusion, dans notre modèle, la distribution des cellules filles des deux premiers blastomères au stade blastocyste ne semble pas associée à l’efficacité de reprogrammation dans les embryons bovins clonés, contrairement aux différences épigénétiques entre cellules donneusesReprogramming, which is the return of a nucleus from a somatic state to a less differentiated state, is a major issue for cell therapy. However, the initial mechanisms governing the reprogramming remain poorly understood. Nuclear transfer (cloning) takes advantage of the unique reprogramming properties of the oocyte cytoplasm, and therefore is an interesting experimental approach to analyze these processes. The aim of this thesis is to study the difference in fibroblasts’ ability to be reprogrammed by taking advantage of a model-situation of differential reprogramming efficiency after cloning in cattle. This model consists of two batches of donor fibroblasts, which form cloned embryos having an 8 fold difference in development to term efficiency. Analysis of donor cells has shown increase ploidy abnormalities in cells of low potential, and transcriptomic similarity between the donor cells, whereas comparison ofcloned embryos transcriptomes showed gene expression reprogramming differences just after embryonic genome activation. Differences in DNA methylation between donor cells were observed in the promoters of candidate genes differentially reprogrammed and in a more comprehensive analysis by RRBS. Finally we studied the distribution of the first two blastomeres’ daughter cells at the blastocyst stage, as an "orthogonal" distribution and development to term of mice cloned embryos are linked (Liu et al., 2012). We have shown the existence of three distributions in the fertilized embryos but haven’t seen any difference of proportions between bovine cloned embryos. In conclusion, in our model, the distribution of the first two blastomeres’ daughter cells at the blastocyst stage does not seem related to the reprogramming efficiency in bovine cloned embryos, unlike epigenetic differences between donor cells
3
Laplante, Pierre. « Effect of MisMatch Repair Deficiency on metastasis occurrence modelized in a syngeneic mouse model ». Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL101.
Texte intégralRésumé :
Contexte : Le cancer colorectal (CCR) déficient en réparation des mésappariements (MMR-D), hypermuté et aux microsatellites instables (MSI) est moins enclin au développement de métastases, et présente un pronostic défavorable après récidive, par rapport au CCR aux microsatellites stables (MSS). Les mécanismes derrière ces deux phénomènes sont encore inexpliqués, principalement en raison du manque d'un bon modèle in vivo de cancer métastatique MSI.Méthodes : Nous avons inactivé le gène Msh2 dans la lignée cellulaire de cancer du sein 4T1luc+, exprimant la luciférase. Le phénotype MMR-D, marqué par une accumulation de mutations ponctuelles à l'échelle du génome et d'insertion-délétion (indels) aux sites de microsatellites, a été établi par des passages sériels in vitro avant l'injection orthotopique dans le coussinet adipeux mammaire inférieur gauche de souris BALB/c. Le développement métastatique a été suivi par imagerie in vivo de l'activité luciférase. Les tumeurs primaires des souris, ayant développé des métastases ou non, ont été séquencées, et des analyses génomiques et transcriptomiques menées. La caractérisation des cellules CD45+ a été réalisée par cytométrie en flux spectrale sur les tumeurs primaires, les sites secondaires (tissu pulmonaire), la moelle osseuse et le sang, avant et après l'implantation tumorale, afin de mieux comprendre les différents microenvironnements associés.Résultats : Une différence significative dans l'apparition des métastases a été observée entre souris porteuses de tumeurs MSS et MSI (100 % contre 82,3 %, p = 0,0456). De plus, les souris présentant des métastases MSI ont montré un développement métastatique moins prononcé à certains sites par rapport aux souris MSS. Le séquençage de l'exome complet (WXS) a révélé que la charge mutationnelle tumorale (mutations non-synonymes/megabase), les indels, ainsi que le MSIscore (pourcentage de microsatellites instables) étaient plus élevés dans les tumeurs MSI comparées aux MSS, mais ces différences n'expliquent pas l'écart de développement métastatique. Les analyses transcriptomiques ont mis en évidence que les tumeurs MSS expriment des programmes associés à la transition épithélio-mésenchymateuse (EMT), tandis que les tumeurs primaires MSI et les tumeurs métastatiques MSI étaient respectivement enrichies en signatures prolifératives et immunitaires. Il est à noter que les tumeurs métastatiques MSI étaient spécifiquement enrichies d'une signature hybride EMT, un indicateur d'agressivité tumorale. Par ailleurs, la cytométrie spectrale a permis d'identifier des populations spécifiques de macrophages et de neutrophiles associés aux tumeurs MSI (TAMs-MSI et TANs-MSI) tant dans les tumeurs primaires que sur les sites métastatiques. Enfin, nous avons découvert que l'infiltration de TANs-MSI (mais pas de TAMs-MSI) au niveau des sites secondaires précédait le développement métastatique et pourrait contribuer à la formation d'une niche pré-métastatique spécifique au MSI.Conclusion : Nous avons établi un modèle de cancer métastatique MSI, reproduisant les caractéristiques principales de la maladie, telles qu'une accumulation de mutation et d'indels élévé, ainsi qu'un potentiel métastatique diminué. Nos résultats montrent que la progression métastatique dans ce modèle MSI est associée à une réduction de l'activité immunitaire à la tumeur primaire, et que les tumeurs métastatiques MSI présentent un enrichissement en signatures prolifératives et en signatures hybrides EMT, pouvant ainsi expliquer le mauvais pronostic observé après récidive chez les patients. De plus, ce modèle a mis en lumière des populations myéloïdes spécifiques aux tumeurs MSI ainsi qu'une niche pré-métastatique potentielle liée au MSI. Ce modèle constitue ainsi un outil précieux pour la recherche fondamentale et translationnelle sur le cancer métastatique MSI, tout en apportant de nouvelles perspectives sur les interactions entre le développement métastatique et le phénotype MSIBackground: It is established that mismatch repair deficient (MMR-D), hypermutated and microsatellite instable (MSI) colorectal cancer (CRC) is less prone to metastasis, and has worse prognosis after recurrence, than microsatellite stable (MSS) CRC. The mechanisms behind both these phenomena are still elusive, mainly due to the lack of a good in vivo model of metastatic MSI cancer.Methods: We generated Msh2 knockouts in the luciferase expressing breast cancer cell line 4T1luc+. The MMR-D phenotype, an accumulation of point mutations genome-wide and insertion-deletion (indels) at microsatellite sites, was established by serial passages in vitro before orthotopic injection in the lower left mammary fat pad of BALB/c mice. Metastasis development was assessed through in vivo imaging of luciferase activity. Primary tumors of mice bearing metastasis, or not, were sequenced, and genomic and transcriptomic analysis were performed. Spectral flow cytometry of CD45+ cells was carried out on primary tumors, secondary site (lung tissue), bone marrow and blood before and after tumor engraftment, to characterize the different microenvironments.Results: A significant difference in metastasis occurrence was noted between the MSS and MSI tumor-bearing mice (100% vs 82.3%, p = 0.0456). Moreover, MSI metastatic mice exhibited less site-specific metastatic development compared to their MSS counterparts. Whole exome sequencing (WXS) revealed that tumor mutation burden (TMB, non-synonymous mutations/megabase), indels, and MSIscore (percentage of instable microsatellites) were elevated in the MSI tumors compared to MSS, but did not explain the difference in metastasis occurrence. Transcriptomic analyses demonstrated that MSS tumors express epithelial-mesenchymal transition (EMT) related programs, while MSI metastatic and MSI localized primary tumor were enriched in proliferative and immune related signatures, respectively. Interestingly, MSI metastatic tumor were uniquely enriched with a hybrid EMT signature, a marker of cancer aggressiveness. Furthermore, spectral cytometry uncovered MSI-specific populations of tumor-associated macrophages and neutrophils (MSI-TAMs and MSI-TANs) at the primary tumor and metastatic sites. Finally, we discovered that MSI-TANs (but not MSI-TAMs) infiltration at secondary site predate metastatic development, and may participate in the creation of an MSI-specific pre-metastatic niche.Conclusion: We successfully developed a model of MSI metastatic cancer, recapitulating the hallmarks of the disease, namely elevated TMB, indels and MSIscore, as well as decreased metastatic potential. We have shown that metastatic development in our MSI model is associated with lower immune activity at the primary tumor, and that MSI metastatic tumors are enriched in proliferative and hybrid EMT signatures, that may explain the poor prognosis after recurrence in patients. At last, this model unveiled MSI-specific tumor-associated myeloid populations, and a potential MSI pre-metastatic niche. We provide here a model for fundamental and translational research on MSI metastatic disease, and provide insights on the crosstalk between metastasis development and the MSI phenotype
4
Bouguyon, Eléonore. « Etude de la signalisation nitrate dépendante du transcepteur NRT1.1 chez Arabidopsis thaliana ». Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20081.
Texte intégralRésumé :
Les plantes sont capables de percevoir dans leur environnement la disponibilité en nitrate (NO3-), un macro-nutriment essentiel. Chez Arabidopsis thaliana, le transporteur de NO3- NRT1.1 constitue un système de perception qui active de nombreuses réponses au NO3-, notamment la régulation de l'expression de gènes et le développement des racines latérales. Dans ce dernier cas, un mécanisme de transduction du signal a été proposé. Celui-ci met en jeu une activité de transport d'auxine par NRT1.1 qui est inhibée par le NO3-. Cependant, le(s) mécanisme(s) moléculaire(s) permettant à NRT1.1 de contrôler un large panel de réponses au NO3- reste(nt) largement inconnu(s). L'objectif de ce travail était donc d'approfondir nos connaissances sur les voies de signalisation du NO3- dépendantes de NRT1.1. Grâce à l'analyse de mutants et de lignées transgéniques exprimant des versions de NRT1.1 présentant des mutations ponctuelles, nous avons pu découpler certaines des réponses NRT1.1-dépendantes et montré que cette protéine peut percevoir/transduire le signal NO3- au travers d'au moins trois mécanismes distincts, possédant des bases structurales différentes au sein de la protéine. D'autre part, ce travail a permis de valider l'hypothèse selon laquelle NRT1.1, en intervenant comme transporteur d'auxine, contrôle directement le développement des racines latérales, et ce indépendamment des autres transporteurs d'auxine qui y sont exprimés. Enfin, nous avons montré qu'en plus de sa régulation transcriptionnelle déjà connue, NRT1.1 est soumis à une puissante et complexe régulation post-transcriptionnelle. En effet, le transcrit NRT1.1 est stabilisé en présence de NO3- dans la racine alors que l'accumulation de la protéine NRT1.1 est réprimée par le NO3- spécifiquement au niveau des primordia de racines latérales. Les résultats obtenus au cours de ce travail ont permis d'élaborer un modèle cohérent du rôle de signalisation joué par NRT1.1, et ouvrent de nombreuses perspectives pour comprendre comment, chez les plantes, un « transcepteur » (transporteur/senseur) peut contrôler une vaste gamme de réponses adaptatives aux facteurs de l'environnementPlants are able to sense the external availability of nitrate (NO3-), a major macro-nutrient. In Arabidopsis thaliana, the NO3- transporter NRT1.1 acts as a sensor that triggers many different adaptive responses, including the regulation of gene expression and lateral root development. In the latter case, a transduction mechanism that involves a NO3--inhibited auxin transport activity dependent of NRT1 has been proposed. However, the molecular mechanism(s) allowing NRT1.1 to control such a large palette of NO3- responses is still largely unknown. Thus the aim of this work was to better understand and characterize the NRT1.1-dependent NO3- signaling pathway(s). Using mutants and transgenic lines expressing point mutated forms of NRT1.1, we uncoupled several of the NRT1.1-dependent responses and thus demonstrated that NR1.1 can sense/transduce NO3- signal through at least three distinct mechanisms at the protein level. This work also largely confirmed the hypothesis that NRT1.1 directly controls lateral root development through its auxin transport activity regardless of the other auxin transporters expressed in lateral root primordia. Finally, we showed that, besides the already well characterized transcriptional NO3--dependent regulation of NRT1.1, this gene is also subjected to complex post-transcriptional regulations. Indeed, on the one hand, NRT1.1 mRNA is stabilized by NO3- in roots whereas, on the other hand, protein accumulation is specifically repressed by NO3- in lateral root primordia. Altogether, these results allowed us to build a comprehensive model of the complex NRT1.1 signaling and open many perspectives to understand how plant “transceptors” (transporter/sensor) can monitor a large variety of adaptive responses to environmental factors
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King, Eoghan. « Caractérisation phénotypique et moléculaire de la réponse du riz au cours de l’interaction avec des espèces de Burkholderia s.l ». Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTG079.
Texte intégralRésumé :
Dans les conditions naturelles, les plantes interagissent avec une grande diversité de microorganismes, entretenant avec elles des types d’interactions variées allant du mutualisme à la pathogénie. Quelque soit le type d’interaction mis en place, les plantes sont capables de reconnaitre des motifs moléculaires microbiens conservés qui induisent l’activation d’une réponse immunitaire dite « non-hôte ». Cette réponse immunitaire basale a largement été étudiée dans le cas des interactions avec des microorganismes mutualistes et pathogènes. Cependant, dans le cas des « symbioses associatives » avec des rhizobactéries ou des bactéries endophytes bénéfiques, regroupées sous le terme de Plant Growth-Promoting Rhizobacteria (PGPR), les réponses immunitaires et physiologiques des plantes ont très peu été décrites. Dans ce contexte, ce projet de thèse a eu pour objectif de décrire les régulations transcriptionnelles de la monocotylédone modèle, le riz, en réponse à l’interaction avec des bactéries bénéfiques -rhizosphériques ou endophytes- et pathogènes du genre Burkholderia sensu lato (s.l.). Ce genre ubiquiste de béta-protéobactéries a la particularité d'avoir été subdivisé en deux genres aux écologies distinctes : le genre Paraburkholderia regroupant des espèces environnementales et bénéfiques des plantes, et le genre Burkholderia sensu stricto (s.s.) qui regroupe des espèces opportunistes et pathogènes de l'homme ainsi que des pathogènes de plantes, mais aussi des espèces phytobénéfiques (comme B. vietnamiensis). L’analyse par RNA-Seq de la réponse transcriptomique du riz à trois souches endophytes, Paraburkholderia kururiensis M130, Burkholderia vietnamiensis TVV75 et Paraburkholderia phytofirmans PsJN, ont permis de mettre en évidence des régulations physiologiques contrastées en fonction de la souche inoculée. De plus, des analyses comparatives de la colonisation des tissus racinaires par ces souches ont pu associer certaines de ces régulations à différents modes de colonisation. Enfin, l’expression de gènes impliqués dans la réponse immunitaire des plantes, identifiés par l’analyse fonctionnelle des transcriptomes, a été mesurée au cours de cinétiques d’interaction avec une plus large diversité de souches. Pour cela, dix souches de Burkholderia s.l., comprenant trois souches pathogènes, ainsi que trois souches PGPR modèles du riz des genres Azospirillum, Herbaspirillum et Pseudomonas ont été choisies. Cette dernière approche a permis de mettre en évidence des régulations transcriptionnelles associées à des types de colonisation, rhizosphérique et endophytique, ou d’interactions, bénéfiques et délétères. Ces travaux s’intègrent dans la caractérisation des bases moléculaires de la réponse des plantes aux microorganismes bénéfiques qui représentent un potentiel important pour le développement de solutions agronomiques durables favorisant la nutrition et la résistance des plantes aux maladiesIn natural conditions, plants interact with a large diversity of microorganisms maintaining with them various types of interaction ranging from mutualism to pathogenesis. Whatever the type of interaction established, the plants are able to recognize conserved microbial molecular motifs which trigger a so-called “non-host” immune response when perceived. This basal immune response has been extensively studied in the case of interactions with mutualistic and pathogenic microorganisms. However, in the case of “associative symbiosis” with beneficial rhizobacteria or bacterial endophytes, grouped under the term Plant Growth-Promoting Rhizobacteria (PGPR), the immune and physiological responses of plants have been scarcely described. In this context, this thesis project aimed at describing the transcriptional regulations of the model monocotyledonous rice, in response to the interaction with beneficial -rhizospheric or endophytic- and pathogenic bacteria of the genus Burkholderia sensu lato (s.l.). This ubiquitous genus of beta-proteobacteria has the particularity of having been subdivided into two genera with distinct ecologies: the genus Paraburkholderia, which groups together environmental and plant-associated species, and the genus Burkholderia sensu stricto (s.s.), which groups together human opportunistic and pathogenic species but also phytobeneficial species such as B. vietnamiensis. RNA-Seq analysis of the transcriptomic response of rice to three endophytic strains, Paraburkholderia kururiensis M130, Burkholderia vietnamiensis TVV75 and Paraburkholderia phytofirmans PsJN, revealed contrasting physiological regulations depending on the inoculated strain; in addition, comparative analyses of root tissue colonization by these strains enabled to associate some of these regulations with different colonization patterns. Finally, the expression of genes involved in the immune response of plants, identified by the functional analysis of transcriptomes, was measured during interaction kinetics with a wider diversity of strains. For this, ten strains of Burkholderia s.l., comprising three pathogenic strains, as well as three model rice PGPR strains of the genera Azospirillum, Herbaspirillum and Pseudomonas were selected. This last approach highlighted transcriptional regulations associated with the types of colonization, rhizospheric and endophytic, or interaction, beneficial and deleterious.This work is part of the characterization of the molecular bases of plants’ response to beneficial microorganisms which represent an important potential for the development of sustainable agronomic solutions favoring nutrition and plant resistance to diseases
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Nociti, Ricardo Perecin. « Transcrição em embriões bovinos produzidos in vitro / ». Jaboticabal, 2018. http://hdl.handle.net/11449/157385.
Texte intégralRésumé :
Orientador: Vera Fernanda Martins Hossepian de LimaResumo: O processo transcricional em embriões extremamente complexo, nosso trabalho estimou o impacto de perturbações nos processos de transcricionais, durante as fases de ativação do genoma embrionário sobre o desenvolvimento embrionário in vitro de embriões; analisamos dados de sequenciamento de rna (RNA-seq) depositados nos bancos públicos (GEO) desde o estágio de oóocito até o dia 19 do desenvolvimento embrionário; Isolamos e caracterizamos a massa celular interna (ICM) e a trofectoderma (TE) do sexo masculino e feminino, oriundos de um mesmo blastocisto produzido in vitro com espermatozoides sexados (X e Y) e com sêmen convencional e caracterizamos e exploramos o transcriptoma desses isolados celulares. Concluímos então que a EGA menor é essencial para o desenvolvimento embrionário bovino, blastocistos possuem a maior atividade transcricional de um total de 6457 genes diferentemente expressos entre os contrastes avaliado encontramos; 2065 genes diferencialmente expressos entre a ICM e a TE, enquanto a ICM está voltada para a manutenção da pluripotência, a TE está voltada ao metabolismo energético. Os nossos dados sugerem que os embriões fêmeas são mais sensíveis ao cultivo in vitro.
Abstract: Transcription process in embryos is a complex process, our work estimated the impact of perturbations in the transcriptional processes during genome activation of vitro produced bovine embryos on their development; we analyzed public data (GEO) from rna sequencing data (RNA-seq) of oocyte up to the 19th day of embryonic development; We’d performed isolation and characterization of male and female inner cell mass (ICM) and trofectoderma (TE) from the same blastocyst produced in vitro with sorted semen (X and Y) and with conventional semen. We did the characterization and exploratory analysis of the transcriptome of these cells. We conclude that minor EGA is essential for bovine embryonic development. Blastocysts possess the highest transcriptional activity of 6457 differentially expressed genes among analyzed contrasts. We found 2065 genes differentially expressed between ICM and TE, while ICM is maintaining pluripotency, TE is focused on energy metabolism. Our data suggest that female embryos are more sensitive to in vitro culture.
Doutor
7
Patin, Constance. « Exploration du dialogue entre le microbiote intestinal et l'hôte chez les enfants nés grands-prématurés à un mois de vie via des méthodes méta-omiques ». Electronic Thesis or Diss., université Paris-Saclay, 2023. http://www.theses.fr/2023UPASQ045.
Texte intégralRésumé :
La naissance prématurée (<37 semaines de gestation) est la principale cause de décès chez les enfants de moins de 5 ans dans le monde. Par conséquent, il est important de comprendre ses conséquences à court et à long terme. Alors que l'association entre la naissance prématurée et la santé à court terme a été largement étudiée, son association avec les résultats à long terme est peu connue. Chez les nourrissons prématurés et très prématurés, le microbiote intestinal est très variable et représente souvent le microbiote trouvé dans l'environnement de l'unité de soins intensifs néonatals.L'objectif de ce travail était de mieux comprendre les interactions entre les profils du microbiote intestinal et l'hôte à l'âge d'un mois chez les nourrissons très prématurés (<32 semaines de gestation ; cohorte EPIPAGE2). Les nourrissons ont été regroupés en fonction des résultats du questionnaire d'âge et d'étape de développement (ASQ) à l'âge de 2 ans.Nous avons réalisé une étude méta-omique sur des échantillons de selles de nourrissons très prématurés à l'âge d'un mois et intégré des données sur le microbiote fécal (séquençage du gène codant l'ARNr 16S), le métabolome (spectrométrie de masse en tandem LC-MS) et le transcriptome de l'hôte (RNA-seq et RT-qPCR).Chez les nourrissons nés grand-prématurés, il est possible de relier des profils de microbiote à différentes voies métaboliques ainsi qu'à des marqueurs immunitaires de l'hôte. Ces combinaisons sont elles-mêmes associées à des évolutions cliniques telles que le transit intestinal ou le neurodéveloppement ultérieur. Le microbiome à 1 mois pourrait être un indicateur non invasif de l'immaturité intestinale. Escherichia et Staphylococcus se sont avérés être les meilleurs indicateurs de maturité et d'immaturité, respectivement. Escherichia pourrait faciliter le processus de maturation intestinale chez les nourrissons prématurésPreterm birth (<37 gestational age) is the leading cause of death in children under 5 years of age worldwide. Therefore, it is important to understand its short-term and long-term consequences. While the association between preterm birth and short-term health has been extensively studied, its link to long-term outcomes remains relatively unknown. In premature and very premature infants, the intestinal microbiota is highly variable and often resembles the microbiota found in the neonatal intensive care unit environment.The aim of this study was to gain a better understanding of the interactions between intestinal microbiota profiles and the host at one month of age in very preterm infants (<32 weeks of gestation; EPIPAGE2 cohort). Infants were grouped based on the results of the Ages and Stages Questionnaire (ASQ) at the age of 2 years, focusing on developmental milestones.We performed a meta-omic study on fecal samples from very-preterm infants at 1 month of age and integrated data on the fecal microbiota (16S rRNA gene sequencing), metabolome (LC-MS), and host transcriptome (RNA-seq).In infants born very prematurely, it is possible to link microbiome profiles to various metabolic pathways as well as host immune markers. These combinations are themselves associated with clinical outcomes such as intestinal transit or later neurodevelopment. The microbiome at 1 month may serve as a non-invasive indicator of intestinal immaturity. Escherichia and Staphylococcus have proven to be the best indicators of maturity and immaturity, respectively. Escherichia might facilitate the process of intestinal maturation in preterm infants
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Jousset, Agnès. « analyse génomique et transcriptomique de bactéries productrices de carbapénèmases ». Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS527.
Texte intégralRésumé :
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-lactaminesMultidrug 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
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Gay, Elise. « Dissection transcriptomique de la biologie de Leptosphaeria maculans lors de ses interactions avec son hôte (le colza) et avec un membre du complexe d'espèces, Leptosphaeria biglobosa Large-scale transcriptomics to dissect two years of the life of a fungal phytopathogen interacting with its host plant ». Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASB015.
