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Desenvolvimento de um pipeline para análise genômica e transcriptômica com base em Web servicesMelo, Henrique Velloso Ferreira 04 September 2009 (has links)
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Previous issue date: 2009-09-04 / Pipeline systems for genomic and transcriptomic analysis aim to create communication bridges among the existing analysis tools, therefore reducing researchers efforts. Most of the pipelines found in the literature lack important features which would be useful to the development of genome or transcriptome sequencing projects. Among them, the capacity of tracking the project results along its development, including the generation of partial reports; the presence of a collaborative environment where the involved laboratories can contribute with new data and chromatograms; the possibility to configure analysis parameters; multiple pipeline support and the possibility to include new tools and modules. In this work, a pipeline prototype was developed to overcome these shortcomings. Sequencing projects progresses are tracked along all over their developments. Chromatograms are progressively received along the development of the project and partial reports over newly received data are generated. The communication with the processing server is done via Web service, which offers a universal language interface, allowing client applications in heterogeneous platforms to submit data and execute operations and queries. Pipelines are configured in XML documents written in a predefined format, through which the researchers choose the tools and parameters to be used. The prototype offers support to multiple pipelines executed simultaneously in the same project. Pipelines are executed in parallel by the means of thread pools, what increases efficiency by distributing the workload in multiprocessed systems. Another feature of the prototype is the extensibility as each pipeline step is wrapped in a module. New modules can be easily inserted in the system through the implementation of a programming interface, therefore without the needing of recompilation. Module insertions are done in a declarative way through XML documents. A client application was also developed in the collaborative platform Sakai, allowing different research groups involved in a sequencing project to create pipelines, view results and exchange information on the project current status. To evaluate the efficiency of the prototype, a case study was carried out. Sequences generated from sequencing of Sphenophorus levis transcriptome were submitted and a pipeline was configured to analyze the data. The case study has pointed out that the prototype is efficient and produces good results. / Sistemas de pipeline para análise de genomas e transcriptomas têm o objetivo de criar pontes de comunicação entre as diferentes ferramentas no intuito de reduzir os esforços do pesquisador no processo de análise. A maioria dos pipelines descritos na literatura carece de funcionalidades importantes para o desenvolvimento de projetos de sequenciamento. Entre elas, a capacidade de acompanhar e gerar resultados parciais das análises ao longo do desenvolvimento do projeto; a presença de um ambiente colaborativo onde os diferentes laboratórios envolvidos possam contribuir com novos dados e cromatogramas; a possibilidade da configuração dos parâmetros da análise; o suporte a múltiplos pipelines com diferentes configurações; e o suporte à inclusão de novos programas e módulos. Neste trabalho, foi desenvolvido um protótipo que supre essas deficiências. O progresso dos projetos é acompanhado ao longo de todo o seu desenvolvimento. Para isso, recebe dados brutos de cromatogramas, realiza análises dos dados parciais e emite relatórios com os resultados. A comunicação com o servidor de processamento é realizada via Web service, oferecendo uma interface na linguagem universal XML que permite que aplicações cliente em plataformas heterogêneas submetam dados e realizem operações e consultas. Os pipelines são configurados através de arquivos XML em formato específico, no qual o pesquisador define os programas a parâmetros a utilizar. O protótipo dá suporte a múltiplos pipelines com execução simultânea em um mesmo projeto. A execução dos pipelines é realizada em paralelo por meio de um pool de threads, o que aumenta a eficiência dividindo a carga de processamento em servidores com mais de um núcleo. Uma aplicação cliente foi desenvolvida na plataforma colaborativa, permitindo que os diferentes grupos de pesquisa envolvidos no sequenciamento criem pipelines, visualizem resultados e troquem informações sobre o andamento do projeto. Outro diferencial do protótipo desenvolvido é a extensibilidade. Cada etapa do pipeline é encapsulada em um módulo. Novos módulos podem ser facilmente inseridos sem a necessidade de recompilação de todo o sistema, bastando para isso que o mesmo implemente uma interface específica. A inserção no sistema é realizada declarativamente em arquivos XML. Um estudo de caso foi realizado com a submissão de cromatogramas a partir do sequenciamento de ESTs (Expressed Sequence Tags) de Sphenophorus Levis. Um pipeline foi configurado para o estudo, e sua execução mostrou que o sistema é eficiente e apresenta bons resultados.