Texte intégralRésumé :
Leptosphaeria maculans est un champignon pathogène responsable de la nécrose du collet chez le colza. Son cycle de vie est long et complexe car composé d’une succession d’étapes de colonisation asymptomatiques puis nécrotiques des différents tissus de l’hôte, depuis la colonisation hémibiotrophe des feuilles à l’automne à la nécrose du collet au printemps. Après ces phases infectieuses dans des tissus végétaux vivants, L. maculans vit en tant que saprophyte dans les résidus de culture. Jusqu’ici, l’étude des déterminants moléculaires de la pathogénie chez L. maculans s’est concentrée sur l’analyse d’une classe spécifique de gènes, codant pour des « effecteurs », et cela durant une seule partie du cycle : l’infection précoce des feuilles. A l’heure actuelle il n’y a pas de vue d’ensemble des gènes impliqués dans la pathogénie durant tout le cycle infectieux. Aussi, le cycle de vie de L. maculans est intimement lié à celui de Leptosphaeria biglobosa, une espèce proche mais moins pathogène. L. biglobosa coinfecte le colza avec L. maculans durant tout leur cycle de vie, mais son impact sur L. maculans est mal connu. Récemment, 420 échantillons biologiques, collectés durant toutes les étapes du cycle de vie de L. maculans, ont été séquencés par RNA-Seq. Ils incluent des conditions in vitro et in planta, à la fois dans un environnement contrôlé (en coinfection ou non avec L. biglobosa) et en conditions d’infection naturelle. Les objectifs de ma thèse étaient : (i) d’identifier le plus exhaustivement possible, par des approches transcriptomiques, les gènes induits chez L. maculans durant l’entièreté de son cycle infectieux et (ii) de déterminer l’impact de l’interaction biotique avec L. biglobosa. J’ai d’abord montré que 9 % des gènes prédits chez L. maculans sont induits spécifiquement lors de l’infection. Ils sont répartis en huit vagues d’expression redécoupant le cycle tel que décrit dans la littérature et révèlent, pour certains stades, une décomposition transcriptomique encore plus complexe, qui dépend de l’organe colonisé ou du type trophique adopté par le champignon. Je démontre aussi l’importance de la régulation épigénétique puisque toutes les vagues d’expression sont enrichies en gènes localisés dans un environnement génomique de type hétérochromatinien. Lorsque le troisième partenaire, L. biglobosa, entre en jeu en début de cycle, l'expression des gènes chez L. maculans stagne et est accompagnée de l’arrêt de la colonisation. Cet effet inhibiteur est cependant restreint à des conditions de co-inoculations rarement observées lors des infections naturelles. Mon projet de thèse constitue ainsi une première description transcriptomique du cycle de vie de L. maculans dans son intégralité et en interaction avec son environnement biotiqueLeptosphaeria maculans is a pathogenic fungus responsible for the stem canker disease of oilseed rape. Its life cycle is long and complex and is composed of a succession of asymptomatic and necrotic colonisation stages of the host tissues, from the hemibiotrophic colonisation of leaves in autumn to the stem canker development in spring. After these infectious stages on living plant tissue, L. maculans survives as a saprophyte within crop residues. So far, the study of the molecular determinants of L. maculans pathogenesis was focused on a specific class of genes, encoding "effector" proteins, and focused on the early infection on leaves. At present there is no global view of the genes involved in the pathogenesis during the entire infection cycle. In addition, the life cycle of L. maculans is closely related to that of Leptosphaeria biglobosa, a closely related, less pathogenic species. L. biglobosa co-infects the oilseed rape plants along with L. maculans throughout the fungal life cycles, but its impact on L. maculans is poorly understood. Recently, 420 biological samples, collected during all stages of the life cycle of L. maculans, have been sequenced by RNA-Seq, including in vitro and in planta conditions, both in controlled environments (whether or not co-infected with L. biglobosa) or under natural infection. The objectives of my thesis were: (i) to identify as exhaustively as possible, using tanscriptomic approaches, the genes induced in L. maculans during its entire infectious cycle and, (ii) to determine the impact of the biotic interaction with L. biglobosa. I first showed that 9% of the predicted genes in L. maculans are induced specifically during infection. These are clustered into eight waves of expression, outlining the cycle as described in the literature and revealing, for some stages, a more complex transcriptomic subdivision, depending on the organ infected or on the lifestyle adopted by the fungus. I also demonstrate the importance of epigenetic regulation since all the waves of expression are enriched with genes located in a heterochromatin environment. When the third partner, L. biglobosa, coinfects the leaves at the beginning of the cycle, the gene expression in L. maculans stagnates and the plant colonisation by L. maculans is inhibited. However, this inhibitory effect is conditioned by strict conditions of co-inoculations, rarely observed during natural infections. My thesis project thus constitutes a first transcriptomic description of the life cycle of L. maculans in its entirety and in interaction with its biotic environment
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Choucair, Fadi. « Exploration du transcriptome spermatique par le séquençage nouvelle génération et le portrait épigénétique de l’infertilité masculine ». Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4058.
Texte intégralRésumé :
L’infertilité masculine est actuellement considérée comme un problème majeur qui pose une situation alarmante sur la santé publique. L’oligozoospermie, l’asthénozoospermie et la tératozoospermie sont les trois anomalies les plus connues des spermatozoïdes. Elles affectent, respectivement, la densité, la motilité et la morphologie des spermatozoïdes. Un spermatozoïde anormal est très souvent corrélé à des altérations génétiques et épigénétiques qui peuvent affecter considérablement le transcriptome. Dans ce sens, le séquençage aléatoire du transcriptome entier des spermatozoïdes ou RNA-seq constitue un outil puissant pour caractériser ces maladies. Jusqu’à présent, il n’existe aucune étude exploitant des données RNA-seq chez des hommes présentant de telles anomalies spermatiques. L’objectif principal de notre étude fût d’identifier des profils distincts des modifications du transcriptome de chaque phénotype d’infertilité pour ainsi révéler des gènes-signatures qui tamponnent une spermatogenèse pathologiquePour ce faire, les transcriptomes des spermatozoïdes de 60 sujets infertiles atteints soit d’oligozoospermie, d’asthénozoospermie ou de tératozoospermie ont été comparés à ceux de 20 patients fertiles. Ces analyses supervisées nous ont conduit à identifier: (i) les gènes clés spécifiques aux différentes anomalies des spermatozoïdes (ii) les voies de signalisation associées, (ii) les différents longs ARNs non codants dérégulés dans ces anomalies. Au niveau de l’oligozoospermie, les transcrits de spermatozoïdes dérégulés étaient associées à divers stades de la spermatogenèse, y compris le cycle cellulaire méiotique, l’assemblage du complexe synaptonémal, la cohésion des chromatides sœurs, les processus métaboliques de piRNA, le processus catabolique protéique dépendant de la voie de l’ubiquitine, à la réponse aux dommages de l'ADN et particulièrement le processus de fécondation. Quant à l’asthenozoospermia, la spermatogenèse, l’assemblage du cil, des voies métaboliques reliées à la spermatogenèse, la chimiotaxie et la physiologie des cellules immunitaires ont été significativement dérégulés. De plus, ce qui nous a intéressé au plus était l’analyse des transcrits sous-exprimés qui a permis l’identification de nombreux transcrits associées aux modifications des histones. Nous avons aussi mis en évidence une sous expression des gènes différentiellement exprimés qui définit la tératozoospermie. Cette sous expression est associée au système ubiquitine-protéasome, à l’organisation du cytosquelette, au cycle cellulaire, à la SUMOylation en réponse aux dommages de l'ADN et aux protéines de réparation ainsi qu’à de nombreux modulateurs épigénétiques. Les gènes signature de l'oligozoospermie ont été liés au processus de fécondation et les composants de la matrice extracellulaire, tandis que ceux de la tératozoospermie sont liés à la spermatogenèse et la morphogenèse cellulaire, alors que les gènes signature de l'asthénozoospermie sont impliqués dans l'assemblage du ribosome et du flagelle. En complément de cette étude, nous avons réalisé une étude très globale du paysage épigénétique du sperme des hommes infertiles. Nous avons, ainsi comparé les niveaux des espèces réactives de l’oxygène (ERO), de méthylation de l’ADN, ainsi que l’intégrité de la chromatine dans les spermatozoïdes de 30 individus infertiles avec ceux de 33 individus fertiles. Nos analyses montrent des niveaux élevés d’ERO chez les individus infertiles. Ces niveaux sont d’une part négativement corrélés avec les niveaux de méthylation globale de l’ADN et d’autre part négativement corrélés avec ceux de la 5-hydroxyméthylcytosine et de la 5-formylcytosine (intermédiaire dans le processus de déméthylation active). Ces derniers suggèrent qu’une infertilité associée au stress oxydatif conditionne l’épigénome du sperme. En conclusion, l’ensemble de notre travail apporte des ressources précieuses et originales dans la compréhension des pathologies de spermeMale infertility is actually considered as a public alarming health problem. The sperm pathologies spectrum ranges between different phenotypes including oligozoospermia, asthenozoospermia and teratozoospermia depending on the sperm conventional parameters abnormalities. Abnormal sperm is characterized by genetic alterations and epigenetic alterations which can affect the transcriptome extensively. These alterations in RNA profiles are retrospectively indicative of aberrant spermatogenic events. RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome. To date, RNA-seq analysis of sperm from infertile men has not been reported. Our objectives are: (i) recognize key clusters, key pathways and specific gene transcripts for different sperm abnormalities; (ii) catalog the spermatozoal lncRNAs in different sperm pathologies; (iii) identify signature genes which are mechanistically important in the cascade of events driving a pathological spermatogenesis; (iii) portray the global epigenetic landscape in sperm from infertile men. Expression data from 60 sperm samples from 3 groups of infertile men (oligozoospermia, asthenozoospermia, and teratozoospermia) were generated on Illumina HiSeq platform, compared to 20 fertiles, and the resulting gene expression patterns were analyzed for functional enrichment. Our supervised analyses identified numerous differentially expressed genes between fertile and infertile men. In oligozoospermia, the deregulated spermatozoal transcripts were associated with various stages of spermatogenesis including meiotic cell cycle, synaptonemal complex assembly, sister chromatid cohesion, piRNA metabolic process, ubiquitin-dependent protein catabolic process, cellular response to DNA damage stimulus and interestingly fertilization. As for asthenozoospermia, spermatogenesis, cilium assembly, metabolic-related pathways, chemotaxis and immune cell physiology were most significantly differentially expressed. Interestingly, numerous transcripts associated with histone modifications were highly down-regulated. With regards to teratozoospermia, we evidenced sperm-specific differentially expressed genes which are involved in the ubiquitin-proteasome, cytoskeleton organization, the cell cycle pathway, SUMOylation of DNA damage response and repair proteins, as well as many putative epigenetic modulators of gene expression.. We also attempted to identify distinct patterns of gene expression changes that were definite to the different abnormal sperm phenotypes in infertile men relative to controls. Signature genes of oligozoospermia were over-enriched by genes involved in fertilization and extracellular matrix components, while signature genes of teratozoospermia were enriched by genes involved in spermatogenesis and cellular components involved in morphogenesis, whilst signature genes of asthenozoospermia were enriched by genes implicated in ribosome and cilium assembly.We complemented this work by a parallel epigenetic analysis of the global epigenetic landscape in infertile men. We compared the levels of reactive oxygen species (ROS), DNA integrity and global epigenetic parameters in sperm from 33 infertile subjects with abnormal semen parameters compared to fertile individuals. We pointed out that infertile men are characterized by strikingly high levels of reactive oxygen species (ROS) which were in part negatively correlated with the global DNA methylation, and positively correlated with the levels of 5-hydroxymethylcytosine and 5-formylcytosine (active demethylation intermediates). These findings suggest that male infertility associated with oxidative stress shapes the sperm epigenetic landscape. In summary, this original work yielded a transcriptional portrait of sperm abnormalities and provided valuable resources that would further elucidate sperm pathologies
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Chaplais, Emmanuel. « Une approche de modélisation de biologie des systèmes sur la spondylarthrite ». Thesis, Versailles-St Quentin en Yvelines, 2015. http://www.theses.fr/2015VERS035V/document.
Texte intégralRésumé :
La Spondyloarthrite (SpA) est un rhumatisme inflammatoire chronique fréquent, avec une prévalence de 0,43 % en France. Elle consiste en une atteinte prédominante du squelette axial, mais aussi des articulations périphériques, et peut conduire à une immobilité du rachis et des articulations sacro-iliaques. Des atteintes extra-articulaires sont fréquentes, telles qu'une uvéite, un psoriasis ou une maladie inflammatoire chronique de l'intestin. Les traitements actuels ne sont que symptomatiques, ciblant principalement les manifestations inflammatoires. L'étiologie de la SpA est multifactorielle avec une composante génétique dominée par l'association forte et bien connue avec l'allèle HLA-B27. Cependant, ce facteur génétique n'est clairement pas suffisant pour induire le développement de la maladie. L'objectif de ce projet de thèse était donc d'identifier d'autres facteurs génétiques à l'origine du développement de la SpA.Mon travail a porté sur l'analyse de deux jeux de données complémentaires, dans une perspective de biologie des systèmes. Dans une première partie, j'ai conduit une analyse de liaison dans 210 familles atteintes de la maladie représentant 1310 personnes génotypées avec des puces Affymetrix 250k. Une nouvelle région significativement liée à la SpA a été détectée en 13q13, avec un intervalle de 1,3 Mb défini par des haplotypes recombinants chez les patients.Ensuite, une analyse transcriptomique des cellules dendritiques dérivées des monocytes de 23 patients HLA-B27+, 23 témoins sains HLA-B27+ et 21 témoins sains HLA-B27-, et stimulées ou non par du LPS, a tenté de distinguer les gènes dont l'expression est modifiée par la maladie de ceux influencés par l'allèle HLA-B27 seul. L'annotation fonctionnelle et une analyse par réseau de gènes ont mis en évidence l'inhibition chez les patients des étapes précoces de la biosynthèse du cholestérolSpondyloarthritis is a frequent chronic inflammatory rheumatism, with a prevalence of 0.43 % in France. This disease presents axial skeleton injuries, but also on peripheral joints, and can results in a total spinal and sacro-iliac motility loss. Extra-articular features including uveitis, psoriasis and inflammatory bowel disease are frequent. Current SpA treatments are only symptomatic, relieving inflammatory symptoms. SpA etiology is largely multifactorial with a genetic component dominated by the long-known strong association with the HLA-B27 allele. This allele, however, is not sufficient for the disease to occur. This thesis project objective was then to identify other genetic factors in the origin of SpA.My work was mainly divided in two complementary data analyses, in a way to get a systems biology approach. The first one consisted in proceed linking analyses on data from Affymetrix genotyping chips gathered from DNA of 1310 people grouped in 210 families. This study allowed notably to detect a new significantly linked region to SpA : 13q13, with an interval of 1.3 Mb. This part of genome is currently being sequenced to allow a better causal SNP identification.Secondly, an Affymetrix HumanGene 1.0 st transcriptomic chips analysis was performed on MD-DCs extracted from 68 people, stimulated or not by LPS during 6 or 24 hours. This cohort was grouped between 23 patients HLA-B27+, 23 healthy controls HLA-B27+ and 21 healthy controls HLA-B27-. I could notice that HLA-B27 allele is farly enough to considerably affect cell transcriptomic profiles, which encourages to include HLA-B27+ healthy controls. Otherwise, a gene network analysis allowed me to highlight on an inhibition of early steps of cholesterol biosyntthesis
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Cologne, Audric. « Exploration du rôle de l'épissage mineur dans le développement embryonnaire : modèle du syndrome de Taybi-Linder) (TALS) ». Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1190/document.
Texte intégralRésumé :
Le Syndrome de Taybi-Linder (TALS) est une maladie génétique rare affectant le développement embryonnaire, caractérisée par un nanisme microcéphalique sévère et un décès précoce des patients. Le gène muté dans ce syndrome est RNU4ATAC, qui encode un petit ARN nucléaire (snRNA) non-codant : U4atac. Ce snRNA est l’une des briques composant le spliceosome mineur, une machinerie nucléaire dédiée à l’épissage des introns U12, un groupe d’introns peu étudié car présent dans ~1 % des gènes seulement. Dans le TALS, ces introns sont fréquemment retenus dans les transcrits matures, l’épissage correct des introns U12 semble donc capital pour le développement embryonnaire. L’étude du profil transcriptomique des patients TALS permet ainsi d’établir les conséquences moléculaires d’un dysfonctionnement du spliceosome mineur, nous permettant d’en apprendre davantage sur les mécanismes d’épissage des introns U12 en condition physiologique ou pathologique, et sur le rôle de l’épissage mineur dans le développement embryonnaire. Cette thèse présente la première analyse approfondie du transcriptome de cellules provenant de patients TALS. Pour mener cette analyse, nous avons développé un pipeline bioinformatique qui, à partir de données RNA-seq de seconde génération, utilise différentes méthodes dédiées à l’étude différentielle de l’expression des gènes ou de la qualité d’épissage entre patients et contrôles. L’épissage étant particulièrement complexe à analyser à partir de reads courts, deux approches complémentaires ont été utilisées : l’une classique, basée sur l’alignement des reads, et l’autre plus originale, basée sur l’assemblage des reads et permettant de détecter plus d’événements d’épissage non-annotés (KisSplice). Une des conséquences attendue d’un dysfonctionnement du spliceosome mineur est une rétention massive des introns U12 dans les ARN matures. Cependant, la détection et la quantification de rétentions d’intron chez les mammifères constituent encore aujourd’hui un challenge bioinformatique. Nous avons donc utilisé une méthode récente dédiée à l’analyse des rétentions d’introns pour caractériser le plus précisément possible le profil transcriptomique des patients TALS. J’ai ainsi participé au développement de KisSplice et de notre outil d’analyse statistique des différentielles d’épissage, kissDE, et mis en évidence certaines caractéristiques de l’épissage mineur, que ce soit en condition physiologique ou pathologiqueThe Taybi-Linder Syndrome (TALS) is a rare genetic disorder of the embryonic development leading to a severe microcephaly, a primordial dwarfism and an early/unexpected death. The mutated gene in this syndrome is RNU4ATAC, which encode a non-coding small nuclear RNA (snRNA) named U4atac, involved in the minor spliceosome. This nuclear machinery is dedicated to the splicing of a small number of particular introns : the U12 introns. Because only about 1 % of the Human’s genes display at least one U12 intron, they have not been extensively study and little is known about their function. In TALS patients’ cells, most of the U12 introns are retained in mature transcripts ; hence, splicing of U12 introns seems important for the embryonic development. Studying TALS patients’ cells transcriptomes both in physiological and pathological conditions should enable us to precisely identify most of the molecular consequences of a minor splicing defect and could shed light on the mechanism linking minor splicing and embryonic development. This thesis is the first work to conduct an in depth analysis of TALS patients’ cells transcriptomes. In order to do a precise analysis, we developed a bioinformatic pipeline that uses multiple methods to detect differentially expressed or spliced genes between patients and controls and from second generation RNA-seq data. Splicing analysis is a very complex task complete with short reads ; hence, we used two complementary approaches. The first one is based on reads alignement to a reference genome, method conventionnally used to work on splicing, and the second one is based on reads assembly (KisSplice), a original method enabling to find more non-annotated splicing events. One of the expected consequences of a minor splicing malfunction is a global U12 introns retention in mature transcripts. However, intron retention detection and quantification in mammals is particulary difficult task in mammals, thus we used a new method dedicated to intron retentions analysis to study the transcriptomic profile of TALS patients. During my thesis, I was one of the developer of KisSplice and kissDE, our differential splicing analysis tool, and I identified important charcteristics of minor splicing either in physiological or pathological conditions
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Soares, VitÃria VirgÃnia MagalhÃes. « IdentificaÃÃo, validaÃÃo e anotaÃÃo funcional de marcadores microssatÃlites em genÃtipos de cajueiro anÃo-precoce (Anacardium occidentale var. nanum) utilizando dados de rna-seq ». Universidade Federal do CearÃ, 2016. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=17697.
Texte intégralRésumé :
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorO cajueiro (Anacardium occidentale L.) à uma planta nativa do Brasil com grande valor comercial. Isso contribui com a geraÃÃo de milhares de empregos diretos e indiretos, especialmente na RegiÃo Nordeste, em Ãpoca de estiagem. Programas de melhoramento genÃtico vem selecionando cultivares de cajueiro melhores adaptados ao ambiente semiÃrido a fim de colocÃ-lo em um mercado cada vez mais competitivo. A busca por marcadores microssatÃlites pode auxiliar os programas de melhoramento no que diz respeito ao acesso à diversidade genÃtica da espÃcie. O presente trabalho tem como objetivo a identificaÃÃo de marcadores SSR em cajueiro com base no transcriptoma de sementes e folhas, bem como sua validaÃÃo por PCR em diferentes genÃtipos comerciais. Utilizando ferramentas de bioinformÃtica foram encontrados motivos SSRs do tipo di- tri- tetra- penta- e hexanucleotÃdeos, onde o motivo do tipo trinucleotÃdeo foi o mais representativo nos transcritos do cajueiro anÃo CCP 76 e comum variando de 60 a 65%, respectivamente. Os transcritos de cajueiro comum e anÃo CCP 76 compartilham um total de 298 marcadores SSR, destes, 29 foram escolhidos para amplificaÃÃo por PCR, os quais 9 mostraram polimorfismo nos genÃtipos testados. As sequÃncias situadas prÃximas aos marcadores SSR codificam proteÃnas, que em sua maioria pertencem a famÃlias gÃnicas. Pode-se concluir que foram encontrados regiÃes contendo marcadores SSRs na regiÃo transcrita de nove genÃtipos de cajueiro, podendo ser uma ferramenta Ãtil nas anÃlises genÃticas e abrindo perspectivas para o papel endÃgeno dos SSRs na funÃÃo proteica.
The cashew tree is a native plant from Brazil with high market value. This contributes to the generation of thousands direct and indirect jobs, especially in the Northeast region, during dry season. Breeding programs have been selecting cashew cultivars that best adapt to semi-arid environment in order to fit it in the increasingly competitive market. The search for microsatellite markers can assist breeding programs regarding to access to genetic diversity of the species. This study aims to identify SSR markers in the cashew tree based on seeds and leaves transcriptome, as well as assess its validation by PCR technique in different commercial genotypes. Using bioinformatics tools, SSRs motifs from types di- tri- tetra- penta- and hexanucleotides were found. Trinucleotide-type motif was the most representative in transcripts both from dwarf cashew CCP 76 and common cashew, ranging from 60 to 65%, respectively. Transcripts from common cashew and dwarf cashew CCP 76 share a total of 298 SSR markers. 29 of these were selected for amplification by PCR, in which 9 showed polymorphism in genotypes tested. The sequences located near to the SSR markers encode proteins, which mostly belong to gene families. It can be concluded that regions containing SSRs markers were found in the transcribed region of nine cashew genotypes, and can be a useful tool in genetic analysis and open up prospects for endogenous role of SSRs in protein function.
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Troca, Heitor Madalena da Silva. « Perfil de expressão gênica de fêmeas de Aedes aegypti durante o cortejo e acasalamento ». Botucatu, 2020. http://hdl.handle.net/11449/192636.
Texte intégralRésumé :
Orientador: Paulo Eduardo Martins RibollaResumo: O Aedes aegypti é o mosquito mais sinantrópico e antropofílico da família Culicidae. Esta espécie sempre coabita com os seres humanos e é extremamente oportunista. A dispersão vetorial está diretamente relacionada à capacidade das fêmeas de encontrar um parceiro com sucesso em um cenário urbano geralmente irregular. No presente trabalho, investigamos alterações transcricionais em fêmeas de Ae. aegypti durante o processo de cortejo e após o acasalamento. Observamos uma alteração substancial na expressão gênica desencadeada logo após o contato com machos de Ae. aegypti, que por sua vez não estavam totalmente correlacionados com as alterações desencadeadas pelo contato. Após analisar genes compartilhados significativamente e diferencialmente regulados entre contato específico e inseminação, a maior parte da mudança transcriptômica observada desencadeada por contato é revertida após o acasalamento, indicando uma situação intermediária entre as condições virgens e de cortejo que supomos ser crucial para o sucesso do acasalamento. Após o contato, vários genes quimiossensoriais relacionados são reprimidos, especialmente proteínas de ligação a odorante. A maioria desses genes retorna a taxas de expressão mais altas após o acasalamento. Nenhum desses genes é significativamente regulado pelo encontro de uma espécie diferente, em nosso estudo, Aedes albopictus. Os resultados apresentados aqui podem ser aplicados a uma abordagem inovadora de controle, focada nos sistemas semioquímicos de... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Aedes aegypti is the most synanthropic and anthropophilic mosquito of Culicidae. This species always cohabits with humans and is extremely opportunistic. Vector dispersal is directly related to the ability of the females on successfully finding a mate in a generally patchy urban scenario. In the present work, we investigate transcriptional changes in Ae. aegypti females during the courtship process and after mating. We observe a substantial alteration in gene expression triggered just upon contact with Ae. aegypti males, which in turn was not fully correlated to the changes triggered by the contact. After analysing shared significant differentially regulated genes between conspecific contact and insemination, the major part of the observed transcriptomic change triggered by contact is reversed after mating, indicating an intermediary situation between naive and mating conditions that we hypothesize to be crucial for mating success. Upon contact, several chemosensory related genes are repressed, especially odorant binding proteins. Most of these genes return to higher expression rates after mating. None of these genes are significantly regulated by the encounter of a different species, Aedes albopictus. The results presented here might be applied to an innovative control approach focusing on the semiochemical systems of mosquitoes in an effort to disrupt undesirable host–insect interaction to reduce the risk of pathogen transmission to humans.
Mestre
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Choucair, Fadi. « Exploration du transcriptome spermatique par le séquençage nouvelle génération et le portrait épigénétique de l’infertilité masculine ». Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4058/document.
Texte intégralRésumé :
L’infertilité masculine est actuellement considérée comme un problème majeur qui pose une situation alarmante sur la santé publique. L’oligozoospermie, l’asthénozoospermie et la tératozoospermie sont les trois anomalies les plus connues des spermatozoïdes. Elles affectent, respectivement, la densité, la motilité et la morphologie des spermatozoïdes. Un spermatozoïde anormal est très souvent corrélé à des altérations génétiques et épigénétiques qui peuvent affecter considérablement le transcriptome. Dans ce sens, le séquençage aléatoire du transcriptome entier des spermatozoïdes ou RNA-seq constitue un outil puissant pour caractériser ces maladies. Jusqu’à présent, il n’existe aucune étude exploitant des données RNA-seq chez des hommes présentant de telles anomalies spermatiques. L’objectif principal de notre étude fût d’identifier des profils distincts des modifications du transcriptome de chaque phénotype d’infertilité pour ainsi révéler des gènes-signatures qui tamponnent une spermatogenèse pathologiquePour ce faire, les transcriptomes des spermatozoïdes de 60 sujets infertiles atteints soit d’oligozoospermie, d’asthénozoospermie ou de tératozoospermie ont été comparés à ceux de 20 patients fertiles. Ces analyses supervisées nous ont conduit à identifier: (i) les gènes clés spécifiques aux différentes anomalies des spermatozoïdes (ii) les voies de signalisation associées, (ii) les différents longs ARNs non codants dérégulés dans ces anomalies. Au niveau de l’oligozoospermie, les transcrits de spermatozoïdes dérégulés étaient associées à divers stades de la spermatogenèse, y compris le cycle cellulaire méiotique, l’assemblage du complexe synaptonémal, la cohésion des chromatides sœurs, les processus métaboliques de piRNA, le processus catabolique protéique dépendant de la voie de l’ubiquitine, à la réponse aux dommages de l'ADN et particulièrement le processus de fécondation. Quant à l’asthenozoospermia, la spermatogenèse, l’assemblage du cil, des voies métaboliques reliées à la spermatogenèse, la chimiotaxie et la physiologie des cellules immunitaires ont été significativement dérégulés. De plus, ce qui nous a intéressé au plus était l’analyse des transcrits sous-exprimés qui a permis l’identification de nombreux transcrits associées aux modifications des histones. Nous avons aussi mis en évidence une sous expression des gènes différentiellement exprimés qui définit la tératozoospermie. Cette sous expression est associée au système ubiquitine-protéasome, à l’organisation du cytosquelette, au cycle cellulaire, à la SUMOylation en réponse aux dommages de l'ADN et aux protéines de réparation ainsi qu’à de nombreux modulateurs épigénétiques. Les gènes signature de l'oligozoospermie ont été liés au processus de fécondation et les composants de la matrice extracellulaire, tandis que ceux de la tératozoospermie sont liés à la spermatogenèse et la morphogenèse cellulaire, alors que les gènes signature de l'asthénozoospermie sont impliqués dans l'assemblage du ribosome et du flagelle. En complément de cette étude, nous avons réalisé une étude très globale du paysage épigénétique du sperme des hommes infertiles. Nous avons, ainsi comparé les niveaux des espèces réactives de l’oxygène (ERO), de méthylation de l’ADN, ainsi que l’intégrité de la chromatine dans les spermatozoïdes de 30 individus infertiles avec ceux de 33 individus fertiles. Nos analyses montrent des niveaux élevés d’ERO chez les individus infertiles. Ces niveaux sont d’une part négativement corrélés avec les niveaux de méthylation globale de l’ADN et d’autre part négativement corrélés avec ceux de la 5-hydroxyméthylcytosine et de la 5-formylcytosine (intermédiaire dans le processus de déméthylation active). Ces derniers suggèrent qu’une infertilité associée au stress oxydatif conditionne l’épigénome du sperme. En conclusion, l’ensemble de notre travail apporte des ressources précieuses et originales dans la compréhension des pathologies de spermeMale infertility is actually considered as a public alarming health problem. The sperm pathologies spectrum ranges between different phenotypes including oligozoospermia, asthenozoospermia and teratozoospermia depending on the sperm conventional parameters abnormalities. Abnormal sperm is characterized by genetic alterations and epigenetic alterations which can affect the transcriptome extensively. These alterations in RNA profiles are retrospectively indicative of aberrant spermatogenic events. RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome. To date, RNA-seq analysis of sperm from infertile men has not been reported. Our objectives are: (i) recognize key clusters, key pathways and specific gene transcripts for different sperm abnormalities; (ii) catalog the spermatozoal lncRNAs in different sperm pathologies; (iii) identify signature genes which are mechanistically important in the cascade of events driving a pathological spermatogenesis; (iii) portray the global epigenetic landscape in sperm from infertile men. Expression data from 60 sperm samples from 3 groups of infertile men (oligozoospermia, asthenozoospermia, and teratozoospermia) were generated on Illumina HiSeq platform, compared to 20 fertiles, and the resulting gene expression patterns were analyzed for functional enrichment. Our supervised analyses identified numerous differentially expressed genes between fertile and infertile men. In oligozoospermia, the deregulated spermatozoal transcripts were associated with various stages of spermatogenesis including meiotic cell cycle, synaptonemal complex assembly, sister chromatid cohesion, piRNA metabolic process, ubiquitin-dependent protein catabolic process, cellular response to DNA damage stimulus and interestingly fertilization. As for asthenozoospermia, spermatogenesis, cilium assembly, metabolic-related pathways, chemotaxis and immune cell physiology were most significantly differentially expressed. Interestingly, numerous transcripts associated with histone modifications were highly down-regulated. With regards to teratozoospermia, we evidenced sperm-specific differentially expressed genes which are involved in the ubiquitin-proteasome, cytoskeleton organization, the cell cycle pathway, SUMOylation of DNA damage response and repair proteins, as well as many putative epigenetic modulators of gene expression.. We also attempted to identify distinct patterns of gene expression changes that were definite to the different abnormal sperm phenotypes in infertile men relative to controls. Signature genes of oligozoospermia were over-enriched by genes involved in fertilization and extracellular matrix components, while signature genes of teratozoospermia were enriched by genes involved in spermatogenesis and cellular components involved in morphogenesis, whilst signature genes of asthenozoospermia were enriched by genes implicated in ribosome and cilium assembly.We complemented this work by a parallel epigenetic analysis of the global epigenetic landscape in infertile men. We compared the levels of reactive oxygen species (ROS), DNA integrity and global epigenetic parameters in sperm from 33 infertile subjects with abnormal semen parameters compared to fertile individuals. We pointed out that infertile men are characterized by strikingly high levels of reactive oxygen species (ROS) which were in part negatively correlated with the global DNA methylation, and positively correlated with the levels of 5-hydroxymethylcytosine and 5-formylcytosine (active demethylation intermediates). These findings suggest that male infertility associated with oxidative stress shapes the sperm epigenetic landscape. In summary, this original work yielded a transcriptional portrait of sperm abnormalities and provided valuable resources that would further elucidate sperm pathologies
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Rosier, Florian. « Au-delà des études d'association à l'échelle du génome : étude transcriptomique et identification des variants régulateurs impliqués dans le développement du sepsis ». Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0542.
Texte intégralRésumé :
En 2017, l'Assemblée mondiale de la Santé et l'Organisation mondiale de la Santé ont fait du sepsis une priorité de santé mondiale en adoptant une résolution pour améliorer, prévenir, diagnostiquer et gérer le sepsis. Cette pathologie tue principalement des personnes déjà fragilisées. Il a été démontré que l'état du patient et la réponse immunitaire de l'hôte est primordiale dans le développement de la pathologie. Mon projet de thèse a tout d’abord consisté à caractériser la réponse transcriptionnelle de cellules du péritoine stimulées au LPS dans un modèle murin du sepsis. Par ailleurs, j’ai recherché les variants génétiques cis-régulateurs potentiellement impliqués dans la mortalité des patients en choc septique par une approche bioinformatique et une validation fonctionnelle expérimentale. Ces 2 projets mettent en évidence, chez l'homme, des mécanismes de la régulation de l’expression de gènes clés dans la survie des patients atteints de sepsisIn 2017, the World Health Assembly and the World Health Organization made sepsis a global health priority by adopting a resolution to improve, prevent, diagnose and manage sepsis. This pathology mainly kills people who are already vulnerable. It has been shown that the patient's condition and the host's immune response are essential in the development of the disease. My thesis project first consisted in characterizing the transcriptional response of peritoneal cells stimulated at the LPS in a mouse model of sepsis. In addition, I researched cis-regulatory genetic variants potentially involved in the mortality of patients in septic shock through a bioinformatics approach and experimental functional validation. These 2 projects highlight, in humans, mechanisms for regulating the expression of key genes in the survival of patients with sepsis
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Pruneau, Ludovic. « Analyse du transcriptome d'Ehrlichia ruminantium agent causal de la cowdriose : mise en évidence des gènes impliqués dans la virulence et les mécanismes d'atténuation et application à l'élaboration d'un vaccin recombinant ». Thesis, Antilles-Guyane, 2012. http://www.theses.fr/2012AGUY0562/document.
Texte intégralRésumé :
AU COURS DE LA THESE, L'ETUDE DU TRANSCRIPTOME DE SOUCHES GARDEL ET SENEGAL VIRULENTES ET ATTENUEES D'E. RUMINANTIUMA ETE REALISEE. UNE ANALYSE DU TRANSCRIPTOME A DIFFERENTS STADES DE DEVELOPPEMENT, A D'ABORD ETE EFFECTUEE POUR LA SOUCHE GARDEL VIRULENTE. AU STADE CORPS RETICULE (FORME INTRACELLULAIRE NON INFECTIEUSE), UNE SUREXPRESSION DES GENES CODANT POUR DES PROTEINES IMPLIQUEES DANS LE METABOLISME, LE TRANSPORT ET L'ECHANGE DE NUTRIMENTS ET DANS LA RESISTANCE AU STRESS OXYDATIF ETAIT OBSERVEE. IL SEMBLERAIT QUEE. RUMINANTIUMMETTE EN PLACE UN PANEL DE MECANISMES POUR SA SURVIE ET SON DEVELOPPEMENT A L'INTERIEUR DE LA CELLULE HOTE. AU STADE CORPS ELEMENTAIRE (FORME EXTRACELLULAlRE INFECTIEUSE), LE GENE DKSA CODANT POUR UN FACTEUR DE TRANSCRIPTION ETAIT SUREXPRIME. CE GENE A ETE MONTRE COMME ETANT IMPLIQUE DANS LA REGULATION DE FACTEURS DE VIRULENCE. IL SEMBLERAIT . DONC, QU'AU STADE CORPS ELEMENTAIRE, IL Y AIT UNE INDUCTION DE MECANISMES DE VIRULENCE. LA COMPARAISON DE L'EXPRESSION DES GENES AU STADE CORPS ELEMENTAIRE ENTRE SOUCHES VIRULENTES ET ATTENUEES A AUSSI ETE EFFECTUEE. NOS RESULTATS ONT MONTRE UNE MODIFICATION IMPORTANTE DE LA MEMBRANE POUR LES SOUCHES VIRULENTES ET ATTENUEES. POUR LES SOUCHES ATTENUEES, IL A ETE MONTRE UNE SUREXPRESSION DES GENES IMPLIQUES DANS LA BIOGENESE MEMBRANAlRE ET UNE SOUS-EXPRESSION·DES PROTEINES DE LA FAMILLE MULTIGENIQUE MAP. CES RESULTATS SUGGERENT QUE LES PROTEINES MAP JOUENT UN ROLE DE LEURRE VIS-A-VIS DE LA REPONSE IMMUNITAIRE PROTECTRICE. DES PROTEINES MEMBRANAlRES HYPOTHETIQUES SONT SUREXPRIMEES A LA FOIS CHEZ LES SOUCHES VIRULENTES ET ATTENUEES. CERTAINES D'ENTRE ELLES SUREXPRIMEES CHEZ LES SOUCHES ATTENUEES SEMBLENT ETRE DE BONS CANDIDATS VACCINAUX ET DEVRAIENT ETRE ETUDIEESTranscriptomic study of gardel and senegal both virulent and attenuated e. ruminantium strains was conducted during my phd. an analysis of transcriptome at different stages of development has been first conducted for virulent gardel strain. at reticulate body stage (intracellular form non-infectious), over-expression of genes coding for proteins involved in metabolism, transport and exchange of nutrients and resistance to oxidative stress was observed. at this stage of development, e. ruminantium seems to activate mechanisms for its survival and development within the host cell. at elementary body stage, dksa the gene encoding for a transcription factor was over-expressed. this gene has been shown to be involved in the regulation of virulence factors. it seems, therefore, at the elementary body stage, e. ruminantium induces its virulence factors. secondly, we compare the transcriptome of elementary body between virulent and attenuated strains. our results showed an important membrane modification of attenuated and virulent strains. for attenuated strains, we observed an over-expression of genes involved in membrane biogenesis and a diminution of expression of map multigenic family. it seems that map proteins subvert the protective immune response. hypothetical membrane proteins are over-expressed in both virulent and attenuated strains. some over-expressed proteins in attenuated strains seem to be good vaccine candidates and willstudied
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Fonseca, Larissa Fernanda Simielli [UNESP]. « Expressão gênica diferencial em tecido muscular de bovinos Nelore divergentes para qualidade de carne ». Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/137800.
Texte intégralRésumé :
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Mais de 80% da carne bovina brasileira é proveniente de animais de origem zebuína. Este produto é considerado de baixa maciez e reduzido teor de gordura entremeada quando comparado à carne de animais de raças taurinas. Investir na melhoria das características organolépticas como marmoreio e maciez da carne torna-se importante do ponto de vista econômico, pois tratam-se de atributos muito apreciados pelo consumidor. A maciez pode ser medida por meio de análises sensoriais, índice de fragmentação miofibrilar ou força de cisalhamento. Nesse último, um aparelho é utilizado para medir a força necessária para o cisalhamento de uma seção transversal de carne e, quanto maior a força dispensada, menor é a maciez apresentada pelo corte de carne. Outra característica relacionada à qualidade da carne é o marmoreio, que pode ser analisada por meio de escala de graduação visual denominada Quality Grade. No entanto, estas metodologias só podem ser utilizadas após o abate do animal. As técnicas de análise do transcriptoma, como o RNA-Seq, permitem a identificação de genes cuja expressão está relacionada com o controle de características quantitativas e podem ser alternativas na busca pelo melhoramento destes atributos. Com isso, o objetivo deste trabalho foi identificar genes diferencialmente expressos utilizando a técnica RNA-Seq, em tecido muscular de bovinos da raça Nelore, visando contribuir para o conhecimento dos fatores genéticos que estão relacionados com a qualidade da carne. Para isso, foi sequenciado o mRNA de 40 amostras divergentes para a maciez da carne (20 com carne dura e 20 com carne macia) e 40 amostras divergentes para marmoreio (20 com alto e 20 com baixo marmoreio). Essas amostras foram escolhidas com base na análise de força de cisalhamento e índice de marmorização realizadas em 132 e 80 amostras de músculo longissimus dorsi respectivamente, coletadas da meia carcaça esquerda de cada animal. As análises dos resultados obtidos pela técnica de RNA-Seq revelaram genes diferencialmente expressos relativos à maciez e marmoreio da carne em gado Nelore. Os genes referentes à maciez estão relacionados ao metabolismo de colágeno, à actina e miosina, ao transporte de cálcio, dentre outros. Por outro lado, nos resultados das análises de marmoreio, os genes que se mostraram diferencialmente expressos estão relacionados à ocitocina, ao transporte de oxigênio, ao crescimento e à síntese de lipídios e proteínas. Portanto, este estudo revelou a possibilidade de se utilizar alguns desses genes diferencialmente expressos como marcadores genéticos em bovinos Nelore para seleção precoce quanto à maciez e marmoreio da carne.
More than 80% of Brazilian beef comes from zebu animals. The Zebu meat show reduced marbling and is considered harder when compared to Taurine meat. The improvement of the organoleptic traits like marbling and meat tenderness is important from an economic point of view, because these attributes are highly appreciated by the consumer. The meat tenderness can be measured by sensory analysis, myofibril fragmentation index or by shear force. This method use an apparatus which measure the force required to shear a cross section of meat. The higher the strength dispensed, lower is the tenderness showed by the cut of meat. Another trait related to meat quality is marbling, which can be analyzed by Quality Grade visual graduation scale. However, these methods can only be performed after the animal was killed. The transcriptome analysis techniques, as RNA-Seq, are able to identify genes whose expression are related to quantitative traits control and can be used as alternative to help the improvement of these traits. Thus, the aim of this study was to identify differentially expressed genes using RNA-Seq, in Nelore cattle muscular tissue, to contribute to the knowledge of the genetic factors that are related to meat quality. We sequenced the mRNA of 40 samples divergently ranked to meat tenderness (20 with hard meat and 20 with tender meat) and sequenced the mRNA of 40 samples divergently ranked to marbling (20 with high and 20 with low marbling). These samples were chosen based on shear force and marbling content analysis held at 132 and 80 longissimus dorsi muscle samples respectively, collected from each left half carcass. The analysis of the results obtained by RNA-Seq technique revealed differentially expressed genes related to meat tenderness and marbling in Nelore cattle. Regarding the meat tenderness, these genes are related to collagen metabolism, actin and myosin, the calcium transport, among others. Moreover, the marbling analysis results, revealed differentially expressed genes related to oxytocin, transport of oxygen, growth and lipids and proteins synthesis. Therefore, this study showed the possibility of using some of these differentially expressed genes as genetic markers in Nellore cattle for early selection to meat tenderness and marbling.
FAPESP: 2013/09190-7
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Kincaid, Smith Julien. « Modification des traits d'histoire de vie au cours de l’hybridation et analyse des mécanismes moléculaires sous- jacents chez les parasites plathelminthes du genre Schistosoma ». Thesis, Perpignan, 2018. http://www.theses.fr/2018PERP0028/document.
Texte intégralRésumé :
Les changements globaux ont en partie pour effet de modifier les aires de répartition géographique des espèces. Les interactions nouvelles entre espèces n’ayant jamais été en contact peuvent potentiellement mener à des cas atypiques de reproduction, notamment l’hybridation. Ce phénomène peut avoir des implications épidémiologiques fortes car il peut conduire à la genèse de pathogènes hybrides. La combinaison du matériel génétique d’espèces distinctes peut conférer de meilleures capacités à la progéniture (vigueur hybride ou hétérosis), pouvant à terme potentiellement mener à des changements adaptatifs et à l'émergence de pathogènes dans des zones non endémiques, ce qui en fait une menace émergente à l’échelle mondiale. Ce travail de thèse se focalise sur la schistosomiase, seconde maladie parasitaire humaine et sa récente émergence en Europe (Corse, France). Après l’identification et la caractérisation génomique d’un parasite hybride entre deux agents distincts de la maladie, S. haematobium chez l’homme et S. bovis chez les bovins, nous avons mené une approche intégrative afin de caractériser à plusieurs échelles les capacités invasives et la virulence de tels parasites. A partir de souches du terrain, nous avons mis en place un protocole d’évolution expérimentale visant à générer des hybrides de première et deuxième générations au laboratoire. Nous avons analysé les modifications de traits d’histoire de vie de ces parasites ainsi que les conséquences moléculaires (génomique et transcriptomique) de ce « clash génomique » et nous montrons que l’hybridation peut être une force évolutive majeure pour les parasitesGlobal changes contribute in modifying species geographical distribution. New interactions between species that have never been in contact before can potentially lead to atypical cases of reproduction, including hybridization. This phenomenon can have strong epidemiological consequences as it can potentially lead to the genesis of hybrid pathogens. The combination of genetic material of distinct species can confer increased capacities to the offspring (hybrid vigor or heterosis), eventually leading to adaptive changes and the emergence of pathogens in non-endemic areas, making them an emerging global threat. This thesis work focuses on schistosomiasis, the second human parasitic disease after malaria and its recent emergence in Europe (Corsica, France). After the identification and genomic characterization of a hybrid parasite between two distinct agents of the disease, S. haematobium in humans and S. bovis in cattle, we conducted an integrative approach to characterize at several scales the invasive capacities and virulence of such parasites. Starting from the field, we set up an experimental evolution protocol aimed at generating first- and second-generation hybrids in the laboratory. We analysed life history trait modifications of these parasites as well as the molecular consequences (genomics and transcriptomics) of this "genomic clash" and we show that hybridization can be a major evolutionary force for parasites
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Cassagne, Myriam. « Etude physiopathologique des complications oculaires observées chez des patients atteints de dermatite atopique traitée par Dupilumab ». Electronic Thesis or Diss., Toulouse 3, 2023. http://www.theses.fr/2023TOU30288.
Texte intégralRésumé :
Le dupilumab a prouvé son efficacité dans le traitement de la dermatite atopique (DA) modérée à sévère. Cependant, la survenue d'événements indésirables oculaires (Ocular Adverse Events, OAE) a été rapporté. Les objectifs de ce travail étaient de décrire l'incidence et la nature des OAE induits par le dupilumab, d'évaluer les facteurs prédisposants potentiels et d'en comprendre les bases moléculaires. Nous avons mené une première étude monocentrique, prospective, en vie réelle, chez des patients adultes atteints de DA traités par dupilumab, qui ont été systématiquement examinés par un ophtalmologiste avant et pendant le traitement. Nous avons inclus 46 patients avec un score clinique de sévérité (SCORing initial médian de DA (SCORAD)) de 46,0 (interquartile : 34,5-55,5). 34,8 % des patients ont présentés des OAE, souvent légers à modérés, mais aboutissant à une interruption du dupilumab pour deux patients. La majorité des patients ont développé ou aggravé une sécheresse oculaire. Six patients (13%) ont développé une conjonctivite, de novo. Les OAE induits par le dupilumab étaient associés à une kératite ponctuée superficielle initiale (Odds ratio (OR) = 6,3 ; intervalle de confiance (IC) à 95% : [1,3-31,6]), un eczéma des paupières (OR = 8,7 ; IC à 95 % : [1,8-40,6]), un antécédent d'allergie alimentaire (OR = 3,8 ; IC à 95 % : [1,002-14, 070]) et/ou des taux sériques d'IgE > 1 000 kU/L (OR = 10,6 ; IC à 95 % : [1,2-91,3]). Nous avons ensuite participé à une étude nationale multicentrique qui a inclus 181 patients. Trente-quatre patients (18,7 %) présentaient une blépharoconjonctivite induite par le dupilumab : soit de novo (n=32 ; 17,6%), soit par aggravation d'une préexistante (n=2 ; 1,1%). La plupart des événements (27/34 ; 79,4 %) étaient modérés. Une analyse multivariée a montré que la DA avec atteinte de la tête et du cou (OR = 7,254 ; IC à 95 % : [1,938-30,07] ; p= 0,004), l'érythrodermie (OR = 5,635 ; IC à 95 % : [1,635-21,50] ; p = 0,007) et la présence d'un syndrome de sec avant le traitement (OR = 3,51 ; IC à 95 % : [3,158-13,90] ; p = 0,031) étaient des facteurs indépendants associés à la blépharoconjonctivite induite par le dupilumab. Le SCORAD ou la réponse au traitement (% d'amélioration du SCORAD à S16), un antécédent de conjonctivite allergique, d'asthme ou de rhinite n'étaient également pas corrélés significativement à la survenue d'OAE. Enfin, nous avons mené une étude bi-centrique, comparant le transcriptome (analysé par biopuces et RT-qPCR) et l'expression de la chimiokine CCL20 (par ELISA) sur des cellules conjonctivales, collectées par empreintes conjonctivales, de patients atteints de DA, avant (M0) et 4 mois après le début du traitement au dupilumab (M4). Trente-six patients ont été inclus et répartis en deux groupes selon leur statut ophtalmologique à M4 : un groupe de 12 ayant développé des OAE (OAE+) et un groupe de 24 qui n'en ont pas développé (OAE-). En analyse multiple transcriptomique, nous avons identifié 52 gènes différentiellement exprimés entre OAE+/M0 et OAE-/M0, 113 entre OAE+/M4 et OAE-/M4, deux entre OAE+/M0 et OAE+/M4, et aucun entre OAE-/M0 et OAE-/M4. L'analyse en IPA (Ingenuity Pathway Analysis) avec un intérêt particulier pour les maladies, a mis en évidence principalement une signature "psoriasis". Parmi les 15 gènes différentiellement exprimés sélectionnés pour la validation par RT-qPCR, seuls 7 étaient significatifs : CCL20, IL-19, NOX1, NOS2, SLC26A4, S100A12 étaient régulés positivement et MUC-7 était régulé négativement entre M0 et M4 chez les patients présentant des OAE (OAE+/M4 vs OAE-/M4). La quantification de la protéine CCL20 par ELISA a confirmé une surexpression entre M0 et M4 pour les deux groupes. En conclusion, le traitement par dupilumab des patients atteints de DA qui présentent des OAE, semble accentuer un profil immunologique orienté Th17, voie particulièrement impliquée dans le psoriasisDupilumab has proven its efficacy in the treatment of moderate to severe atopic dermatitis (AD). However, the occurrence of ocular adverse events (OAEs) has been reported. The objectives of this work were to describe the incidence and nature of OAEs induced by dupilumab in AD patients, to evaluate potential predisposing factors, and to understand their molecular bases. We conducted a first single-center, prospective, real-life study in adult AD patients treated with dupilumab, who were systematically examined by an ophthalmologist before and during treatment. We included 46 patients with a median initial SCORing of AD (SCORAD) of 46.0 (interquartile range: 34.5-55.5). 34.8% of patients experienced OAEs, often mild to moderate, but leading to interruption of dupilumab for two patients. The majority of patients developed or worsened dry eye. Six patients (13%) developed conjunctivitis de novo. Dupilumab-induced OAEs were associated with dry eye with superficial punctuate keratitis (Odds ratio (OR) = 6.3 95% confidence interval (CI): [1.3-31.6]), eyelid eczema (OR= 8.7; 95% CI: [1.8-40.6]), a history of food allergy (OR = 3.8; 95% CI: [1.002-14.070]) and/or serum IgE levels > 1.000 kU/L (OR = 10.6; 95% CI: [1.2-91.3]). We then participated to a national multicenter study which included 181 patients. Thirty-four patients (18.7%) presented blepharoconjunctivitis induced by dupilumab: either de novo (n=32; 17.6%) or by worsening of a pre-existing condition (n=2; 1.1%). Most events (27/34; 79.4%) were moderate. Multivariate analysis showed that AD with head and neck involvement (OR = 7.254; 95% CI [1.938-30.07]; p = 0.004), erythroderma (OR = 5.635; 95% CI [1.635] -21.50]; p = 0.007) and the presence of dry eye syndrome before treatment (OR = 3.51; 95% CI [3.158-13.90]; p = 0.031) were independent factors associated with dupilumab-induced blepharoconjunctivitis. The SCORAD or the response to treatment (% improvement in SCORAD at W16), a history of allergic conjunctivitis, asthma or rhinitis were also not significantly associated with the occurrence of OAE. Finally, we conducted a bicentric study, comparing the transcriptome (analyzed by DNA microarrays and RT-qPCR) and the expression of the chemokine CCL20 (by ELISA) of conjunctival cells, collected by conjunctival impression, on AD patients before (M0) and 4 months after dupilumab beginning (M4). Thirty-six patients were included and divided in two groups, according their ophthalmological status at M4: one group of 12 who developed OAE (OAE+) and another group of 24 who did not (OAE-). In multiple analysis of the full transcriptome, we found 52 differentially expressed genes (DEG) between OAE+/M0 and OAE-/M0, 113 DEG between OAE+/M4 and OAE-/M4, two DEG between OAE+/M0 and OAE+/M4, and none DEG between OAE-/M0 and OAE-/M4. Ingenuity Pathway Analysis enrichment with a special interest in diseases, mainly indicated a psoriasis signature. Among the 15 DEG selected for RT-qPCR validation, only 7 were significant: CCL20, IL-19, NOX1, NOS2, SLC26A4 and S100A12 were up-regulated, whereas MUC-7 was downregulated in OAE+/M4 vs OAE-/M4 patients. CCL20 protein quantification by ELISA confirmed an over-expression between M0 and M4 for the two groups. In conclusion, most cases of dupilumab-induced blepharoconjunctivitis are de novo. Pre-existing factors (dry eye syndrome, AD with eyelid, head and neck) are associated with an increased risk of OAE. Dupilumab seems to switch AD immunological profile of OAE patients from Th2 to Th17, particularly involved in psoriasis
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Mazzitelli, Jean-Yves. « Évaluation de la toxicité de molécules médicamenteuses par une étude des réponses comportementales, physiologiques et transcriptomiques d’un mollusque dulçaquicole (Radix balthica) et d’un plathelminthe (Schmidtea polychroa) ». Thesis, Toulouse 2, 2017. http://www.theses.fr/2017TOU20014/document.
Texte intégralRésumé :
Les médicaments sont fréquement retrouvés dans les effluents de STEP relargués dans l’environnement aquatique. Dans le but de prévenir et de mieux comprendre les impacts des médicaments sur les écosystèmes aquatiques, il semble pertinent d’évaluer les perturbations comportementales, physiologiques et transcriptomiques des psychotropes sur les organismes aquatiques. Dans ce contexte, l’objectif de cette étude a été d’évaluer les perturbations induites par 4 psychotropes (oxazépam, carbamazépine, cyamémazine et sertraline) chez deux organismes, Radix balthica et Schmidtea polychroa. Pour ce faire, des embryons de Radix au stade trochophore et des planaires adultes ont été exposés à chaque psychotrope (du μg/L jusqu’à 100 μg/L). Il en ressort que les psychotropes allongent la durée du développement embryonnaire du Radix et perturbent le déplacement, la reproduction et la digestion, mais pas la régénération de la planaire. D’un point de vue transcriptomique, nous avons réalisé le séquençage du transcriptome en conditions différentielles chez le Radix. Ainsi, nous avons obtenu d’une part les séquences du transcriptome et d’autre part, après analyse en différentiel, 144 contigs différentiellement exprimés par l’oxazépam parmi lesquels 94 ont été vérifiés en RT-qPCR chez des Radix exposés à chaque psychotrope. Il ressort de cette analyse que les psychotropes impactent principalement la voie de signalisation Notch, mais aussi les voies de biosynthèse des polyamines et des catécholamines. Les psychotropes modulent aussi l’expression de gènes codant des protéines de la Matrice Extra Cellulaire (MEC), comme la Matriline ou encore la Dentine sialophosphoprotéine. Chez la planaire, nous avons analysé l’expression de 87 gènes impliqués dans différentes fonctions. Il ressort de cette étude que les 4 psychotropes modulent l’expression de nombreux gènes impliqués dans la mobilité ciliaire et musculaire et dans les systèmes nerveux, reproducteur, excréteur et digestifPharmaceuticals are often found in WWTP effluents realesed in surface water. In order to prevent and to understand the pharmaceuticals impact on aquatic ecosystems, it seems relevant to evaluate behavioural, physiological and transcriptomic disturbances of psychotropics on freshwater organisms. The aim of this study was thus to analyse disturbances of 4 psychotropics (oxazepam, carbamazepine, cyamemazine and sertraline) on 2 freshwater organisms, Radix balthica and Schmidtea polychroa. In our experiments, both Radix embryos at the trochophore stage and mature planarian were exposed to each psychotropic (from 1 to 100 μg/L). This psychotropic exposure results in an increase of the duration of Radix embryonic development and a disturbance of the mobility, the reproduction and the digestion but not the regeneration of planarian. Regarding the transcriptomic impact, we performed RNA sequencing in differential conditions of Radix embryos exposed or not to oxazepam. On one hand, this analysis allowed us to obtain the transcriptome sequences and, on the other hand, 144 contigs differentially expressed among which 94 genes were verified by RT-qPCR. The results showed that psychotropics impact mainly the Notch signalling pathways, but also the biosynthetic pathways of polyamines and catecholamines. Psychotropics also disturb the gene expression encoding some Extra Cellular Matrix (ECM) protein such as Matrilin and Dentin sialophosphoprotein. Regarding the molecular study of the psychotropics impact on planarian, we analysed the expression of 87 genes involved in different functions. The results showed that the 4 psychotropics modulate expression of genes involved in ciliary and muscular motility and in the nervous, reproductive and excretory systems. Genes from the digestive system are also impacted by the psychotropics. All the observed impacts on the 2 organisms suggest a possible disturbance on the population fitness and therefore on the freshwater ecosystems
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González, Mula Almudena. « Mode de vie d'Agrobacterium tumefaciens dans la tumeur ». Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS130.
Texte intégralRésumé :
Le phytopathogène Agrobacterium tumefaciens est l'agent causal de la maladie appelée galle du collet, et est capable d'infecter plus de 90 familles de plantes dicotylédones. Cette ∝-protéobactérie appartient à la famille Rhizobiaceae. A. tumefaciens est un complexe de différentes espèces regroupées en 10 génomovars (G1 à G8 et G13). A. tumefaciens C58 appartient au groupe du G8. Son génome est constitué de 4 réplicons : 1 chromosome circulaire, 1 chromosome linéaire et des 2 plasmides dispensables : pAt (pour A.tumefaciens) et pTi (pour Tumor inducing, qui est requis pour la virulence). Pour explorer de nouveaux aspects du mode de vie d’A. tumefaciens, et en particulier l'interaction entre la bactérie et sa plante hôte, deux approches différentes ont été utilisées pour identifier, caractériser et analyser les gènes qui pourraient jouer un rôle dans l'adaptation des bactéries à la tumeur. Une expérience de l'évolution par des passages en série de trois souches différentes de l'agent pathogène sur la plante hôte Solanum lycopersicum a été effectuée afin de clarifier la dynamique évolutive du génome au cours de l'infection. Parallèlement, une étude de différents transcriptomes (in planta et in vitro) a été réalisée et étudiée pour élucider des gènes bactériens candidats impliqués dans l'interaction de la bactérie avec la plante et divers composés produits dans la tumeur. Ce travail tente de donner une vue plus générale du processus d'adaptation de la bactérie à la niche écologique qui est la tumeurAgrobacterium tumefaciens is the causal agent of the plant disease called crowngall, and it’s able to infect more than 90 families of dicotyledonous plants. It is an α-Proteobacterium and belongs to the Rhizobiaceae family. A. tumefaciens is a complex of different species grouped in 10 genomovars (G1 to G8, and G13). A. tumefaciens C58 belongs to the G8 group. Its genome consists in 4 replicons: 1 chromosome circular, 1 chromosome linear and 2 dispensable plasmids: pAt (for A. tumefaciens) and pTi (for Tumor inducing), which is required for virulence. To explore new aspects of the A. tumefaciens lifestyle, and in particular the interaction between the bacteria and its plant host, two different approaches have been used to identify, characterize and analyze genes that could play a role in the adaptation of the bacteria to tumor lifestyle. An evolution experiment by serial passages of three different strains of thepathogen on the host plant Solanum lycopersicum has been carried out to clarify the evolutionary dynamics of the genome during the course of infection. In parallel, a study of different transcriptomes (in planta and in vitro) was performed and studied to elucidate bacterial candidate genes involved in the interaction of the bacteria with the plant and various compounds produced in the tumor. This work attempts to give a more general view of the process of adaptation of the bacteria to the ecological niche that is the tumor
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Chaput, Catherine. « Effet d'un niveau élevé de bêta-hydroxybutyrate (BHB) au jour 45 post-partum sur la qualité transcriptomique et épigénétique des embryons ». Master's thesis, Université Laval, 2020. http://hdl.handle.net/20.500.11794/66285.
Texte intégralRésumé :
En début de lactation, la vache subit un stress important occasionné par l’impossibilité de combler l’ensemble de ses besoins énergétiques par sa consommation exogène. Cette période spécifique se caractérise par une balance énergétique négative, entraîne une utilisation excessive des réserves corporelles de l’animal et représente un défi métabolique important. Ironiquement, depuis maintenant plus de 40 ans, le système incite les producteurs laitiers à effectuer l’insémination au jour 60 post-partum, c’est-à-dire au moment où la vache rencontre un déficit métabolique. Ce déficit au moment de la conception aurait un impact chez la progéniture, notamment au niveau épigénétique. Ce projet consiste à documenter l’effet de la balance énergétique négative sur la qualité de l’embryon et, en l’occurrence, à proposer des pistes afin d’améliorer la fertilité des bovins laitiers. La mesure du bêta-hydroxybutyrate (BHB) a été effectuée à partir d’échantillons sanguins entre 45 et 60 jours post-partum sur dix-huit vaches de race Holstein. Selon la mesure obtenue, la vache fut classée comme étant faible ou élevée en BHB, afin d’avoir au moins six vaches par groupe. Après un processus de stimulation ovarien, chaque vache fut inséminée et les embryons, récoltés. Pour chaque vache, deux embryons ont été transférés dans deux primipares, afin de déterminer subséquemment la persistance des marqueurs dans le matériel biologique. Grâce à la plate-forme EmbryoGENE, il fut possible de déterminer l’expression génique ainsi que l’état de méthylation de l’ADN des embryons récoltés. Les résultats obtenus soutiennent l’existence d’une altération du métabolisme énergétique au niveau embryonnaire, notamment par la modification de la voie de signalisation de mTOR ainsi que celle des sirtuines. Cette altération semble se traduire par une dysfonction mitochondriale et une inhibition de la transcription, entraînant un freinage au niveau cellulaire, probablement dû à la programmation de l’embryon à utiliser ses réserves lipidiques lors de conditions importantes de stress.In early lactation, the cow undergoes an important stress generated by the impossibility of filling its entire energetic needs by exogenous consumption. This is characterized by a negative energy balance, excessive use of animal body reserves and represents an important metabolic challenge. Ironically, for more than 40 years now, the system has been encouraging dairy farmers to inseminate on day 60 postpartum, when the cow has a metabolic deficit. This deficit at the time of conception could impact the offspring, especially at the epigenetic level. This project is meant to document the effect of the negative energy balance on the quality of the embryo and to identify ways to improve the fertility of dairy cows. The beta hydroxybutyrate (BHB) measure was done from blood samples between day 45 and 60 postpartum on eighteen Holstein cows. According to the measure obtained, each cow was classified as low or high in BHB, so as to have at least six cows per group. After an ovarian stimulation process, each cow was inseminated and the embryos were harvested. For each cow, two embryos were transferred in two primiparous cows in order to subsequently determine the persistence of the markers in the biological material. With the EmbryoGENE platform, it was possible to determine the gene expression as well as the methylation status of DNA embryos. The results obtained support the existence of an alteration of the energetic metabolism at the embryonic level, especially by the modification of the signaling pathway of mTOR as well as those of the sirtuins. This alteration appears to result in mitochondrial dysfunction and inhibition of transcription, leading to a reduced activity at a cellular level, probably due to programming of the embryo to use its lipid reserves during severe stress conditions.
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Gréau, Lilian. « Réponse du peuplier et de ses communautés microbiennes rhizosphériques et endophytes exposés au phénanthrène ». Electronic Thesis or Diss., Université de Lorraine, 2023. https://docnum.univ-lorraine.fr/ulprive/DDOC_T_2023_0096_GREAU.pdf.
Texte intégralRésumé :
Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants dans les sols dont l'élimination peut être effectuée par les plantes et les communautés microbiennes associées (phytoremédiation). Bien que les HAP puissent être absorbés et transportés dans les tissus végétaux, le rôle potentiel des endophytes dans la biodégradation des HAP et l'atténuation de leur toxicité a été sous-estimé au détriment des microorganismes rhizosphériques qui ont été largement étudiés. Dans ce contexte, il semble nécessaire d'évaluer la réponse de la plante et de son microbiome à la présence de HAP pour mieux comprendre l'impact global de la contamination du sol. La réponse du peuplier (Populus x canadensis) à un gradient de huit concentrations de phénanthrène (PHE), un HAP modèle à 3-cycles, a été étudiée en utilisant des stratégies complémentaires combinant l'analyse du transcriptome (par 2 approches : expression différentielle des gènes (DEGs) et utilisation de l'outil DRomics), des mesures d'activités enzymatiques en réponse au stress oxydatif et le suivi de la croissance des plantes. La biomasse des plantes était de plus en plus faible le long du gradient. La voie de l'éthylène mais aussi la biosynthèse des phénylpropanoïdes/flavonoïdes ont été stimulées par la présence de PHE. Les taux de transcrits et les niveaux d'activités de plusieurs gènes/enzymes impliqués dans la réponse au stress oxydatif ont également été stimulés. De plus, une concentration élevée de PHE altérait l'expression de gènes de défenses contre les stress biotiques dans les racines, tandis que l'activité photosynthétique était accrue. Ensuite, nous avons étudié l'impact du gradient de PHE sur les communautés microbiennes rhizosphériques et endophytiques par séquençage Illumina MiSeq. La contamination par les HAP a modifié les structures des communautés bactériennes et fongiques dans le sol alors que seules les communautés fongiques ont été impactées dans les racines. L'abondance relative de nombreux taxons a diminué en présence de HAP. L'exploration des fonctions bactériennes liées aux cycles biogéochimiques a révélé que plusieurs fonctions telles que la dégradation de la matière organique étaient négativement impactées par le PHE. A l'inverse, les dégradeurs potentiels de HAP ont été favorisés dans le sol exposé à ce contaminant. Enfin, le potentiel des endophytes pour atténuer le stress des plantes induit par le PHE a été étudié. Des isolats endophytes fongiques et bactériens ont été obtenus à partir de racines de peupliers cultivés dans un sol contaminé par le PHE. Leur capacité à promouvoir la croissance des plantes et à tolérer/biodégrader le PHE a été mise en évidence in vitro et les isolats les plus intéressants ont été réinoculés au peuplier. Parmi les différentes combinaisons testées, un consortium bactérien-fongique est apparu comme un candidat prometteur pour des perspectives de phytoremédiation. Globalement, l'utilisation d'une approche unique associant un gradient de contamination et des outils omiques, a permis de mettre en évidence l'impact d'une contamination par le PHE sur la physiologie du peuplier et ses communautés microbiennes associées. Ce travail est le premier à étudier les réponses couplées d'une plante avec ses microorganismes associés à la contamination par les HAP en utilisant une plante couramment utilisée en phytoremédiation et un sol présentant une contamination historique avec des communautés microbiennes adaptéesPolycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants in soils. Their removal can be performed by plants and associated microbial communities (phytoremediation assisted by microorganisms). Although PAHs can be taken up and transferred in plant tissues, the role of endophytes in PAH-biodegradation and mitigation of PAH-toxicity has been underestimated at the expense of rhizospheric microorganisms that have been extensively studied. In this context, it is necessary to evaluate the response of both the plant and its associated microbiome to the presence of PAHs to better understand the overall impact of soil contamination. The response of Populus x canadensis to a gradient of eight concentrations of phenanthrene (PHE), a model 3-cycles PAH was investigated using complementary strategies combining transcriptome analysis (using both differentially expressed genes (DEGs) and DRomics tools), oxidative stress enzyme quantification and plant growth monitoring. Plant biomass decreased along the gradient. Ethylene pathway but also phenylpropanoid/flavonoid biosynthesis were triggered in a PHE concentration dependent manner. Poplar response to oxidative stress was highlighted both at the transcript and enzymatic levels. Moreover, high PHE concentrations impaired plant defenses against biotic stressors in roots while photosynthetic activity was elicited. Then, we investigated the impact of the PHE gradient on both rhizospheric and endophytic microbial communities by Illumina MiSeq sequencing. PAH contamination modified both bacterial and fungal community structures in soil while only fungal communities were impacted in roots. The relative abundance of many taxa decreased in the presence of PHE. The exploration of bacterial functions linked to biogeochemical cycles revealed that several functions such organic matter degradation was negatively impacted by PHE. Conversely, potential PAH-degraders were favoured in the soil exposed to this contaminant. Finally, the potential of endophytes to mitigate plant stress mediated by PHE was investigated. Fungal and bacterial endophytic isolates were retrieved from poplar roots grown in PHE contaminated soil. Their ability to promote plant growth and tolerate/biodegrade PHE was evidenced in vitro and the most interesting isolates were re-inoculated to poplar. Among the different combinations tested, a bacterial-fungal consortium appeared as a promising candidate for phytoremediation perspectives. Overall, the use of a unique approach associating a contamination gradient and multi-omics, highlighted the impact of a PHE contamination on poplar physiology and its associated microbial communities. This work is the first to investigate plant-microbial responses to PAH contamination using a plant commonly used in phytoremediation and an aged contaminated soil with adapted microbial communities
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Coqueiro, Danila Souza Oliveira 1984. « Expressão gênica diferencial induzida por eliciadores (quitosana e ácido salicílico) nos patossistemas citros-Xanthomonas citri subsp. citri e citros-Xylella fastidiosa = Differential gene expression induced by elicitors (chitosan and salicylic acid) in citrus-Xanthomonas citri subsp. citri and citrus-Xylella fastidiosa pathosystems ». [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314472.
Texte intégralRésumé :
Orientadores: Marcos Antonio Machado, Alessandra Alves de SouzaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Avaliou-se as alterações transcricionais em laranja 'Pera' (Citrus sinensis L. Osb.) promovidas por quitosana (CHI) e ácido salicílico (SA), utilizando RNA-seq, e o efeito destes compostos no controle do cancro cítrico (Xanthomonas citri subsp. citri) e da clorose variegada dos citros (CVC - Xylella fastidiosa). As plantas foram tratadas com CHI ou SA e após 48h e 24h, respectivamente, foram coletadas amostras foliares para avaliar seus transcriptomas. Para a avaliação dos eliciadores sobre o cancro cítrico e a CVC, as plantas foram tratadas com CHI ou SA e após 48h e 24h, respectivamente, inoculadas com as duas bactérias separadamente. A partir de 24h da inoculação, foram coletadas amostras foliares para avaliar a curva de crescimento de ambas as bactérias, a redução da severidade e/ou incidência das doenças e respostas de defesa da planta por RT-qPCR. Com os resultados do transcriptoma, observou-se que mais genes foram induzidos pelo tratamento com SA do que com CHI. O tratamento com SA aumentou a expressão de genes que participam da via de sinalização do SA na planta (WRKY50, PR2 e PR-9) e genes da biossíntese do etileno e ácido jasmônico (ACS 12, fator de transcrição contendo domínio AP2 e OPR3). Além disso, promoveu a indução de genes relacionados ao metabolismo secundário, processos redox e estresse biótico. No tratamento com CHI, foi observada maior indução de genes relacionados ao metabolismo secundário. Para ambos os tratamentos, a via da auxina foi reprimida. No experimento para controle do cancro cítrico, observou-se que ambos os eliciadores promoveram reduções na severidade e incidência da doença. Entretanto, a CHI pareceu não interferir diretamente na formação do biofilme pela bactéria, mas pode ter dificultado a multiplicação de X. citri na planta. O SA retardou a entrada da bactéria na planta e, aparentemente, inibiu mais a formação do biofilme bacteriano do que a CHI. Comparações da expressão gênica entre os eliciadores reforçam a ideia de que a CHI tem maior potencial de induzir resistência ao cancro cítrico do que SA. No experimento para o controle da CVC, observou-se que a CHI induziu importantes genes da via do SA (NPR1, TGA, EDS1) e etileno (EIN-3, PR-4) 24h após a inoculação. Aplicações exógenas de SA potencializaram o seu efeito endógeno na planta, pois houve indução de NPR1, TGA e PRs. Entretanto, não foi possível estabelecer uma relação clara entre a multiplicação de X. fastidiosa, a incidência da doença e o uso da CHI e SA em laranja 'Pera', já que na maioria das avaliações não houve redução da população bacteriana em amostras foliares e não houve redução da incidência em plantas tratadas. Com base nos resultados, observou-se que CHI e SA induziram diversos genes envolvidos em respostas de defesa em laranja 'Pera'. Entretanto, essas respostas podem ser moduladas diferencialmente a depender do patógeno que afeta a planta, pois os eliciadores foram eficientes no controle da X. citri, um patógeno que coloniza o mesófilo da planta, entretanto não foram efetivos no controle da X. fastidiosa, um patógeno que coloniza o xilema da planta, embora respostas de defesa tenham sido expressas nos momentos iniciais (24h) após a inoculação com X. fastidiosa
Abstract: This study was carried out to evaluate transcriptional modification in sweet orange 'Pera' (Citrus sinensis L. Osb.), promoted by chitosan (CHI) and salicylic acid (SA), using RNA-seq, and the effect of these compounds on citrus canker (Xanthomonas citri subsp. citri) and citrus variegated chlorosis (CVC - Xylella fastidiosa). Plants were treated with CHI or SA and after 48h and 24h, respectively, leaf samples were collected to assess the transcriptome. In the experiments for disease assessment, the plants were treated with CHI or SA and after 48h and 24h, respectively, inoculated. Starting from 24h after inoculation, leaf samples were collected to evaluate the multiplication of the pathogens (X. citri and X. fastidiosa), reduction of the severity and / or incidence and plant defense responses by RT-qPCR. Based upon the transcriptome results, it was observed that more genes were induced by SA than by CHI. SA treatment increased the expression of genes that participate in the SA signaling pathway in the plant (WRKY50, PR2 and-PR9), and genes involved in the biosynthesis of ethylene and jasmonic acid (ACS 12, transcription factor containing AP2 and OPR3 domain). Besides these, SA promoted induction of genes of secondary metabolism, redox processes and biotic stress. The treatment with CHI exhibited higher induction of genes related to secondary metabolism. For both treatments, the auxin pathway was suppressed. In the experiment for the control of citrus canker, it was observed that both elicitors reduced the severity and incidence of the disease. However, CHI seems not to interfere directly in biofilm formation, but may have hindered the multiplication of X. citri in the plant. The SA slowed down the entry of the bacteria into the plant and, apparently, inhibited the formation of biofilm more efficiently than the CHI. Comparisons of gene expression between elicitors reinforce the idea that CHI has higher potential to induce resistance to citrus canker than SA. In the experiment for the control of CVC, it was observed that the CHI induced important genes of the SA (NPR1, TGA, EDS1) and ethylene (EIN-3, PR-4) pathways 24h after inoculation. Exogenous applications of SA potentiated its endogenous effect in the plant, since there was induction of EDS-1, NPR1, TGA and PRs. However, it was not possible to establish a clear relationship between the multiplication of X. fastidiosa, the incidence of the disease and the use of CHI and SA in 'Pera' sweet orange, since most of the assessments did not show reduction of bacterial populations in leaf samples and there was no reduction of the incidence in treated plants. Based upon the results of this study, it was observed that CHI and SA induced several genes involved in defense responses in 'Pera' sweet orange. However, these responses can be modulated differentially depending on the pathogen that affects the plant. This fact was demonstrated in this study, as elicitors were effective in controlling X. citri, a pathogen that colonizes the mesophyll of the plant, but were not effective in controlling X. fastidiosa, a pathogen that colonizes the xylem of the plant, although defense responses were expressed in the early stages (24 h) after inoculation with X. fastidiosa
Doutorado
Bioquimica
Doutora em Biologia Funcional e Molecular
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Martins, Luciana Moreira. « Análise do transcriptoma de Podalia orsilochus (Cramer, 1775) ». Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-26082016-103637/.
Texte intégralRésumé :
Os insetos são capazes de sobreviver em diversos ecossistemas do planeta e, mesmo estando constantemente expostos à ameaça de infecção microbiana, permanecem livres de infecções na maior parte do tempo. Essa capacidade de sobrevivência aliada à larga distribuição dos insetos em regiões totalmente diferentes tem estimulado a pesquisa de novos agentes terapêuticos nesta classe devido à descoberta de diversos componentes de mecanismos inespecíficos de combate à infecção, sendo possível sua aplicação no controle de diversas doenças. Todavia, apesar de um grande número de moléculas de defesa ter sido identificado a partir de vários insetos, pouca informação sobre suas aplicações está disponível. Desta forma, o presente trabalho elucida o perfil transcriptômico geral e dos genes de defesa do tegumento de Podalia orsilochus durante sua fase larval. Como consequência, os transcritos e os dados obtidos permitirão o auxílio em pesquisas posteriores, seja para comparação, citação, conhecimento biológico e das respostas de defesa ou das relações de filogenia do animal.The insects are able to survive in diverse ecosystems on earth, and even being constantly exposed to the threat of microbial infections, remain free of infection for most of the time. This survivability combined with the wide distribution of insects in totally different regions has stimulated the search for new therapeutic agents in this class due to the discovery of several components of nonspecific mechanisms to fight infection, and possible implementation in the control of various diseases. However, despite a large number of defense molecules have been identified from various insects, little information is available on their applications. Thus, this paper elucidates the general transcriptomic profile and integument of defense gene Podalia orsilochus during their larval stage. As a result, the transcripts and the data obtained will aid in further research, to compare, reference, biological knowledge and defense or animal phylogeny relationships.
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Viala, Vincent Louis. « Análise combinada do transcriptoma de glândula de veneno e do proteoma do veneno da espécie Pseudonaja textilis (Elapidae : Serpentes) ». Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-13062014-104311/.
Texte intégralRésumé :
As toxinas de veneno de serpentes têm como principal função alterar a homeostase das presas para fins de alimentação ou defesa. O estudo aprofundado da composição do veneno de serpentes é importante para a produção de soros antiofídicos mais eficientes, para a descoberta de novos fármacos e na compreensão de processos biológicos, ecológicos e evolutivos. As pesquisas com toxinas têm mostrado uma versatilidade natural, refinada pela evolução, na diversificação de funções em famílias de proteínas recrutadas de suas funções endógenas, por meio de sucessivas duplicações e acumulo de mutações levando a uma evolução acelerada. A miríade de toxinas disponíveis e sua diversidade de funções ainda não foram completamente descritas. A combinação das análises em larga escala do transcriptoma de novo da glândula de veneno e do proteoma do veneno permite elaborar um perfil mais completo do toxinoma do veneno, permitindo inclusive um aumento na sensibilidade da detecção de toxinas pouco representadas e inesperadas nos venenos. O objetivo geral deste estudo foi analisar o toxinoma do veneno de uma das mais perigosas espécies australianas, a Pseudonaja textilis (Elapidae). Foi possível identificar no veneno as toxinas: fatores de coagulação de veneno do complexo protrombinase, subunidades de fosfolipases A2 (PLA2) da neurotoxina textilotoxin e PLA2 de atividade procoagulante, neurotoxinas tipo three-finger toxin (3FTx), inibidores de protease do tipo-kunitz textilinin, e pela primeira vez, uma nova variante de 3FTx, lectinas tipo C, CRiSP além de indícios de toxinas de lagarto Heloderma e outras proteínas candidatas a toxinas como calreticulin e dipeptidase 2. Metaloproteinases, pouco estudadas em Elapidae, foram clonadas e detectadas no veneno por ensaios de fracionamento e imunoreatividade. A análise do transcriptoma identificou novas isoformas e variantes de toxinas, principalmente das 3FTx e dos inibidores de serinoproteases, assim como transcritos de toxinas que não foram detectadas no veneno e que merecem mais investigações. O quadro de sintomas com acidentes em humanos é bem explicado pelas toxinas identificadas, porém, em seu habitat natural, as toxinas pouco conhecidas e até então não descritas devem ter funções importantes e específicas na predação. Identificar esta diversidade de variantes é importante para entender o modo de ação das toxinas.Snake venom toxins alter prey homeostasis for feeding or defense. In depth studies of venom composition are important for better antivenom production, for new drugs lead and discovery and for better understanding of biological, ecological and evolutionary processes. Research on toxins have shown the natures way of innovating, refined by evolution, diversifying functions of protein families recruited from their endogenous function to the venom gland by successive gene duplication and mutation accumulation, leading to an accelerated evolution. A myriad of available toxins and diversity of functions is still available for discovery. Combining high throughput techniques such as venom gland de novo transcriptomics and venom proteomics, one can assess and observe a more complete profile of the snake toxinome, additionally allowing an upscale in low represented and unexpected toxin detection. The aim of this project was to investigate the venom toxinome of one of the most dangerous Australian species, Pseudonaja textilis (Elapidae). The toxins identified in it venom was: protrombinase complex coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and procoagulant PLA2, neurotoxic three-finger toxins (3FTx), Kunitz-type protease inhibitor textilinin, and for the first time, a new long 3FTx, C-type lectins, CRiSPs, as well as evidences of lizard toxins from Heloderma genus and other toxin candidates calreticulin and dipeptidase 2. Metalloproteinases, little investigated in Elapidae, was cloned and detected in the venom after fractionation and immunoassay. The transcriptome revealed new toxin variants and isoforms, specially 3FTx and serine protease inhibitors, as well as transcripts from toxins not detected in the venom that deserves further investigation. Human accident symptoms are well explained by the identified toxins, however, in its natural environment, little known and undescribed toxins must have specific and important role in predation. Identifying this diversity is important to better understand toxins ways of action.
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Lima, Juliana Joice Pereira 1987. « Physiological and molecular studies during acquisition of longevity in soybean (Glycine max (L.) Merrill) seeds / ». Botucatu, 2016. http://hdl.handle.net/11449/140140.
Texte intégralRésumé :
Orientador: Edvaldo Aparecido Amaral da SilvaCoorientador: Olivier Leprince
Banca : Henk W.M Hilhorst
Banca: José Márcio Rocha Faria
Banca: Julia Buitink
Banca: Olivier H.L.Leprince
Resumo: Soja é uma das mais culturas oleaginosas usadas para alimentação animal e humana bem como para uma larga aplicação industrial. Dada a sua capacidade de fixar nitrogênio atmsférico, é fundamental para o desenvolvimento de uma agricultura sustentável. Produzir sementes altamente vigorosas é a chave para aumentar a eficiência da produção da cultura. Longevidade de semente é a capacidade de sobreviver no estado seco por períodos prolongados e representa uma importante característica de qualidade da semente. Nesta pesquisa o objetivo foi obter insights sobre processos moleculares que regulam a aquisição de longevidade em sementes de soja. Com o sequenciamento de nova geração da Illumina, o RNA foi sequenciado a partir de sete estágios diferentes durante a aquisição de longevidade, gerando entre 14 e 38 milhões de reads. Estes reads foram alinhados com os modelos de genes de Glycine max Wm82.a2.v1 preditos no genoma de soja. Transcritos diferencialmente expressos (DET) foram correlacionados com o aumento da longevidade. Análise de enriquecimento da ontologia do gene daqueles DET revelaram uma siginificante sobre representação de termos associados com resposta a estresse e processamento e modificação de RNA. Processo biológico fotossíntese foi relacionado à baixa longevidade. Heat Shock Factors (HSF) e vários fatores de transcrição associados com resposta a estresse biótico (WRKY e NFXL1) são genes candidatos com possíveis papéis na longevidade de semente e merecem uma caracterizaçã... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Soybean is one of the most important oil crop species used for food and feed as well as a range of industrial applications. However, producing highly vigorous seeds is a key lever to increase crop production efficiency. Low physiological seed quality, which is more prone to occur under tropical environment, leads to poor stand establishment and decreased in yields. Seed longevity is the ability to survive the dry state for prolonged periods of time and represents an important trait for seed quality. Here, the objective was to obtain insights into the mechanisms regulating the progressive acquisition of longevity. Using Illumina high-throughput sequencing, RNA was sequenced from seven different stages during the acquisition of longevity, generating between 14 and 38 million of reads. These reads were aligned to the Glycine max Wm82.a2.v1 gene model. Differentially expressed transcripts (DET) were correlated with the increase in seed longevity. Transcriptome and GO enrichment analyses of these DET revealed a significant over-representation of terms associated with response to stress and RNA processing and modification. Photosynthesis biological process was related to low seed longevity. HSF and several TF associated with biotic defense (WRKY3 and NLFX1) are candidate genes whose putative role in seed longevity deserve further characterization. We also performed the determination of the content of non-reducing soluble sugars, and we observed that the accumulation of non-reducin... (Complete abstract click electronic access below)
Résumé: Le soja est l'une des plus importantes espèces de cultures d'huile utilisée aussi bien en nourriture que dans diverses gammes d'applications industrielles. C'est pourquoi produire des graines vigoureuses est un levier essentiel pour augmenter efficacement de la production de la récolte. La qualité de graine physiologiquement faible, qui est plus à même de se produire sous un environnement tropical, mène à un pauvre établissement des plantes ainsi qu'à une diminution du rendement. La longévité d'une graine est la capacité de celle-ci à survivre à la sécheresse durant de longues périodes et représente une caractéristique importante sur la qualité d'une graine. Ici, l'objectif était d'obtenir une idée sur les mécanismes en régulant l'acquisition progressive de la longévité. En utilisant le séquençage à haut-débit, ARN a été séquencé en sept différentes étapes durant l'acquisition de longévité, générant entre 14 et 38 millions de reads. Ces reads ont été alignés sur les modeles de gene de Glycine max Wm82.a2.v1. Les transcripts différentiellement exprimés (DET) sont corrélée avec l'augmentation de la longévité de la graine. L'analise d'enrichissement via GO de ces DET ont révélé une importante surreprésentation des termes associés à la réponse au stress et traitement et modification de l'ARN. Le processu biologique Photosynthèse était liée à une faible longévité des semences. HSF (heat shock factor) et plusieurs facteurs de transcription associés à la défense biotique (WRKY 3 et ... (Résumé complet accès életronique ci-dessous)
Doutor
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Kurissio, Jacqueline Kazue. « Análise da expressão gênica celular e viral durante a infecção in vitro pelo herpesvírus canino 1 ». Botucatu, 2017. http://hdl.handle.net/11449/148734.
Texte intégralRésumé :
Orientador: João Pessoa Araújo JuniorResumo: A análise de transcriptoma é essencial para determinar a relação entre as informações codificadas num genoma, a sua expressão e variação genotípica. O transcriptoma é o conjunto completo de transcritos e a quantidade gerada, relacionado a um estágio de desenvolvimento específico ou condição fisiológica da célula. Estes transcritos podem ser mapeados utilizando genomas como referência, para investigação de expressão do genes. Para isso, exige procedimentos de mineração de grandes volumes de dados de RNA-Seq para extrair conhecimentos biológicos. As ferramentas de bioinformática foram desenvolvidas para facilitar e agilizar essas análises, transformando dados em informações. Assim, alguns desses recursos foram empregados na análise de dados gerados pelo RNAseq, a partir de RNAm de cultura celular MDCK infectada por herpesvírus canino tipo 1 (1CaHV-1 - Canid alphaherpesvirus type 1) em diferentes momentos pós infecção. Dessa forma, foi realizado um dual RNAseq, em que foi avaliado tanto a expressão gênica celular do hospedeiro como do patógeno viral, no curso da infecção. Para isso, foram analisados o transcriptoma das atividades celulares e os processos envolvidos no ciclo de infecção viral, até o momento 32h-pi. Assim, foram identificados as atividades de respostas celulares à infecção viral, mecanismos regulatórios induzidos pelo vírus, transcrição de genes virais imediatos, iniciais e tardios. Dentre eles, foi verificado a elevação da expressão do gene COX-2 induzido pela... (Resumo completo, clicar acesso eletrônico abaixo)
Doutor
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Van, Leen Eric. « On the morphogenesis of the D. melanogaster pupa : a study on gene patterning and tissue folding ». Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS387.
Texte intégralRésumé :
Au cours du développement, la coordination des comportements cellulaires est essentielle à la formation d’organes complexes et fonctionnels. L’analyse de ces processus cellulaire est essentielle pour comprendre comment les tissues se forment au cours du développement. Pour ce faire, il est tout d’abord primordial d’identifier les gènes dont l’expression est corrélée avec chacun de ces processus cellulaires. Avec pour modèle la formation de l’épithélium dorsal (le notum) de la pupe de drosophile, mon travail de thèse a visé à identifier les mécanismes moléculaires qui gouvernent la régulation spatiale de la morphogenèse de l’échelle cellulaire à l’échelle de l’organisme. Dans un premier temps, j’ai mis en œuvre une analyse de transcriptomique spatiale qui m’a permis d’identifier de nouveaux gènes impliqués dans la morphogenèse du notum. Dans un second temps, j’ai développé un microscope confocal rotatif avec l’aide de la plateforme d’imagerie de l’Institut Curie. En appliquant cette nouvelle méthode au cours du développement de la pupe jusqu’au stade adulte, j’ai pu observer la morphogenèse de l’aile et du notum de manière simultanée. J’ai ainsi identifié un nouveau mouvement morphogénétique du notum entre 45-50 hAPF qui semble indépendant de la morphogenèse de l’aile dans le temps et dans l’espace. Enfin j’ai montré que ce mouvement est contrôlée par l’expression de sérine-protéases qui libèrent l’attachement de l’épithélium à la cuticule. Ce travail de thèse représente un apport important à une compréhension fine et intégrée de la régulation de la morphogenèse et de la coordination des dynamiques cellulaires au cours du développementIn order to achieve complex shapes during development, multicellular organisms need to coordinate cellular behaviors to form complex and functional organs. Identifying genes that are expressed in patterns that correlate with cellular processes is therefore primordial. Using the dorsal epithelium (the notum) of drosophila pupa as a model, my thesis aimed at uncovering the molecular mechanisms which control the spatial regulation of morphogenesis at the cell and tissue scale. First, I developed spatial transcriptomics which enabled the identification of new expression patterns involved in notum morphogenesis. Second, I developed, in collaboration with the imaging platform of Institut Curie, Rotating Sample Confocal Microscopy. Using this technique, I was able to simultaneously observe the morphogenesis of the notum, hinge and wing blade. This enabled the discovery of a new morphogenetic movement in the notum between 45-50hAPF. My results suggest that this extensive folding and elongation of the notum is independent of folding in the wing. Furthermore, I demonstrated that the expression of serine proteases regulate the attachment of the tissue to the cuticle which triggers the onset of the folding and determines the final shape of the tissue. Overall, this work increases our understanding of the spatial regulation of morphogenesis and contributes to the knowledge on how the extracellular matrix can regulate tissue shape
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Bonnefont, Cécile. « Analyses génomiques fonctionnelles de la résistance aux mammites : études de deux lignées divergentes de brebis sélectionnées sur la concentration cellulaire du lait ». Thesis, Toulouse, INPT, 2011. http://www.theses.fr/2011INPT0081/document.
Texte intégralRésumé :
Les mammites sont des inflammations de la mamelle provoquées principalement par des bactéries. Elles représentent un problème majeur en élevage laitier. Elles sont caractérisées par de fortes augmentations de la concentration des cellules somatiques (CCS) dans le lait. Le score des CCS (SCS) est fortement corrélé à la présence d'infections mammaires, ainsi il est utilisé en sélection pour améliorer la résistance aux mammites. Pour comprendre les mécanismes mis en place lors d'une sélection sur les SCS, deux lignées divergentes de brebis ont été produites à partir de géniteurs ayant des valeurs extrêmes de cet index. Le principal objectif de ce travail est d'identifier et de comprendre les mécanismes qui confèrent une plus grande résistance ou sensibilité aux mammites provoquées par des staphylocoques. Nos travaux ont porté principalement sur des analyses transcriptomiques de trois types cellulaires majeurs, après contact avec des staphylocoques : les cellules inflammatoires du lait recueillies après infection, principalement composées de neutrophiles, les cellules dendritiques, comme cellules présentatrices d'antigènes et les cellules épithéliales mammaires, première barrière de défense contre l'infection. Nous avons montré que la migration des cellules immunitaires et les processus inflammatoires se traduisent par des activations de voies différentes chez les brebis des deux lignées divergentes. Nous avons aussi identifié des gènes fonctionnels candidats pour expliquer la différence de sensibilité aux mammites. En parallèle, nous avons étudié l'association entre le polymorphisme de l'ADN et la présence d'abcès mammaires liés aux mammites dans un dispositif cas-contrôle visant à détecter des gènes à effet majeur. Les études ont permis de mettre en évidence une zone chromosomique d'intérêt située sur le chromosome OAR5. Les deux approches de génétique moléculaire (analyses du transcriptome et du polymorphisme du génome) ont permis d'identifier des gènes fonctionnels et positionnels candidats pour comprendre les mécanismes associés à la résistance aux mammitesMastitis is udder inflammation. It is one of the major health issues in dairy cattle and sheep as they produce economic losses for the dairy industry. Mastitis is characterized by an increase in milk inflammatory somatic cells. The somatic cell score (SCS) is well correlated to clinical and subclinical mastitis. To enhance insights into the genetic mechanisms involved in such a selection on SCS, two divergent lines of sheep were generated based on extreme breeding values for SCS. The main objective of this work was to identify and improve the understanding of mechanisms that are responsible for a better resistance to Staphylococcus mastitis. Our study was based on transcriptomic analysis of three cell types after Staphylococcus contact: milk inflammatory cells, mainly composed of neutrophils, dendritic cells and mammary epithelial cells. We showed that immune cell migration and inflammatory processes were activated by different pathways in the two divergent lines. We also identified functional candidate genes that may partly explain the differences of mastitis susceptibility. In parallel, we analysed the association of DNA polymorphism with the presence of mammary abscesses caused by mastitis. The study highlighted a chromosomal region on OAR5 that is associated to the presence of mammary abscesses. Both approaches of molecular genetics as transcriptomics and genomics analyses enabled identification of candidate functional and positional genes for the better understanding of the mechanisms associated to mastitis resistance
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Wolf, Julien de. « Remise en question de la procédure de perfusion pulmonaire ex vivo par modification du liquide de perfusion avec dialyse continue dans un modèle porcin ». Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL086.
Texte intégralRésumé :
La Perfusion Pulmonaire Ex Vivo (PPEV) est une technique innovante permettant d'améliorer la fonction des poumons de donneurs à critères élargis, augmentant ainsi le nombre d'organes disponibles pour la transplantation. En ventilant et perfusant les poumons en normothermie, cette méthode permet de récupérer des poumons de qualité incertaine et de les rendre aptes à la transplantation. Cependant, la PPEV prolongée peut entraîner œdème, réponses inflammatoires et accumulation de déchets métaboliques. Pour pallier ces limitations, mes travaux de thèse ont exploré l'effet de l'intégration d'un hémodialyseur dans le circuit de PPEV afin de réguler la composition du liquide de perfusion et maintenir la viabilité pulmonaire, en évaluant les effets biologiques sur 6 et 12 heures dans un modèle porcin.Méthodes Les expérimentations ont été menées conformément aux directives de l'UE et aux réglementations françaises, approuvées par le comité éthique COMETHEA. Seize porcs ont été répartis en quatre groupes de PPEV : sans changement de liquide de perfusion, remplacement partiel horaire (protocole TORONTO), dialyse pédiatrique et dialyse adulte. La dialyse pédiatrique utilisait une membrane avec une surface efficace de 0,2 m² et un seuil de filtration de 30 kDa, tandis que la dialyse adulte utilisait une membrane avec une surface efficace de 1,8 m² et un seuil de filtration de 40 kDa. La première étude a été réalisée sur une durée de 6 heures avec les 4 groupes, et la seconde sur 12 heures avec les groupes remplacement partiel horaire et dialyse pédiatrique.Les paramètres physiologiques et métaboliques ont été mesurés, les cytokines ont été dosées par Luminex/Multiplex, et les profils d'expression génique ont été évalués par séquençage d'ARN.Résultats L'analyse des paramètres physiologiques a montré une stabilité de la compliance pulmonaire, de la pression artérielle pulmonaire et des échanges gazeux sans distinction significative entre les groupes. Les procédures de dialyse ont corrigé les déséquilibres électrolytiques et métaboliques, stabilisant les concentrations de lactate et de glucose. Cependant, les cytokines inflammatoires (TNFα, IL-6, IL-8, IL-10) ont montré une augmentation après trois heures, avec des niveaux plus élevés dans le groupe de dialyse pédiatrique.Les analyses d'expression génique ont révélé que la PPEV est associée, quelque soit le groupe, à l'activation des voies inflammatoires, de la survie cellulaire, et de la prolifération. Par contre, la dialyse pédiatrique a induit des profils d'expression prédictifs d'une plus forte activation endothéliale et d'une plus forte signalisation cytokinique par rapport au remplacement partiel horaire.Conclusion L'ajout d'un circuit de dialyse au protocole de PPEV permet un meilleur équilibre des électrolytes et du métabolisme du liquide de perfusion. Cependant, cette approche est associée à une augmentation des cytokines inflammatoires, ce qui pourrait avoir des implications négatives pour la transplantation pulmonaire. Malgré des perspectives prometteuses, des évaluations supplémentaires et des améliorations sont nécessaires avant une application clinique, notamment l'utilisation de membranes d'adsorption améliorées et l'ajout de nutriments pour optimiser le système de perfusion. Ces résultats soulignent l'importance des analyses génomiques fonctionnelles pour comprendre la réponse biologique à la PPEV et guider les améliorations futuresEx Vivo Lung Perfusion (EVLP) is an innovative technique that enhances the function of donor lungs with extended criteria, thus increasing the number of organs available for transplantation. By ventilating and perfusing the lungs at normothermia, this method allows for the recovery of lungs of uncertain quality and makes them suitable for transplantation. However, prolonged EVLP can lead to edema, inflammatory responses, and the accumulation of metabolic waste. To address these limitations, my thesis work explored the effect of integrating a hemodialyzer into the EVLP circuit to regulate the composition of the perfusion fluid and maintain lung viability, evaluating the biological effects over 6 and 12 hours in a porcine model.MethodsThe experiments were conducted in accordance with EU guidelines and French regulations, approved by the COMETHEA ethics committee. Sixteen pigs were divided into four EVLP groups: without perfusion fluid change, hourly partial replacement (TORONTO protocol), pediatric dialysis, and adult dialysis. Pediatric dialysis used a membrane with an effective surface area of 0.2 m² and a filtration threshold of 30 kDa, while adult dialysis used a membrane with an effective surface area of 1.8 m² and a filtration threshold of 40 kDa. The first study was conducted over 6 hours with all four groups, and the second over 12 hours with the hourly partial replacement and pediatric dialysis groups.Physiological and metabolic parameters were measured, cytokines were assayed by Luminex/Multiplex, and gene expression profiles were evaluated by RNA sequencing.ResultsAnalysis of physiological parameters showed stability in lung compliance, pulmonary arterial pressure, and gas exchange without significant differences between groups. The dialysis procedures corrected electrolyte and metabolic imbalances, stabilizing lactate and glucose concentrations. However, inflammatory cytokines (TNFα, IL-6, IL-8, IL-10) increased after three hours, with higher levels in the pediatric dialysis group.Gene expression analyses revealed that EVLP is associated, regardless of group, with the activation of inflammatory pathways, cell survival, and proliferation. In contrast, pediatric dialysis induced expression profiles predictive of stronger endothelial activation and cytokine signaling compared to hourly partial replacement.ConclusionThe addition of a dialysis circuit to the EVLP protocol allows for better electrolyte and metabolic balance in the perfusion fluid. However, this approach is associated with an increase in inflammatory cytokines, which could have negative implications for lung transplantation. Despite promising prospects, further evaluations and improvements are necessary before clinical application, including the use of enhanced adsorption membranes and the addition of nutrients to optimize the perfusion system. These results highlight the importance of functional genomic analyses to understand the biological response to EVLP and guide future improvements
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Mendoza, Elkin Fernando Rodas. « Identificação de genes-alvos na patogenicidade de Xanthomonas citri subsp. citri com enfoque no sistema de secreção tipo III / ». Jaboticabal, 2016. http://hdl.handle.net/11449/144186.
Texte intégralRésumé :
Orientador: Jesus Aparecido FerroCoorientador: Flávia Maria de Souza Carvalho
Coorientador: Roberto Hirochi Herai
Banca: Henrique Ferreira
Banca: José Belasque Júnior
Banca: Marcos Túlio de Oliveira
Banca: Alessandro de Mello Varani
Resumo: Xanthomonas citri subsp. citri (Xac) é o agente causal do cancro cítrico, uma das principais doenças que acometem a citricultura mundial. Atualmente não há uma maneira eficiente de controle do cancro, e novos métodos devem ser desenvolvidos para o tratamento desta doença. Assim, o estudo dos mecanismos utilizados pela Xac durante o processo infeccioso pode revelar novos alvos para o desenvolvimento de compostos farmacológicos que possam eliminar ou controlar o patógeno. Neste estudo, a técnica de RNA-Seq foi utilizada para a identificação de genes diferencialmente expressos (GDE) na Xac em condições in vivo e in vitro. Para isso, cinco variedades de citros com níveis diferentes de suscetibilidade ao cancro cítrico, e meios de cultura indutores de fatores de virulência foram utilizados. Muitos dos genes que codificam para proteínas relacionadas ao sistema de secreção tipo 3 (T3SS), enzimas extracelulares, resposta ao estresse oxidativo, transportadores de ferro e fósforo foram induzidos pela Xac nas condições in vivo. No entanto, in vitro, os perfis de expressão para estes mesmos genes foram diferentes. Estes dados permitiram compreender melhor o ambiente intracelular do hospedeiro, e como este se relaciona com os mecanismos de ativação dos fatores de virulência e patogenicidade de Xac. Neste sentido, os dados apresentados neste estudo mostraram que o T3SS é o principal fator de virulência expresso por esta bactéria em condições in vivo. Além disso, nossos resultados sugerem t... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker, a major disease affecting citrus worldwide. Currently there is no effective way of cancer control, and new methods must be developed for the treatment of this disease. Thus, the study of the mechanisms used by Xac during the infectious process can reveal new targets for the development of pharmacologic compounds that can eliminate or control the pathogen. In this study, RNA-Seq technique was used to identify Xac differentially expressed genes (DEG) in vivo and in vitro conditions. For this purpose, five citrus varieties with different levels of susceptibility to citrus canker and culture mediums inducing virulence factors were used. Many of the genes encoding proteins of the type 3 protein secretion system (T3SS), extracellular enzymes, oxidative stress response, iron and phosphorus transport were induced in Xac in vivo conditions. However, the expression profiles for these same genes were different than observed in vitro conditions. These data allowed us to better understand the intracellular environment of the host, and how this relates to the activation mechanisms of pathogenicity and virulence factors in Xac. In this context, the data presented in this study show the T3SS as the main virulence factor expressed by the bacteria in vivo conditions. Furthermore, our results also suggest that low concentrations of inorganic phosphorus (Pi) and nitrogen, that bacteria sense in the plant apoplast, are ... (Complete abstract click electronic access below)
Doutor
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Azevedo, Mariana da Silva. « Competência organogênica in vitro das linhagens MT-Rg1 e MT-pro em tomateiro (Solanum lycopersicum L. cv Micro-Tom) ». Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-04012017-093959/.
Texte intégralRésumé :
Diversos estudos elucidaram mecanismos envolvidos com a organogênese in vitro, porém pouco é conhecido a respeito da fase de aquisição de competência, fundamental para que a regeneração ocorra. Alguns genes já foram identificados por interferirem na fase de aquisição de competência em tomateiro (Solanum lycopersicum), mas ainda existem diversas lacunas a serem esclarecidas. Para investigar a expressão de genes e o controle hormonal na fase de aquisição de competência, foram utilizados os mutantes de tomateiro, sob o background genético da cultivar Micro-Tom (MT), MT-Rg1 e MT-pro (procera), os quais afetam positiva ou negativamente a organogênese in vitro, respectivamente. Embora a resposta constitutiva a giberelina no mutante MT-pro seja conhecida, a identidade molecular do gene RG1 permanece indefinida. O mutante MT-Rg1 apresenta aumento tanto na formação de gemas caulinares quanto de raízes e reduz o tempo necessário para a indução desses órgãos, devido à diminuição do período para a aquisição de competência. A partir do estabelecimento das fases de aquisição de competência e indução da organogênese in vitro para MT e MT-Rg1, foram identificados genes diferencialmente expressos entre estes genótipos. Entre estes genes, CELL DIVISION CYCLE ASSOCIATED 7 e LACCASE 1A estão regulados positivamente em MT-Rg1 e todos estão fortemente relacionados à fase de aquisição de competência, e a alterações na proliferação de células do protoxilema durante o início da organogênese. Por outro lado, a resposta constitutiva à giberelina no mutante MT-pro reduz a formação de gemas caulinares e raízes e aumenta a formação de calos in vitro, sem afetar o tempo requerido para a indução de gemas caulinares e raízes. De forma oposta a MT-Rg1, o gene CDCA7 apresenta expressão reduzida durante a fase de aquisição de competência em MT-pro, diminuindo o número de células do protoxilema em divisão. Outro fator importante para a divisão celular no mutante MT-pro é o aumento da expressão do gene WUS, causando um aumento da proliferação das stem cells, que são células indiferenciadas relacionadas à formação de novos órgãos. Esta proliferação celular inadequada, somada a uma alteração desfavorável na homeostase das citocininas, justifica o efeito negativo do alelo pro na formação de gemas caulinares, o que permitiu a criação de um novo modelo para organogênese in vitroSeveral studies have enabled the discovery of mechanisms to achieve in vitro organogenesis; however, little is known about the phase of acquisition of competence, essential for regeneration. A few genes have been identified to interfere in the acquisition of the competence phase in tomato (Solanum lycopersicum), but there are still many gaps to be filled. We have used the mutants, under the genetic background of the Micro-Tom cultivar, MT-Rg1 and MT-pro (procera), which positively or negatively affect in vitro organogenesis, respectively, to investigate gene expression and the hormonal control in the phase of acquisition of competence. Despite the fact that the constitutive gibberellin response in the procera mutant is well-established, the molecular identity of RG1 gene remains unknown. The MT-Rg1 mutant presents an increase in the formation of both shoot and roots and a reduced period for the induction of these organs, because of the reduced time required for acquisition of competence.We searched for the identity of differentially expressed genes between MT and MT-Rg1 after the establishment of the competence acquisition phase and organogenesis induction stages. Among those genes, CDCA7 and LAC1A are upregulated in MT-Rg1 and these genes appear to be strongly related with the acquisition of competence phase and changes in proliferation of protoxylem cells during early organogenesis. The constitutive response to gibberellin in the MT-pro mutant decreases the formation of shoot and roots and increase in vitro calli formation, without reducing the induction phase of shoots and roots. Unlike MT-Rg1, MT-pro reduces the CDCA7L expression during the acquisition of competence phase, causing a reduction of the protoxylem dividing cells. Another important factor for cell division in MT-pro mutant is the increased expression of the WUS gene, leading to an abnormal proliferation of stem cells. Thereby, this abnormal cell proliferation, in addition to an unfavorable change in the cytokinin homeostasis, justify the negative effect of the pro allele in the shoot formation, which enabled the proposal of a new model for in vitro organogenesis
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Kirsch, Anaïs. « Signatures moléculaires impliquées dans la transformation des cellules Bhas 42 induite par les silices amorphes synthétiques ». Thesis, Université de Lorraine, 2020. http://www.theses.fr/2020LORR0015.
Texte intégralRésumé :
Les nanoparticules de silice amorphe synthétique (SAS) sont utilisées dans de nombreuses applications industrielles. De par leur structure amorphe, les SAS ne sont pas considérées comme cancérogènes chez l'Homme, contrairement aux silices de structures cristallines. Pourtant, des études in vivo et in vitro ont proposé que certaines SAS pourraient potentiellement être cancérogènes. Le test de transformation des cellules Bhas 42 a été développé afin d’évaluer les effets transformant des agents chimiques. Une étude menée à l’INRS a mis en évidence la capacité des SAS précipitées NM-200 et NM-201 et des SAS pyrogénées NM-202 et NM-203 à induire la transformation cellulaire. Afin d’étudier les effets moléculaires induits par les SAS conduisant à la transformation des cellules Bhas 42, nous avons effectué une analyse transcriptomique non supervisée, après exposition des cellules Bhas 42 à la SAS NM-203, et à des agents chimiques de référence, la silice cristalline Min-U-Sil 5® (témoin positif particulaire), la terre à diatomée (DE), une microparticule de silice amorphe (témoin négatif particulaire) et le 12-O-tétradécanoylphorbol-13-acétate (TPA), un agent promoteur de tumeur. Les résultats montrent un lien entre la transformation des cellules Bhas 42 induite par la SAS pyrogénée NM-203, la silice cristalline Min-U-Sil 5® et le TPA et la modification de l’expression de gènes et de voies de signalisation impliquées dans la prolifération, l’adhésion cellulaire, la réparation de l’ADN et l’inflammation. De plus, nous avons identifié une signature commune de 21 gènes impliqués dans les stades précoces de la transformation cellulaire induite par les trois agents chimiques. Parmi eux, 12 gènes ont été sélectionnés en fonction de leur niveau d'expression (sur ou sous-exprimés) 48 heures après traitement avec les agents chimiques. Des altérations géniques similaires ont été obtenues lorsque des cellules Bhas 42 sont traitées avec les SAS précipitées NM-200 et pyrogénées NM-202. En conclusion, ce travail met en évidence un lien entre la formation de foyers de transformation induits par le traitement des cellules Bhas 42 avec les SAS, la silice cristalline Min-U-Sil 5® et le TPA et des modifications des profils d'expression de gènes et de voies de signalisation. La mise en évidence de la signature des 12 gènes représente une liste de "bio-marqueurs" potentiels et communs de la transformation cellulaire induite par les SAS, la silice cristalline Min-U-Sil 5® et le TPASynthetic amorphous silica nanoparticles (SAS) are used in many industrial applications. Due to their amorphous structure, SAS are not considered carcinogenic in humans, unlike silica with crystalline structures. However, in vivo and in vitro studies have proposed that certain SAS could potentially be carcinogenic. The Bhas 42 cell transformation test was developed to assess the transforming effects of chemical agents. A study carried out at the INRS highlighted the capacity of the precipitated SAS NM-200 and NM-201 and the pyrogenic SAS NM-202 and NM-203 to induce cell transformation. In order to study the molecular effects induced by SAS which lead to the transformation of Bhas 42 cells, we performed an unsupervised transcriptomic analysis, after exposure of Bhas 42 cells to SAS NM-203, and to reference chemical agents, crystalline silica Min-U-Sil 5® used as a positive particulate control, diatomaceous earth (DE), an amorphous silica microparticle used as a negative particulate control and 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter. The results show a link between the transformation of Bhas 42 cells induced by pyrogenic SAS NM-203, crystalline silica Min-U-Sil 5® and TPA and the modification of the expression of genes and signaling pathways involved in proliferation, cell adhesion, DNA repair and inflammation. In addition, we have identified a common signature of 21 genes involved in the early stages of cell transformation induced by the three chemical agents. The genes composing the common signature are involved in cell proliferation and adhesion. Among them, 12 genes were selected according to their expression level (over-expressed or under-expressed) 48 hours after treatment with chemical agents. Similar gene alterations have also been obtained when Bhas 42 cells are treated with NM-200 precipitated SAS and NM-202 pyrogenic. In conclusion, this work highlights a link between the formation of transformation foci induced by the treatment of Bhas 42 cells with SAS, crystalline silica Min-U-Sil 5® and TPA and changes in expression profiles of genes and signaling pathways. The signature of the 12 genes represents a list of potential and common "biomarkers" of cell transformation induced by SAS, crystalline silica Min-U-Sil 5® and TPA
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Arista, Gautier. « Génomique comparative et fonctionnelle de familles de gènes liés au métabolisme secondaire de la vigne (Vitis vinifera) et de ses proches parents ». Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ010/document.
Texte intégralRésumé :
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 ColmarGrapevine (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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Crivelente, Horta Maria Augusta 1981. « Análise do transcriptoma de Trichoderma harzianum para a bioprospecção de enzimas hidrolíticas = Analysis of Trichoderma harzianum transcriptome for bioprospecting of hydrolytic enzymes ». [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316510.
Texte intégralRésumé :
Orientadores: Anete Pereira de Souza, Sindélia Freitas AzzoniTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Buscando contribuir com o desenvolvimento da tecnologia de produção do etanol de segunda geração, o presente estudo analisa o transcriptoma de T. harzianum IOC-3844 utilizando técnicas de sequenciamento high-thoughput. O principal objetivo dessas análises foi identificar, caracterizar e catalogar os transcritos expressos por T. harzianum relacionados com a degradação de substratos complexos, como o bagaço de cana de açúcar, revelando o conjunto de genes envolvidos na degradação da biomassa. A análise do transcriptoma do fungo Trichoderma harzianum sob condições que induzem a degradação da biomassa permitiu a identificação de sequências de genes potencialmente eficazes no processo de biodegradação, uma etapa essencial à compreensão do processo de hidrólise enzimática. O sequenciamento resultou em 246 milhões de sequências com 72 pb, o que corresponde a 14,7 GPB analisados. Após a montagem , 32.494 contigs foram gerados, submetidos à identificação e classificados de acordo com sua identidade. Todas as sequências de contigs foram comparados com o banco de dados do NCBI, Gene Ontology (GO terms), Enciclopédia de Genes Kyoto (KEGG), Carbohydrate Active-Enzymes (CAZYmes). Foram identificados 487 CAZymes no transcriptoma, inclusive aquelas ligadas as reações químicas de despolimerização de celulose e hemicelulose. As sequências classificadas como atividade catalítica (6.975) e atividade reguladora (143) podem estar envolvidas com esse tipo de reação.A análise permitiu definir o principal conjunto de genes envolvidos na degradação da celulose e de hemicelulose do T. harzianum , e genes acessórios relativos à despolimerização de biomassa. Uma análise dos níveis de expressão permitiu determinar os conjuntos de genes diferencialmente expressos em diferentes condições de cultivo. Os resultados obtidos acrescentam conhecimento sobre a constituição do genoma, as atividades de expressão gênica do fungo Trichoderma harzianum e fornece informações importantes a respeito dos mecanismos genéticos de degradação de biomassa que o fungo utiliza. As informações obtidas poderão ser utilizadas para outras espécies de fungos filamentosos com potencial para a biodegradação
Abstract: In order to contribute to the development of second-generation ethanol technology, this study analyzes the transcriptome of T. harzianum IOC-3844 using high-thoughput sequencing techniques. The main objective of this analysis was to identify, characterize and catalog the transcripts expressed by T. harzianum related to the degradation of complex substrates such as sugar cane bagasse, revealing the set of genes involved in the degradation of biomass. The analysis of the transcriptome of the fungus Trichoderma harzianum under conditions that induce the degradation of biomass allowed the identification of genes potentially effective in the biodegradation process, an essential step for understanding the enzymatic process. Sequencing resulted in 246 million sequences with 72 bp, which corresponds to 14.7 GBP analyzed. After assembly, 32,494 contigs were generated, identified and classified according to their identity. All sequence contigs were compared with NCBI database, Gene Ontology (GO terms), Kyoto Encyclopedia of Genes (KEGG), Carbohydrate Active-Enzymes (CAZYmes). 487 CAZymes were identified in the transcriptome, including those related to reactions of cellulose and hemicellulose depolymerization. Sequences classified as catalytic activity (6,975) and regulatory activity (143) may be involved with this type of reaction. This analysis define the set of genes involved in the degradation of cellulose and hemicellulose of T. harzianum, and accessories genes related to depolymerization of the biomass. An analysis of expression levels was used to calculate the set of differentially expressed genes in different culture conditions. The results add to knowledge about the composition of the genome and gene expression activity of the fungus Trichoderma harzianum, and provides important information regarding the genetic mechanisms of biomass degradation that the fungus uses. The information obtained may be used for other species of filamentous fungi with potential for biodegradation
Doutorado
Genetica Vegetal e Melhoramento
Doutora em Genética e Biologia Molecular
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Nguyen, Phuong Anh. « Biocontrôle de la flore fongique phytopathogène et/ou mycotoxinogène : étude des mécanismes moléculaires et physiologiques impliqués dans les interactions microbiennes antagonistes, optimisation des compétences des agents biologiques de contrôle identifiés ». Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT086/document.
Texte intégralRésumé :
Fusarium verticillioides est une moisissure phytopathogène et mycotoxinogène dont l’occurrence est fréquente dans le sol et dans de nombreux céréales et végétaux et plus particulièrement dans le maïs et le blé. La contamination des cultures par F. verticillioides conduit à des maladies variées et est souvent associée avec une accumulation de mycotoxines. Les fumonisines B1 (FB1) and B2 (FB2) sont les toxines les plus dangereuses produites par F. verticillioides. Des conséquences létales de la consommation de produits alimentaires contaminés par les fumonisines ont été reportées chez les animaux et l’homme. Différentes démarches ont été utilisées pour lutter contre cette moisissure et ses toxines mais souvent liées à l’utilisation de fongicides chimiques par ailleurs reconnus pour leurs effets néfastes pour l’homme, les animaux et l’environnement. Des pratiques alternatives ont été développées pour préserver les récoltes en respectant les écosystèmes et la santé humaine et animale. L’utilisation d’amendements organiques est apparue comme une stratégie intéressante de contrôle des maladies des plantes en assurant un apport de PGPM (Plant Growth Promoting Microorganisms) et agents de biocontrôle. Notre étude a donc eu pour but de caractériser les communautés microbiennes, et plus particulièrement celles d’intérêt pour le biocontrôle, d’amendements organiques et sols amendés ainsi que de tester leur potentiel antifongique envers F. verticillioides. Les mécanismes de biocontrôle ont été étudiés en utilisant des approches métabolomiques et transcriptomiques par séquençage haut débit. L’étude des communautés microbiennes dans les amendements et les sols amendés a été réalisée par pyroséquençage de marqueurs qui sont des portions du gène ribosomal 16S et de la séquence ITS2. Le microbiote des amendements est essentiellement constitué par les familles des Pseudonocardiaceae, Bacillaceae et Trichocomaceae qui incluent différents PGPM et agents antifongiques. Par ailleurs les amendements semblent favoriser l’installation des familles d’intérêt dans les sols tout en limitant celle des pathogènes tels les Nectriaceae qui comprennent des Fusarium pathogènes. Les tests d’antagonisme ont montré que les sols amendés réduisaient la croissance de F. verticillioides de façon plus importante que le sol de référence en inhibant la production de microconidies. La production de fumonisines a également été fortement réduite en présence des métabolites produits par le microbiote des sols amendés (plus de 68 % et 92 % pour FB1 and FB2 respectivement). Des souches d’actinomycètes isolées des amendements, et identifiées comme appartenant au genre Streptomyces, ont montré une activité antifongique envers F. verticillioides. Parmi celles-ci, Streptomyces AV05 a été sélectionnée pour les tests supplémentaires en raison de son fort potentiel d’inhibition vis-à-vis de F. verticillioides. Des comparaisons des endométabolomes et transcriptomes des 2 souches cultivées seules ou en confrontation ont été réalisées et de nombreuses différences ont été remarquées pour chacune entre les 2 modalités. Ainsi, 29 métabolites impliqués dans les modifications du métabolome de F. verticillioides en co-culture avec Streptomyces AV05 ont été identifiés et différentes voies métaboliques semblent avoir été affectées. L’étude du transcriptome des 2 souches a donné des résultats allant dans le même sens que celle du métabolome. En effet des changements significatifs de niveau d’expression ont été observés au niveau transcriptomique pour 800 gènes de F. verticillioides et 115 gènes de Streptomyces AV05 en situation de confrontation. L’identification de ces gènes est en cours et plusieurs d’entre eux semblent impliqués dans les modifications des profils métaboliques observées chez les 2 souches. L’intégration des données métabolomiques et transcriptomiques devrait permettre d’améliorer a compréhension des mécanismes mis en jeu au cours de la confrontationFusarium verticillioides is a phytopathogenic and mycotoxigenic filamentous fungus that can be found with high occurrence in soils and in a wide range of cereals and vegetables, particularly in corn and wheat. The F. verticillioides contamination in crops leads to various diseases and is usually associated with an accumulation of mycotoxins. Fumonisin B1 (FB1) and fumonisin B2 (FB2) are the most dangerous mycotoxins produced by Fusarium verticillioides. Lethal consequences caused by fumonisins have been reported in animals and in human due to the consumption of contaminated food products. To deal with this pathogenic fungus and its mycotoxins, several approaches have been applied but they are usually based on the use of chemical agents that are reported to be unsafe for humans, animals and ecosystems. Alternative practices that maintain the quality and the abundance of crops while preserving the ecosystems and human and animal health have been developed. The application of organic amendments has been reported as an interesting strategy for controlling diseases by providing an abundant source of PGPM (Plant Growth Promoting Microorganisms) and biocontrol agents. The aim of our study was to characterize the microbial communities of organic amendments and amended soils focusing on the microbial families of interest for biocontrol and to assess the antifungal potential of amended soils and their microbiota towards F. verticillioides. Mechanisms of biocontrol were studied using metabolomics and transcriptomics approaches. Evaluation of microbial communities in amendments and amended soils using pyrosequencing of the 16S rDNA and the ITS genes showed that the amendments contained mainly the families of Pseudonocardiaceae, Bacillaceae and Trichocomaceae that were believed to include various PGPM and antifungal agents. Furthermore, the amendments were expected to promote the families of interest in soil and also to limit those of pathogens such as Nectriaceae that might contain many pathogenic Fusarium. Antifungal assays showed that the amended soils reduced the F. verticillioides growth better than the reference soil by inhibiting the microconidia production. The fumonisin production was strongly reduced by the metabolites produced by the amended soils’ microbiota (up to 68 % and 92 % for FB1 and FB2 respectively). Some actinomycete isolates from these amendments were identified as Streptomyces and they demonstrated antifungal activity against F. verticillioides. Among the Streptomyces strains, Streptomyces AV05 was selected for further studies because of its strong inhibition towards F. verticillioides. The interaction between the Streptomyces strain AV05 as antagonist agent and F. verticillioides was investigated. The study of the endometabolome and the transcriptome of the two microorganisms was carried out in two different conditions: strains cultivated alone or in confrontation. Many modifications have been noticed into the endometabolome of the 2 microorganisms during the direct confrontation and 29 metabolites involved in the endometabolome alteration of F. verticillioides in co-culture with Streptomyces AV05 were identified. Many fungal metabolic pathways were suggested to have been affected. The results of the study of the mRNA of the 2 strains appeared to be in accordance with the metabolomics results. A change in the transcriptomic level was observed: 800 genes of F. verticillioides and 115 genes of Streptomyces AV05 were found to be expressed differently when the strains were cultivated alone or in confrontation. The identification of these genes is in progress. Many of them are expected to be responsible for the change in the metabolic profiles observed in the bacterial-fungal interaction. The integration of the metabolomic and transcriptomic informations could improve the understanding of the mechanisms of biocontrol during direct confrontation
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Silva, Raphael Tavares da. « Desenvolvimento de uma abordagem computacional para a tradução in silico de variantes de splicing detectadas no transcriptoma humano ». reponame:Repositório Institucional da FIOCRUZ, 2012. https://www.arca.fiocruz.br/handle/icict/6406.
Texte intégralRésumé :
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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.
Um dos mecanismos capaz de aumentar a diversidade do proteoma de eucariotos é o splicing alternativo nos pré-mRNAs. Este mecanismo celular ocorre durante a transcrição dos genes, sendo ocasionado por um ou mais dos seguintes eventos: retenção de íntrons, uso alternativo de sítio de splice 5', uso alternativo de sítio de splice 3' e uso alternativo de éxons. Análises recentes de Bioinformática utilizando experimentos de RNA-Seq mostram que aproximadamente 90% dos genes humanos produzem mais de um transcrito decorrente de eventos de splicing alternativo. O impacto do splicing alternativo no proteoma humano vem sendo alvo de algumas abordagens de Bioinformática, sendo esperado que uma grande porção de tais transcritos alternativos possa alterar o conteúdo da cadeia polipeptídica obtida após a sua tradução. Devido à sua importância, diversos trabalhos já foram desenvolvidos com o objetivo de facilitar a identificação de eventos de splicing alternativo a partir de dados provenientes de cDNA, bem como sua associação com a estrutura das proteínas de suas isoformas. Entretanto, são poucas as abordagens que realizaram a tradução in silico do transcriptoma humano na busca por variantes de splicing e a utilização de dados oriundos de sequenciadores de segunda geração (NGS) ainda é muito pouco explorada para tratar do tema. Desta maneira, o presente projeto tem como objetivo a aplicação de uma nova abordagem para a identificação e tradução de variantes de splicing alternativo usando dados de NGS. Foram utilizadas leituras da plataforma de sequenciamento Roche/454 oriundas de estudos de câncer para um enriquecimento de nosso banco de dados original que continha previamente mRNAs completos e ESTs. Após o enriquecimento, a metodologia empregada pelo nosso grupo conseguiu detectar 4.574 variantes de splicing inéditas em nosso banco. O novo banco gerado foi traduzido levando a criação de um repertório proteico contendo 159.638 sequências polipeptídicas não redundantes. Na busca por variantes inéditas utilizando dados de proteômica, foram identificadas três possíveis nos genes humanos tubulina 2b, tubulina 4b e actina. Dados de sequenciamento da plataforma Illumina também foram utilizados para uma avaliação da sua contribuição em número de variantes e sequências polipeptídicas traduzidas em nosso repertório. Encontramos que a nossa abordagem foi capaz de anotar 53% mais sequências polipeptídicas quando comparada ao repertório de ENSEMBL Gene. Desta forma, acreditamos que o presente projeto pode auxiliar no melhoramento da anotação de peptídeos encontrados por técnicas de proteômica, bem como no descobrimento de novos marcadores moleculares.
Alternative splicing of pre-mRNAs is one of the mechanisms capable to increase the proteome diversity in eukaryotes. This cellular mechanism occurs during the transcription of genes and is associated with one or more of the following events: intron retention, 5’ alternative splice, 3’ alternative splice and exon skipping. Recent Bioinformatics analysis using RNA-Seq experiments showed that approximately 90% of human genes produce more than one transcript due to alternative splicing events. The impact of alternative splicing in the human proteome has been the focus of some Bioinformatics approaches and is expected that the majority part of these alternative transcripts can alter the polypeptide chain produced after its translation. Due to its importance, many studies have been developed focused on facilitating the identification of alternative splicing events based on cDNA data, as well as to study the protein structure of its isoforms. However, few studies performed the in silico translation of the human transcriptome to search for new splicing isoforms using Next Generation Sequencing (NGS) data. In this way, our project aims to the development of a new approach to identify and translate alternative splicing isoforms using NGS data. Roche/454 reads of cancer studies were used to enrich our initial database, which was previously populated with full-length mRNAs and ESTs data. After the enrichment step, the methodology developed by our group could detect 4,574 new splicing variants in our database. The enriched database was translated, producing a protein repository with 159,638 non-redundant polypeptide sequences. Searching for new isoforms using experimental proteomic data, three possible new isoforms were identified for the human genes tubulin 2b, tubulin 4b and actin. Illumina sequencing data was used to assess its contribution for the number of new isoforms and the translated polypeptide sequences on our database. We realized that our approach was capable to annotate 53% more polypeptide sequences when compared with the ENSEMBL Gene repository. In this way, we believe that our project can support the improvement of peptide annotation found by proteomic techniques, as well as to discover new molecular markers.
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Loe-mie, Yann. « Contribution bioinformatique à l' analyse du transcriptome humain ». Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4002/document.
Texte intégralRésumé :
Dans la première partie j'ai analysé des jeux de données de RNA-seq de transcriptome de petits ARNs disponibles dans les bases de données publiques. J'y ai observé 2 points intrigants : - une grande partie des lectures (bien que courtes) ne peux pas être alignée sur le génome de référence sans discordance et cette fraction non-alignable est parfois majoritaire. - de nombreuses lectures ont des tailles autours de 15-18nt qui ne correspondent à aucun type de petits ARNs connues, cette fraction est également majoritaires dans certains cas. Ces expériences sont souvent conçues pour la détection des miRNAs et l'analyse bioinformatique de ces données passent toujours par un alignement sur le génome de référence ou sur des séquences connues pour donner des petits ARNs. J'ai donc simplement éliminé la contrainte d'alignement dans l'analyse de ces données et effectué un regroupement des lectures par similarité (à la manière des ESTs). Ce regroupement donne une vision différente des données dans laquelle la notion de position génomique n'est plus centrale et ouvre la possibilité d'y découvrir des phénomènes non-standard. La deuxième partie est tirée d'une collaboration avec le laboratoire U675 INSERM. J'ai fait l'analyse bioinformatique des gènes dérégulés par la répression par RNAi du gène REST dans une lignée de neuroblastome de souris (N18). Ce gène est un facteur de transcription qui réprime les gènes neuronaux dans les cellules non neuronales. Ce répertoire de gènes dérégulés est potentiellement constitué de gènes clefs dans la biologie des neuronesIn first part of this thesis I have analysed small RNA-seq transcriptome data. I have noticed : - a large fraction of reads can't be aligned perfectly on reference genome - lot of reads are very short (15-18 nt) and don't match on previously known functionnal small RNAs. These experiments are designed for miRNA discovery and bioinformatics analysis of these data use alignments on genome or on known small RNA precursors sequences. I have eliminated the alignment and I have clustered these sequences. This clustering let me to observe these data with a new view in wich the genomic location is not central and open the gate to discover unconventional events. The second part is the analysis of deregulate genes by the silencing of the gene REST/NRSF in mouse N18 cell line. This gene is a transcription factor and it works as a repressor of neuronal genes in non neuronal cells. This deregulate genes repertoire potentially contains key genes in neuron biology. We found in this repertoire a network of genes centered on SWI/SNF complex including SMARCA2. This gene was associated to schizophrenia (SZ) in association studies and structural variation studies. In this network we found another genes associated to SZ. We show that these genes exhibit positive evolution in primate compare to rodents
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Grison, Stéphane. « Etude des effets multigénérationnels d'une exposition chronique à faible dose d'uranium par analyses omiques ». Thesis, Université Clermont Auvergne (2017-2020), 2018. http://www.theses.fr/2018CLFAC043/document.
Texte intégralRésumé :
Pour enrichir les connaissances scientifiques sur les effets biologiques des radionucléides et risques des contaminations chroniques sur la descendance, une étude multigénérationnelle in vivo d’exposition a été réalisée à doses non toxiques d'uranium. Ce modèle, a permis de suivre les effets biologiques de l’uranium sur trois générations de rats (F0, F1 et F2) par des analyses cliniques et le suivi de marqueurs biologiques. Dans cette étude, des analyses métabolomiques, transcriptomiques et épigénomiques ont été réalisées à partir d’échantillons de sang, d’urine et de rein.Pour la première génération des rats contaminés (F0), des différences dépendant du sexe des animaux sont observables par l’analyse des niveaux d’expression géniques (ARNm et micro-ARN) dans les reins, des profils métabolomiques et biochimiques dans les reins, l’urine et le sang. Aucune modification épigénétique des profils de méthylation de l’ADN rénal n’est à noter. Pour les deux générations suivantes (F1 et F2), un effet multigénérationnel dépendant aussi du sexe des rats est observable au niveau des profils métabolomiques urinaires et rénaux ainsi qu’au niveau des profils épigénétiques de méthylation de l'ADN des reins. Une baisse de poids corporel et des reins a aussi été observée pour la troisième génération de rats chez les mâles (F2).En conclusion, les travaux de cette thèse montrent qu’une contamination chronique à faible dose d'uranium entraine des effets biologiques sur plusieurs générations de rats. Ils sont observables à différents niveaux moléculaires des systèmes de régulation cellulaires et dépendent du sexe des rats. Ces effets, étroitement liés à des systèmes biologiques intégrés, sont utiles à la compréhension des mécanismes biologiques des expositions à l'uranium et à l’évaluation des risques de nocivités à long termes. Dans le domaine de la radioprotection, ces résultats justifient la nécessité de considérer les dimorphismes sexuels des individus et les conséquences des expositions sur les générations à venirIn order to deepen scientific knowledge regarding biological effects of radionuclides and associated risk to offspring, an in vivo multigenerational study of chronic exposure to a non-toxic dose of uranium was performed by monitoring three generation of rats (F0, F1 and F2). Clinical parameters and biological markers, including metabolomics, transcriptomics and epigenomics high throughput analysis were conducted in blood, urine and kidney samples.For the first generation of contaminated rats (F0) sex-differences to uranium effects were observed in kidney for gene expression (mRNA, miRNA) and in kidney, urine and blood for biochemical parameters and metabolomics profiles. No epigenetic modification of DNA methylation profiles was shown in kidney. For the next two generations (F1, F2), a multigenerational sex-specific effect is observed for both metabolomics and renal DNA methylation profiles of contaminated rats. Moreover, for the last generation of male rats (F2), a decrease of both total body and kidney weight was shown.In conclusion, low-dose chronic contamination of rats to uranium leads to multigenerational effects. Including sex-differences, they can be shown at different molecular levels of the cellular system. Depending of integrated system biology, data of this thesis are useful in the understanding of biological mechanisms of uranium effect and risk of delayed harmful effect. In the field of radiation protection, these results prove the requirement of considering sexual dimorphisms and consequences of such exposures to offspring
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Cardoso, Kiara Carolina 1979. « Transcriptoma da glândula venenífera da serpente Bothrops alternatus (urutu) e caracterização molecular e bioquímica parcial da dipeptidilpeptidase IV ». [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312156.
Texte intégralRésumé :
Orientador: Stephen HyslopTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: O estudo do transcriptoma de bibliotecas de cDNA da glândula venenífera de serpentes, realizado a partir da análise de ESTs (expressed sequence tags), tem se mostrado útil na identificação de genes expressos neste tecido, inclusive no gênero Bothrops, responsável pela maioria dos acidentes ofídicos no Brasil. Neste trabalho utilizamos uma abordagem transcriptômica para analisar a composição gênica da glândula venenífera da serpente Bothrops alternatus, uma espécie encontrada no sudeste e sul do Brasil, Uruguai, norte da Argentina e leste do Paraguai. Também clonamos e caracterizamos parcialmente a enzima dipeptidilpeptidase IV (DPP IV), uma enzima que cliva peptídeos com prolina ou alanina na penúltima posição em sua porção N-terminal e que tem sido detectada em diversas peçonhas ofídicas. A construção de bibliotecas de cDNA usando métodos convencionais de clonagem, sequenciamento e análise bioinformática resultou em 5,350 ESTs que foram reunidas em 838 contigs and 4512 singletons. Pesquisas a partir de bancos de dados relevantes (BLAST) mostraram 30% de hits e 70% no-hits. Os transcritos relacionados a toxinas correspondem a 23% do total de transcritos e 78% dos hits, respectivamente. A análise por ontologia gênica (GO) detectou genes relacionados ao metabolismo geral, transcrição, tradução, processamento, degradação de polipeptídeos, funções estruturais, e regulação celular. Os principais grupos de toxinas identificados foram metaloproteinases (81%), peptídeos potenciadores da bradicinina/peptídeos natriuréticos do tipo C (8,8%), fosfolipases 'A IND. 2' ('PLA IND. 2'; 5,6%), serinoproteinases (1.9%) e lectinas do tipo C (1,5%). As metaloproteinases eram quase queexclusivamente da classe PIII, com poucas da classe PII e nenhuma da classe PI. As 'PLA IND. 2' eram todas ácidas; nenhuma 'PLA IND. 2' básica foi detectada. Outras toxinas encontradas incluíram a L-aminoácido oxidase, proteínas secretadas ricas em cisteína, DPP IV, hialuronidase, toxinas three-finger e ohanina. Foram identificadas duas proteínas não-tóxicas, a tioredoxina e a Dusp6 (fosfatase de dupla especificidade) que mostraram alto grau de similaridade a proteínas semelhantes de outras serpentes. Também foram observados polimorfismos de nucleotídeos únicos (SNPs - single-nucleotide polymorphisms), microssatélites, transposons e repetições invertidas, todos os quais podem contribuir de alguma forma para a multiplicidade de toxinas na glândula. Estes resultados mostram que a glândula venenífera de B. alternatus possui as principais classes de toxinas encontradas em estudos transcriptômicos e proteômicos de outras espécies botrópicas. ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: Transcriptomic studies of snake venom gland cDNA based on the analysis of expressed sequence tags (ESTs) have been useful in identifying the genes expressed in this organ in a variety of species, including the genus Bothrops, which is responsible for most venomous snakebites in Brazil. In this work, we used a transcriptomic approach to analyze the gene composition of the venom gland of Bothrops alternatus (urutu), a species found in southeastern and southern Brazil, Uruguay, northern Argentina e eastern Paraguay. We also cloned and partially characterized dipeptidylpeptidase IV (DPP IV), an enzyme that cleaves peptides with proline or alanine as the penultimate residue in the N-terminal region and has been identified in several snake venoms. A cDNA library constructed using conventional methods of cloning, sequencing and bioinformatic analysis yielded 5,350 ESTs that formed 838 contigs and 4512 singletons. Databank BLAST searches yielded 30% hits and 70% no-hits. Toxin-related transcripts accounted for 23% of the total transcripts and 78% of the hits. Gene ontology analysis detected genes related to general metabolism, transcription, translation, processing, polypeptide degradation, structural functions and cellular regulation. The main toxin groups identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases 'A IND. 2' ('PLA IND. 2'; 5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively class PIII, with few class PII and no class PI enzymes. The 'PLA IND. 2' were all acidic; no basic 'PLA IND. 2' were detected. Other toxins identified included L-amino acid oxidase, cysteine-rich secretory proteins, DPP IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and a dual specificity phosphatase (Dusp6), shared high sequence homology with similar proteins from other snakes. Single-nucleotide polymorphisms (SNPs), microsatellites, transposons and inverted repeats were also observed and may contribute to the toxin diversity of the gland. These results show that the venom gland of B. alternatus contains the major toxin classes identified in transcriptomic and proteomic studies of other Bothrops species. The predominance of class PIII metalloproteinases agrees with the hemorrhagic activity of this venom, while the low content of serine proteinases and C-type lectins could account for the less intense coagulopathy observed after envenoming by this species. The lack of basic 'PLA IND. 2' agrees with the lower myotoxicity of this venom compared to other Bothrops species. ... Note: The complete abstract is available with the full electronic digital thesis or dissertations
Doutorado
Farmacologia
Doutor em Farmacologia
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Rodó, Morera Jordi. « Transcriptomic analysis of white and brown adipose tissue during non-shivering thermogenesis ». Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667915.
Texte intégralRésumé :
L’obesitat i la diabetis tipus 2 (DT2) són dues malalties estretament relacionades que representen un greu problema de salut, social i econòmic per la seva alta prevalença a tot el món. Ambdues malalties també es relacionen amb altres patologies que presenten una mortalitat elevada. Les teràpies disponibles actualment no són del tot efectives. Per tant, el desenvolupament de noves estratègies terapèutiques per a l'obesitat i la DT2 és crucial.
El teixit adipós s'ha definit com un òrgan que té un paper central en el control del balanç energètic. El descobriment de funcions endocrines i termogèniques dels adipòcits han tornat a centrar l’interès en l’estudi d’aquest teixit. La termogènesi no associada a tremolor està descrita per dur-se a terme al teixit adipós marró dels ratolins, però sota certs estímuls, com l'exposició prolongada al fred, cèl·lules amb una morfologia similar als adipòcits marrons (adipòcits beix) apareixen en alguns dipòsits de teixit adipós blanc de rosegadors i humans, un procés conegut com a browning. L’activació a través de l’exposició al fred de la termogènesi no associada a tremolor en humans augmenta el consum energètic en repòs, la sensibilitat a la insulina i millora el metabolisme de la glucosa. No obstant, es necessiten més estudis d'expressió gènica per aprofundir en els mecanismes moleculars subjacents a la millora induïda per fred a través de la termogènesi no associada a tremolor, així com per determinar les diferències entre l'activació del teixit adipós marró (BAT) i el browning del teixit adipós blanc (WAT).
En aquesta tesi doctoral, es van examinar els canvis transcriptómica dels dipòsits adiposos blancs epidídimal i inguinal (eWAT i iWAT, respectivament), així com la del dipòsit adipós marró interscapular (iBAT) de ratolins exposats a 22ºC o 4ºC durant 4 dies. L’exposició al fred va augmentar l’activitat metabòlica i termogènica de l’iWAT. En aquest dipòsit, els gens relacionats amb la glucòlisi, el cicle de l'àcid tricarboxílic, la lipòlisi i la degradació d'alguns aminoàcids van presentar una expressió incrementada per mantenir la potència protonmotriu per generar calor. A més, l'expressió de gens relacionats amb la termogènesi també va augmentar molt, demostrant una inducció del browning induïda per fred en el iWAT. S'ha descrit que el dipòsit d’eWAT és resistent al browning. Per tant, l’activació metabòlica observada d’aquest dipòsit va ser suau en comparació amb la d’iWAT, i en aquest dipòsit no es va observar cap millora rellevant de la termogènesi no associada a tremolor. Finalment, l’iBAT ja presentava nivells d’expressió de gens termogènics elevats, ja que els ratolins no estaven estabulats a termo-neutralitat.
L’observació que els gens termogènics i metabòlics presentaven un patró d’expressió similar entre les mostres va recolzar l’ús d’eines d’anàlisi de patrons per descriure Atp4b i 1700040L02Rik com a nous gens potencialment implicats en la termogènesi. La sobreexpressió d’Atp4b i 1700040L02Rik en el teixit adipós mitjançant l’ús de vectors AAV va produir una reducció del guany de pes corporal, una disminució del pes de l'eWAT i del fetge, la millora de la hipertròfia dels adipòcits blancs i la reducció de la esteatosi hepàtica. Tots aquests resultats van ser potencialment fruit de la termogènesi millorada detectada en iWAT. En general, aquests resultats indiquen un possible nou paper anti-obesogènic d’aquests gens.
Els resultats d’aquesta tesi han contribuit a una millor comprensió de la inducció de la termogènesi no associada a tremolor en els dipòsits de teixit adipós de ratolins. Entre els diferents dipòsits de teixit adipos, l’anàlisi exploratori dels nivells d’expressió gènica va determinar que el iWAT era el dipòsit que va respondre de manera més significativa a l’exposició al fred. A més, tal com es va observar en l’anàlisi de l’enriquiment de vies i d’ontologia gènica, aquesta resposta va ser altament coordinada, presentant un elevat nombre de gens relacionats amb rutes metabòliques que presentaven una expressió altament incrementats. Igualment, l’estudi detallat de les vies metabòliques va destacar una gran inducció de la termogènesi no associada a tremolor, revelar que els mecanismes de producció i de consum d’energia estaven altament sincronitzats. Aquest estudi detallat del teixit adipós també va permetre la identificació de nous gens potencialment implicats en la termogènesi no associada a tremolors.Obesity and type 2 diabetes (T2D) are two closely related diseases that represent a serious health, social and economic problem due to their high prevalence worldwide. Both diseases are also associated with other pathologies that present high mortality. The currently available therapies are not entirely effective. Thus, the development of new therapeutic strategies for obesity and T2D is crucial. Adipose tissue has been defined as an organ that plays a central role in the control of energy balance. The proved endocrine and thermogenic functions of adipocytes has renewed interest in the study of this tissue. Non-shivering thermogenesis has been described as occurring in brown adipose tissue of mice, but under certain stimuli, such as prolonged cold exposure, brown fat-like cells (beige adipocytes), appear in some white adipose tissue depots of rodents and humans. The activation of non-shivering thermogenesis in humans through cold-exposure increases resting energy expenditure, whole-body glucose disposal, insulin sensitivity, and ameliorates glucose metabolism independently of BMI. However, more gene expression studies to gain insight into the molecular mechanisms underlying the cold-induced enhancement of non-shivering thermogenesis, as well as to determine differences between BAT activation and browning of WAT, are needed. In this study, the transcriptomic response of epididymal and inguinal white adipose depots (eWAT and iWAT, respectively) as well as that of the interscapular brown adipose depot (iBAT) of mice either exposed to 22ºC or 4ºC for the period of 4 days were examined. Cold exposure increased the metabolic and thermogenic activity of iWAT. In this depot, genes related to glycolysis, tricarboxylic acid cycle, lipolysis, and the degradation of some amino acids presented a high upregulation to maintain the protonmotive power to generate heat. Moreover, the expression of thermogenic-related genes was also highly increased, demonstrating a cold-induced browning of iWAT. The eWAT depot has been reported to be resilient to browning. Thus, the observed metabolic activation of this depot was mild in comparison with that of iWAT, and no relevant enhancement of non-shivering thermogenesis was observed in this depot. Finally, iBAT already presented high expression levels of thermogenic genes because mice were not housed at thermoneutrality. The observation that genes related to thermogenesis and metabolism presented a similar expression pattern among samples endorsed the utilization of pattern matching analysis tools to unravel Atp4b and 1700040L02Rik as novel genes potentially involved in thermogenesis. The overexpression of Atp4b and 1700040L02Rik in adipose tissue by means of AAV vectors produced a body weight gain reduction, decreased eWAT, and liver weight, amelioration of white adipocytes hypertrophy, and reduced hepatic steatosis potentially as a result of the detected enhanced thermogenesis in iWAT. Overall, these results indicate a new potential anti-obesogenic role for these genes. The results from this thesis contributed to a better understanding of the induction of non-shivering thermogenesis in adipose tissue depots in mice. Among the different adipose depots, exploratory data analysis of the gene expression levels of mice exposed from 22ºC to 4ºC determined that iWAT was the depot that responded most significantly to cold exposure. Moreover, as observed in the pathway enrichment and gene ontology analysis, this response was highly coordinated, presenting a high number of genes related to metabolic pathways highly affected. The detailed study of the metabolic pathways led to the detection of a high induction of non-shivering thermogenesis, revealing that both energy production and energy consumption mechanisms were highly synchronized. This in detail study of the adipose tissue also allowed the identification of novel genes potentially involved in non-shivering thermogenesis.
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Cardoso-Silva, Cláudio Benício 1982. « Análise do transcriptoma e de sequências genômicas de variedades comerciais de cana-de-açúcar = Transcriptome and genomic sequences analysis of commercial sugarcane varieties ». [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316503.
Texte intégralRésumé :
Orientadores: Anete Pereira de Souza, Renato Vicentini dos SantosTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A cana-de-açúcar é uma das espécies de maior importância econômica no mundo devido ao seu potencial bioenergético. No entanto, o seu alto nível de complexidade genética é um desafio para a aplicação de ferramentas moleculares no melhoramento. Os recentes avanços das tecnologias de sequenciamento e genotipagem indicam o potencial de aumentar o nosso entendimento sobre a genética e a biologia molecular desta espécie. As sequências genômicas e de transcriptomas são valiosa fonte de informação para o desenvolvimento de ferramentas moleculares que permitam a identificação de regiões no genoma que estejam relacionadas com características de interesse para o melhoramento. O uso das novas tecnologias de sequenciamento de alto desempenho tem grande potencial de impacto nestas pesquisas. A presente tese objetivou analisar o transcriptoma de seis variedades comerciais e dados genômico da variedade R570, com a finalidade de identificar genes potencialmente úteis para o desenvolvimento de marcadores moleculares. A partir do método RNA-Seq, foram geradas mais de 400 milhões de sequências, as quais permitiram obter um total de 72.269 transcritos representados por uma única isoforma montados com auxílio do programa Trinity. Estes transcritos foram alinhados com sequências de Viridiplantae, gramíneas, e exclusivamente contra proteínas de sorgo, arroz, milho e transcriptoma de cana-de-açúcar, depositados em banco de dados público. Esta análise permitiu identificar o conjunto de genes de cana-de-açúcar compartilhados com outras gramíneas, bem como levou à identificação de novos transcritos que não haviam sido catalogados para cana-de-açúcar, além de longos RNAs não codificantes. Os transcritos foram anotados no Cluster of Orthologous Groups (COG) e no Gene Ontology (GO), com posterior análise de enriquecimento dos termos GO, a partir da qual foram anotados os transcritos, possivelmente relacionados a genes que conferem características de importância agronômica. No transcriptoma foram identificados mais de 700 mil SNPs e aproximadamente cinco mil regiões microssatélites. Analisando um total de 32 Mbp de sequências genômicas da variedade R570 foram identificados 4.342 microssatélites, com frequência média de sete SSR/Kb. As sequências geradas e exploradas neste trabalho são valiosa fonte de informações para entender a arquitetura genética da cana-de-açúcar, principalmente para o desenvolvimento de marcadores moleculares, os quais podem ser utilizados no mapeamento genético
Abstract: The sugarcane is one of the most economically important species in the world, due to their energy potential. However, high level of genetic complexity has been a major challenge for the use of molecular tools applied to improvement of this crop. Recent advances in sequencing and genotyping technologies indicate the potential to increase our understanding of the genetics and molecular biology of this specie. The genomic and transcriptomic are valuable sources of information for the molecular tools development that allow identification of regions in the genome that are related to characteristics of interest for the improvement. The high-throughput sequencing technologies have great impact of this research. This thesis aimed to analyze the transcriptome of six commercial varieties and genomic sequencing from R570 variety, in order to identify genes potentially useful for the molecular markers development. From RNA-Seq method were generated over 400 million sequences, which allowed obtain a total of 72,268 transcripts representing a single isoform assembled by Trinity. These transcripts were aligned against Viridiplantae, grasses, and exclusively against sorghum, rice and maize proteins, and sugarcane transcriptome available in the public database. This analysis allowed identifying a set of shared genes with other grasses, new transcripts that had not yet been cataloged for sugarcane and long non-coding RNAs. The transcripts were also annotated using the COG (Cluster of Orthologous Groups) and GO (Gene Ontology) database, followed by enrichment analysis for GO terms, from which it was possible to identify genes that play roles, possibly related to traits of agronomic importance. In the transcriptome were identified over 700 thousands SNPs and five thousands microsatellites regions. In the genomic sequences from R570 variety, in a total of 32 Mbp were identified 4,342 microsatellites, with an average frequency of seven SSR / Kb. The sequences generated and explored in this work is a valuable source to understand the genetic architecture of the sugarcane, mainly for molecular markers development, which can be used in genetic mapping
Doutorado
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular
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Camargo, Eduardo Leal Oliveira 1981. « Influência do nitrogênio na formação e qualidade da madeira de eucalipto ». [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314277.
Texte intégralRésumé :
Orientador: Gonçalo Amarante Guimarães PereiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O nitrogênio (N) é o principal macronutriente necessário para o desenvolvimento das plantas e acúmulo de biomassa. O gênero Eucalyptus é a principal fonte de biomassa para as indústrias de base florestal, principalmente a de papel e celulose. Suas altas taxas de crescimento, propriedades da madeira e repostas a manejo silviculturais tem grande potencial para utilização na geração de biocombustíveis e químicos. Neste novo cenário, a projeção é de uma crescente demanda de biomassa e, em função disso, é necessário associar novas estratégias aos programas de melhoramento genético, que resultem não somente no aumento da produtividade, como também na adequação de caracteres da qualidade da madeira. A qualidade da madeira depende de diversas propriedades do xilema secundário que são influenciadas por fatores genéticos e ambientais, como a disponibilidade água e nutrientes. Neste estudo foi demonstrado que a fertilização nitrogenada causa alterações na formação e qualidade do xilema secundário de eucalipto. Plantas de um clone comercial de Eucalyptus urophylla x E. grandis foram submetidas a adubações contrastantes de N. Análises histológicas de regiões caulinares demonstraram que a deposição de lignina foi reduzida pela fertilização em excesso de N, enquanto um aumento consistente foi observado nas plantas cultivadas em solução deficiente em N. Esses resultados foram corroborados por análises químicas. Pela análise de sequenciamento de RNA foram identificados 1.469 genes diferencialmente expressos e exibindo um padrão de expressão relacionado à quantidade de N disponível. Dentre esses se destacam genes relacionados ao metabolismo de nitrogênio e fenilpropanóides. Os resultados possibilitaram entender os mecanismos envolvidos na manutenção das altas taxas de lignificação sob deficiência de N, bem como apontar possíveis genes candidatos relacionados com a qualidade da madeira de eucalipto
Abstract: The nitrogen (N) is the main macronutrient required for plants development and biomass accumulation. The genus Eucalyptus is the principal source of biomass to forest industries, mainly to the pulp and paper. Their fast growth, valuable wood properties and ease of management have great potential to generate biofuel and chemicals. In this scenario, the projections lead to a high demand of biomass, resulting in the need to associate new strategies of genetic improvement programs, which results in a higher productivity with a better wood quality. The wood quality depends from many properties of secondary xylem that are influenced by genetics and environment factors, like water and nutrients availability. In this study, we demonstrate in Eucalyptus that N fertilization promotes changes on secondary xylem formation and quality. Plants from a commercial clone of Eucalyptus urophylla x E. grandis were submit to contrasting N fertilizations. The histological analyses from stem regions demonstrate that lignin deposition were reduced by high N fertilization, whereas a consistent increased were observed on those cultivate at N deficient treatment. These results were corroborate by chemical analyses. By the RNA sequencing analyses, it was identified 1,469 differently expressed genes, exhibiting an N-dependent expression pattern. Between those are genes related to N and phenylpropanoid metabolisms. The results allowed us to understand the mechanism involved on the maintenance of the high rates of lignifications under N depletion, and also to point some possible candidates genes involve on Eucalyptus wood quality
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular
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Mandelli, Fernanda. « Estudos dos mecanismos de adaptaçãoo ao estresse oxidativo da bactéria termófila Thermus filiformis = Evaluation of the adaptation mechanisms to the oxidative stress of the thermophilic bacterium Thermus filiformis ». [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/256571.
Texte intégralRésumé :
Orientadores: Adriana Zerlotti Mercadante, Fabio Marcio SquinaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Espécies reativas de oxigênio (ERO) e nitrogênio (ERN) são geradas dentro das células pela exposição a agentes endógenos e exógenos, estas espécies, quando em níveis normais, encontram-se envolvidas na produção de energia, regulação do crescimento celular, sinalização intercelular e síntese de substâncias biológicas importantes. Por outro lado, se produzidas em excesso, podem provocar oxidação lípidica, de proteínas ou do DNA causando o que conhecemos por estresse oxidativo. Para combater o excesso de espécies reativas, os organimos produzem moléculas antioxidantes tais como os carotenoides e enzimas como superóxido dismutase e catalase. No entanto, é difícil apontar as estratégias de adaptação dos micro-organismos em resposta a diferentes condições de estresse através do estudo individual de moléculas produzidas. Diante do exposto, esta pesquisa teve por objetivo elucidar o genoma, proteoma e transcriptoma bem como a produção de carotenoides da bactéria termófila Thermus filiformis quando submetida à algumas condições de cultivo: presença e ausência de H2O2 e temperatura de crescimento abaixo (63 ?C) e acima (77 ?C) do seu ótimo (70 ?C). Para tanto, o genoma e transcriptoma foram analisados com o emprego de tecnologias de sequenciamento de última geração e ferramentas computacionais, e a proteômica e os carotenoides foram caracterizados por cromatografia líquida e espectrometria de massas. Além disso, devido à conhecida capacidade antioxidante e alto potencial de aplicabilidade na indústria farmacêutica, cosmética e de formulação de alimentos, foi feita a clonagem, expressão e caracterização da enzima superóxido dismutase de Thermus filiformis (TfSOD). A TfSOD apresentou atividade enzimática utilizando como cofator tanto manganês quanto ferro e termoestabilidade a até 80 ?C. O sequenciamento de DNA produziu um total de 9.680.471 reads pareados e uma montagem com n50 = 85,2Kb, n90 = 17,1kb, contig de maior tamanho com 275,5kb e um tamanho total de 2,46MB. A predição genética resultou em 2.403 genes codificadores de proteínas. Na análise de transcriptoma, 97,1% dos genes codificadores de proteínas preditos apresentaram expressão com valores detectáveis de RSEM (RNA-Seq by Expectation-Maximization). Através da análise do transcriptoma foram identificados 37% e 5,86% dos genes diferencialmente expressos (p-valor<0,05) nos ensaios com diferentes temperaturas e com e sem adição de H2O2, respectivamente. Através da análise do proteoma, no ensaio com diferentes temperaturas, foi encontrado um total de 27,7% proteínas diferencialmente expressas com um FDR (False Discovery Rate) < 0,05%, sendo 20% significativamente diferentes (p-valor<0,05, teste T) e, no ensaio com e sem adição de H2O2, um total de 28,3% com um FDR < 0,05%, sendo 6% significativamente diferentes (p-valor<0,05, teste T). Algumas diferenças foram observadas na produção de carotenoides de acordo com cada condição de cultivo. Quanto ao perfil de carotenoides, nas condições a 70 ?C e a 77 ?C os carotenoides majoritários foram termozeaxantina-15 e termozeaxantina-13, enquanto que para condição a 63 ?C foram termozeaxantina-15 e zeaxantina livre. A amostra cultivada a 70 ?C sem adição de H2O2 apresentou a maior quantidade de carotenoides totais (1.516 ?g/g), por outro lado o extrato rico em carotenoides que apresentou maior capacidade de desativação do radical peroxila (50,5) foi o da amostra com adição de H2O2. Os resultados do presente estudo mostram que os principais processos afetados pela mudança de temperatura e adição de peróxido de hidrogênio foram: catabolismo, transcrição e tradução de proteínas. Observou-se também que a alteração na temperatura teve uma maior influencia na expressão diferencial de genes e proteinas do que a adição de peróxido. Através das análises do trancriptoma e do proteoma de T. filiformis foram identificadas enzimas termo-estáveis com potencial de aplicação industrial, como por exemplo alfa-amilases, alfa-galactosidases e esterases. Além disso, o extrato rico em carotenoides dessa bactéria apresentou capacidade de desativar o radical peroxila superior à capacidade de extratos de frutas e até mesmo de padrões de carotenoides
Abstract: Reactive oxygen (ROS) and nitrogen (RNS) species are produced in the cells by exposure to endogenous and exogenous agents, these species, when at normal levels, are involved in energy production, cell growth regulation, intercellular signaling and synthesis of important biological substances. On the other hand, if overproduced, can cause lipid, protein and DNA oxidation, leading to what is known as oxidative stress. To combat excessive reactive species, organisms produce antioxidant molecules such as carotenoids and enzymes such as superoxide dismutase and catalase. However, it is difficult to point out the adaptation strategies of microorganisms in response to different stress conditions through the study of individual molecules. Therefore the aim of this research was to elucidate the genome, proteome and transcriptome, as well as the carotenoid production of Thermus filiformis when submitted to the some cultivation conditions under stress: without and with hydrogen peroxide and temperature below (63 ?C) and above (77 ?C) the optimum (70 ?C). In order to achieve this aim, the genome and transcriptome were analyzed using next generation technology and computational tools, and proteome and carotenoids were characterized by liquid chromatography and mass spectrometry. Moreover, due to its known antioxidant capacity and potential application on pharmaceutical, cosmetics and food formulations, a superoxide dismutase from Thermus filiformis (TfSOD) was cloned, expressed and characterized. The TfSOD showed cambialistic characteristics, once it had enzymatic activity with either manganese or iron as cofactor and thermostability until 80 ?C. The DNA sequencing produced a total of 9,680,471 paired reads and the produced assembly had an n50 = 85.2Kb, n90 = 17.1kb, the largest contig size = 275.5kb and total size of 2.46MB. Gene prediction resulted in 2,403 protein coding genes. In the transcriptome analysis, 97.1% of predicted protein coding genes showed detectable expression with RSEM values (RNA-Seq by Expectation-Maximization). Through the computational analysis of T. filiformis transcriptome 37% and 5.86% of the genes significantly different (p-value < 0.05) in the assays with different temperatures and with and without H2O2 were identified, respectivelly. In the total proteome analysis a total of 27.7% proteins were differentially expressed with a FDR (False Discovery Rate) < 0.05%, being 20% significantly different (p-value < 0.05, T-test) in the temperature assay and 28.3% proteins with a FDR (False Discovery Rate) < 0.05%, being 6% significantly different (p-value < 0.05, T-test) in the H2O2 assay. Some changes were observed in the carotenoid production according to the cultivation condition. Regarding to the carotenoid profile, the major carotenoids under conditions at 70 ?C (without and with H2O2) and at 77 ?C were thermozeaxanthin-15 and thermozeaxanthin-13 while at 63 ?C were thermozeaxanthin-15 and free-zeaxanthin. The sample cultivated at 70 ?C without H2O2 showed the highest amount of total carotenoid (1516 ?g/g of dry mass), on the other hand the sample with the highest antioxidant capacity was the one cultivated at 70 ?C with H2O2. The carotenoid rich extract of all conditions studied showed a peroxyl scavenging capacity higher than those carotenoid rich extracts from some fruits and from some carotenoid standards, demonstrating the potential applicability of T. filiformis extracts in industry. The results of this study show that the main processes affected by temperature change and addition of H2O2 were: catabolism, transcription and protein translation. It was also observed that the change in temperature has greater influence on the differential expression of genes and proteins than the H2O2 addition. Through trancriptome and proteome analysis of T. filiformis thermostable enzymes have been identified with potential industrial applications, such as alpha-amylases, alpha-galactosidases and esterases. Moreover, the extract rich in carotenoids of this bacterium had a greater peroxyl radical scavenging capacity than the capacity of fruit extracts and even carotenoids standards
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos
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Coradini, Alessandro Luis Venega 1988. « Domesticação genética da levedura oleaginosa Lipomyces starkeyi e estudo da expressão de genes ligados ao metabolismo de xilose e lipídeos durante cultivo em frascos agitados ». [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/266081.
Texte intégralRésumé :
Orientadores: Telma Teixeira Franco, Ana Carolina DeckmannDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: A crise energética mundial aliada aos problemas ambientais tem despertado grande preocupação mundial, aumentando assim a busca por combustíveis mais "limpos" que possam substituir os já existentes. Dentre estes substitutos podemos destacar os chamados biocombustíveis, como o etanol e o biodiesel. No caso da produção de biodiesel, diversas fontes de lipídios são consideradas, incluindo óleos vegetais, gorduras animais e óleos reciclados. O uso de microrganismos oleaginosos foi apresentado como uma fonte alternativa de óleos e gorduras de baixo custo (Meng et al., 2009). O óleo produzido por microrganismos pode ter como substrato qualquer fonte de carbono, incluindo bagaços e resíduos agrícolas em geral, evitando tanto a competição por áreas de plantio como agregando valor a dejetos de outros setores agrícolas. A esta abordagem dá-se o nome de "biocombustíveis de segunda geração". Neste contexto, as leveduras surgem como fortes candidatas à produção de biodiesel, uma vez que diversas ferramentas de cultivo e manipulação já foram implantadas especialmente para o uso de leveduras em processos industriais e biotecnológicos. Entretanto, nem todas as espécies de leveduras são adequadas para a produção de óleo, uma vez que a capacidade de acumular grandes quantidades de lipídeos varia significativamente entre as espécies. As leveduras do gênero Saccharomyces, por exemplo, apresentam de 8-15 de lipídeos totais em relação à massa seca. Outras, como as espécies Rhodotorula graminis, Rhodotorula gracilis e Lipomyces starkeyi, apresentam cerca de 30%, 60% e 65% de lipídeos totais, respectivamente. Entretanto, o emprego de microrganismos selvagens (i.e., não domesticados) limita o controle dos parâmetros que afetam o processo produtivo, uma vez que as bioconversões de interesse dependem de ajustes metabólicos desconhecidos, e a falta de conhecimento genético destes microrganismos dificulta a aplicação de técnicas de manipulação genética. Assim, o presente estudo teve como objetivo "domesticar" a linhagem DSM70826 da levedura Lipomyces starkeyi tornando-a de fácil manipulação genética e tornando-a uma plataforma para posterior introdução de características que possam melhorar o seu desempenho quanto ao acúmulo de lipídeos para produção de biodiesel. Uma vez que foi realizado o sequenciamento do genoma desta levedura durante trabalho anterior conduzido em nosso laboratório, também objetivamos utilizar este banco de dados para identificar e estudar alguns genes considerados relevantes aos processos fermentativos de xilose e também ao acúmulo de lipídeos, auxiliando a elucidar as principais rotas metabólicas envolvidas nestes processos
Abstract: The global energy crisis combined with environmental problems has aroused great worldwide concern, increasing the search for "cleaner" fuels that can replace existing ones. Among these substitutes the so-called biofuels, such as ethanol and biodiesel can be highlighted. In the case of biodiesel, different fat sources are considered, including vegetable oils, animal fats and recycled oils. The use of oleaginous microorganisms was presented as an alternative source of low cost oils and fat (Meng et al., 2009). The oil produced by microorganisms can use any substrate as carbon source, including bagasse and agricultural waste in general, avoiding the competition for growing areas and adding value to waste of other agricultural sectors. This approach is called "second generation biofuels". In this context, yeasts appear as strong candidates for biodiesel production, since many manipulation and cultivation tools have been implemented especially for the use of yeast in industrial and biotechnological processes. However, not all species of yeasts are suitable for the production of oil, since the ability to accumulate large amounts of lipids varies significantly between species. Yeasts of the genus Saccharomyces, for example, show 8-15% of total lipids in the dry mass. Others, such as the species Rhodotorula graminis, Rhodotorula gracilis and Lipomyces starkeyi, have about 30 %, 60% and 65% of total lipids, respectively. However, the use of wild microorganisms (i.e., non-domesticated) limits the control parameters that affect the production process, since bioconversions of interest depend of unknown metabolic adjustments, and lack of genetic knowledge of these microorganisms hinders the application of genetic manipulation techniques. Therefore, the present study aimed to "domesticate" the yeast strain Lipomyces starkeyi DSM70826 making it easy genetic manipulation and making it a platform for subsequent introduction of features that can improve your performance on lipid accumulation for production of biodiesel. Since the genome sequencing of this yeast was performed during previous work conducted in our laboratory, we also aim to use this database to identify and study some genes considered relevant to xylose fermentation processes and also to lipid accumulation, helping to elucidate the main metabolic pathways involved in these processes
Mestrado
Engenharia de Processos
Mestre em Engenharia Química
48
Fuzita, Felipe Jun. « Molecular physiology of digestion in arachnida : functional and comparative-evolutionary approaches ». Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-08122014-100929/.
Texte intégralRésumé :
Spiders and scorpions are efficient predators arachnid (PA) consuming preys larger than themselves. Few studies reported, molecularly, the digestion in PA. This work describes a biochemical, transcriptomic and proteomic analysis of the midgut and midgut glands (MMG) and digestive juice (DJ) from Nephilengys cruentata and Tityus serrulatus MMG. Cathepsin L, B, D and F, legumain, trypsin, astacin, carbohydrases and lipases were identified by these approaches. Peptide isomerase and ctenitoxins, which are venom proteins were identified, showing a correlation among digestive and venom enzymes. Summarily, PA relies in multi peptidase system mainly constituted of astacins for extracellular prey liquefaction and cathepsin L for intracellular digestion, describing a molecular model for digestion. Probably, during evolution, gene duplication led a diversification of astacins in the derived groups of Arachnida, like spiders, distinctly from what is observed in basal groups like scorpions. These data on Arachnida digestion will allow detailed multi disciplinary studies.Aranhas e escorpiões são aracnídeos predadores eficientes (AP) consumindo presas maiores que eles mesmos. Poucos estudos descrevem molecularmente a digestão em AP. Neste trabalho caracterizamos bioquimicamente, por transcriptoma e proteoma o intestino e glândulas digestivas (IGD) e suco digestivo (SD) de Nephilengys cruentata e o IGD de Tityus serrulatus. Catepsinas L, B, D e F, legumaína, tripsinas, astacinas, carboidrases e lipases foram identificadas. Peptídeo isomerase e ctenitoxina foram identificadas no IGD. Estas proteínas podem indicar uma correlação entre enzimas digestivas e do veneno. Portanto, AP apresentam várias peptidases principalmente astacinas para liquefazer a presa extraoralmente e catepsinas L para digestão intracelular, descrevendo um modelo molecular para a digestão. Provavelmente, durante a evolução, eventos duplicação gênica levaram à diversificação das astacinas aracnídeos derivados, como as aranhas, diferentemente dos grupos basais, como os escorpiões. Estes dados sobre a digestão em Arachnida permitirão estudos multidisciplinares.
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Sínico, Thais Elise. « Avaliação da interação entre ácaros Brevipalpus yothersi Baker (1949) e o vírus da leprose dos citros C (CiLV-C) ». Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/11/11138/tde-01082018-183208/.
Texte intégralRésumé :
Ácaros Brevipalpus yothersi Baker (1949) são vetores do citrus leprosis virus C (CiLV-C), o mais comum causador da leprose dos citros. Esta é considerada, atualmente, a doença viral de maior importância na cultura dos citros no Brasil e ocorre também em países da América do Sul e Central, além do México, na América do Norte. A doença prejudica a vida útil da planta, devido à queda de folhas e frutos, e seca de ramos, podendo levá-la à morte quando o ataque é demasiadamente severo. O controle e manejo da leprose são basicamente atribuídos ao uso de acaricidas, demandando dos produtores um acréscimo significativo no orçamento por defensivos agrícolas e potencial risco ao ambiente. Nos últimos anos, análises de expressão gênica (transcriptoma), envolvendo ácaros praga da agricultura, resultaram em informações importantes como o envolvimento de genes específicos no processo de resistência a acaricidas e desenvolvimento biológico. No entanto, ainda pouco se sabe sobre a interação do ácaro B. yothersi com o CiLV-C, tornando o estudo de transcriptoma muito significativo para a obtenção de informações aprofundadas sobre o atípico padrão vírus-vetor do patossistema leprose. Assim, através do sequenciamento de RNA (RNAseq), foi investigado o perfil de expressão diferencial entre ácaros B. yothersi virulíferos e avirulíferos. O RNAseq foi realizado em sistema Illumina HiSeq 2500, e os dados analisados com base em linguagem R e ferramentas do software Bioconductor. Os reads sequenciados foram mapeados no genoma de B. yothersi e foi feita análise de expressão utilizando DESeq2. Foram observados 5.690 genes diferencialmente expressos (GDE), sendo 2.736 transcritos induzidos em ácaros virulíferos. Os GDE foram analisados em banco de dados do NCBI, buscando por proteínas similares em artrópodes. Dentre os transcritos induzidos em B. yothersi, potencialmente em resposta ao CiLV-C, foram encontrados genes relacionados ao processo de detoxificação de xenobióticos, metabolismo primário e imunidade, com possível envolvimento na interação vírus-vetor. A expressão de 23 genes foi verificada por RT-PCR quantitativo em tempo real (RT-qPCR). As análises indicaram que os genes envolvidos em detoxificação são induzidos durante a interação entre o ácaro da leprose e o CiLV-C.The false spider mite Brevipalpus yothersi Baker (1949) is recognized as vector of citrus leprosis virus C (CiLV-C), the most common causal agent of citrus leprosis disease. Currently, it is considered the viral disease of major importance in citrus in Brazil and occurs in countries of South and Central America, as well as in Mexico, North America. It reduces the lifespam of the plants due to leaf and fruit drop, dried branches, and can lead to death when the attack is severe. The management of leprosis is based primarily on the chemical control of the mite vector, increasing significantly the cost of production and harming the environment. On the last years, gene expression (transcriptome) analyzes involving phytophagous mites resulted in important data, such as the involvement of specific genes in the process of resistance to acaricides and biological development. However, there is little information about B. yothersi-CiLV-C interaction, making the transcriptome a very interesting tool to obtaining data regarding the atypical virus-vector relationship of the leprosis pathosystem. RNA sequencing (RNAseq) was used to investigate the differential expression profiles between viruliferous and aviruliferous B. yothersi mites. The RNAseq was performed using the Illumina HiSeq 2500 system, the data were analyzed using the R language and Bioconductor software packages. The sequenced reads were mapped on the genome of B. yothersi and expression analysis was performed in DESeq2. We identified 5.690 differentially expressed genes (DEGs), of which 2.736 transcripts were induced in viruliferous mites. The DEGs were analyzed in NCBI database, searching for similar proteins in arthropods. Among the transcripts induced in response to CiLV-C, there are genes related to the detoxification of xenobiotics, primary metabolism, immunity and possible involvement in the virus-vector interaction. Expression of 23 genes was verified by quantitative real-time RT-PCR (RT-qPCR). The analyzes indicate that detoxification genes are induced during the interaction between the false spider mite and CiLV-C.
50
Amorim, Fernanda Gobbi. « Clonagem e expressão heteróloga da hialuronidase e/ou novas toxinas obtidas a partir do transcriptoma da glândula da peçonha de Tityus serrulatus ». Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/60/60134/tde-02052016-153125/.
Texte intégralRésumé :
As hialuronidases de peçonhas animais são capazes de clivar o hialuronan presente na matriz extracelular, facilitando a difusão das toxinas no tecido da vítima. Essas enzimas têm sido negligenciadas, devido à instabilidade enzimática e à baixa concentração na peçonha. Assim, a expressão heteróloga das hialuronidases permite a sua obtenção em quantidades que viabilizam seu estudo estrutural e funcional. Associado a isso, o transcriptoma propicia a identificação de novas toxinas e componentes de baixa proporção na peçonha. Portanto, o presente trabalho realizou o transcriptoma da glândula de peçonha do escorpião Tityus serrulatus e a clonagem e expressão heteróloga da hialuronidase. No transcriptoma foram obtidos 558 ESTs, dos quais 61,8% correspondem às toxinas, dentre elas neurotoxinas com ação em canais iônicos, metaloproteinas, hipotensinas, peptídeos antimicrobianos, dentre outros. Foram também identificadas novas toxinas como o neuropeptídeo e Ts16.1, descritos pela primeira vez para o gênero Tityus. Dentre os transcritos obtidos, foi identificado apenas um clone correspondente ao C-terminal incompleto de uma hialuronidase de T. serrulatus. Consequentemente, foi produzido o gene sintético contendo a sequência da TsHyal-1, obtida em bancos de dados, no vetor de expressão pPICZ?A para expressão heteróloga em P. pastoris. A rTsHyal-1 foi expressa em escala laboratorial em meio não suplementado (BMM) em pH 7,0 durante 96 h da indução com alimentação diária de metanol a 0,75%. A rTsHyal-1 foi produzida na sua forma solúvel e ativa (838,31 UTR/mg) e resultou em um rendimento proteico de 250 mg/L de material expresso. O secretoma do meio de expressão mostrou que além da rTsHyal-1, a P. pastoris também secreta proteínas nativas associadas à ligação do ATP, metabolismo de carboidrato e resposta ao estresse oxidativo. A rTsHyal-1 foi parcialmente purificada em troca catiônica fraca e apresentou atividade específica de 1.097,45 UTR/mg. A rTsHyal-1 apresenta massa molecular de 49,5 kDa e o tratamento com PNGase F seguido da análise por espectrometria de massas (MALDI-TOF) indicou que uma possível N-glicosilação de 4,5 kDa. Adicionalmente, o sequenciamento dos digestos trípticos da enzima realizado no MALDI-TOF e pelo Q-Exactive resultaram em 46,8% de cobertura da sequencia da proteína. A rTsHyal-1 apresenta especificidade pelo substrato hialuronan, seguido da condroitina C, A e B e apresentou atividade ótima em pH 6,0 e a 40°C. Adicionalmente, o ensaio do MTT indicou que a enzima recombinante não apresenta citotoxicidade in vitro. Os resultados obtidos determinaram as melhores condições para a expressão heteróloga da rTsHyal-1, que corresponde à primeira hialuronidase recombinante de escorpião expressa em P. pastoris com atividade enzimática preservadaThe venom hyaluronidases of animals are able to cleave hyaluronan present in the extracellular matrix, acting as a spreading factor for the toxins into the tissues of the victim. These enzymes have been neglected due to their instability and low concentration in the venom. Thus, heterologous expression of hyaluronidases permits the obtainment of sufficient amount for structural and functional studies. Moreover, the transcriptome allows the identification of new toxins or components with low proportion in the venom. In this way, this study aimed the construction of the transcriptome of Tityus serrulatus venom gland and cloning/heterologous expression of hyaluronidase. In the transcriptome, 558 ESTs were identified and 61.8% corresponded to toxins, such as neurotoxins with action on ion channels, metalloproteins, hypotensins, antimicrobial peptides, among others. In addition, new toxins were identified, comprising one neuropeptide and Ts16.1, described for the first time to the genus Tityus. Among the obtained transcripts, one identified clone corresponded to an incomplete C-terminal of a hyaluronidase. Consequently, a synthetic gene was synthesized containing the sequence of TsHyal-1 (obtained from databases) in the pPICZ?A vector for heterologous expression in P. pastoris. The rTsHyal-1 was expressed at laboratorial scale in unsupplemented medium (BMM) at pH 7.0 for 96 h, after induction time, with daily feeding of 0.75% methanol. The rTsHyal-1 was produced in soluble and active form (838.31 UTR/mg) and resulted in a protein yield of 0,266 mg/mL in final expressed material. Besides, the secretome of the medium showed that P. pastoris also secretes native proteins bound with ATP, proteins related to carbohydrate metabolism and oxidative stress response. The rTsHyal-1 was partially purified in a weak cation exchange and presented specific activity of 1097.45 UTR/mg. The rTsHyal-1 has molecular mass of 49.5 kDa and the treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, the sequencing of tryptic digests performed in the MALDI-TOF and Q-Exactive resulted in 46.8% of protein sequence coverage. The rTsHyal-1 presents substrate specificity for hyaluronan followed by chondroitin C, A and B and showed an optimum activity at pH 6.0 and 40°C. Furthermore, the MTT assay indicated that the recombinant enzyme does not display in vitro cytotoxicity. These results validate the biotechnological process of the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpions expressed in P. pastoris system with enzymatic activity preserved.