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Comparative analyses of the salivary gland secretomes from related species of the gall midge family CecidomyiidaeAl-Jbory, Zainab January 1900 (has links)
Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / C. Michael Smith / The tools for arthropods with sucking-mouth parts to attack hosts are mainly in the saliva. For plant-sucking insects, these salivary secretions are primarily produced in the salivary glands. Secreted proteins (also referred to as salivary gland secretomes) are among the important components in the saliva of sucking insects. Gall midges (Cecidomyiidae), a large family of plant-sucking insects, apparently secrete proteins (some of them are effector proteins) into host tissues, inducing various forms of plant outgrowth (galls). Three major insect pest species in the genera Mayetiola, the stem gall midges, are known to produce saliva that can reprogram plant cells and manipulate the host plant growth, causing serious damage to the plants of small grains. The three pest species are the Hessian fly (Mayetiola destructor), the barley midge (Mayetiola hordei), and the oat midge (Mayetiola avenae). Another economically important species of this gall midge family is the wheat midge (Sitodiplosis mosellana). It is a major insect pest of spring wheat and feeds on wheat heads, causing damage to the developing wheat seeds.
A global analysis of the salivary gland secretome of first instar larvae of the Hessian fly, (a member of Mayetiola and) a model species for studying insect-plant interactions, has previously revealed a large number of genes encoding Secreted Salivary Gland Proteins, so called SSGPs. For comparison, we conducted analyses on transcripts encoding SSGPs from salivary glands of the first instar larvae of the wheat midge, barley midge, and oat midge.
In the first chapter, a transcriptomic analysis of wheat midge has been conducted. In this analysis, a total of 3,500 cDNA clones were sequenced, and 1,301 high quality sequences were obtained and approximately 25% of the cDNAs (with high quality sequences) encoded SSGPs. The SSGPs were grouped into 97 groups based on sequence homology. Among the SSGP-encoding transcripts, 206 encoded unique proteins with no sequence similarity to any known protein and 29 encoded proteins similar to known proteins including proteases, serpines, thioesterases, ankryins, and feritins. The compositions of SSGP transcripts from the wheat midge were then compared with that of Hessian fly. The analyses have identified many common characteristics between the species. Despite these commonalities, no sequence similarity was found between SSGPs from wheat midge and those from Hessian fly, suggesting that SSGPs from these two insect species perform different functions to manipulate host plants.
The second chapter contains results of comparative transcriptomic analyses on the barley and oat midges. A total of 2570 cDNA clones were sequenced from the barley midge, and 743 were high quality cDNA sequences, and the analysis identified 458 cDNA clones encoding SSGPs, of these, 178 encoded unique proteins (also called unigenes). Transcripts encoding SSGPs were grouped into 51 groups based on sequence homology. A total of 3226 cDNA clones were sequenced from oat midge, and 718 cDNA sequences were high quality and used for further analysis. The analysis identified 450 cDNA clones encoding SSGPs. Among the SSGP-encoding transcripts, 194 are unigenes, which were placed into 50 groups.
The compositions of SSGP transcripts from the barley and oat midges were then compared with that of Hessian fly. The analysis identified five groups containing 102 (57.3%) unigenes from barley midges and seven groups containing 107 (55.1%) unigenes from oat midges which encode SSGPs that are conserved among the three species. The SSGPs conserved among the three midges are from family one (SSGP-1), family 4 (SSGP-4), family 11 (SSGP-11), and family 71 (SSGP-71). The SSGPs conserved among the three species indicate conserved functions such as a role in plant manipulation.
Some SSGP unigenes were found to be conserved between only two species. Specifically, there were eight gene groups which are conserved between two species. Within these eight groups 19 (10.7%) unigenes from the barley midge and 25 (12.9%) unigenes from the oat midge were found to be conserved between only the barley and oat midges, whereas no homologues have been found in the Hessian fly. The remaining unigenes encode SSGPs that are unique to different midge species. The highly divergent SSGP groups that have been identified with no homology among the three midges indicate potential roles of these SSGPs in host specification.
Due to the important roles of effector proteins in insect-plant interactions for gall midge species and since no insect effector protein have been identified directly from infested plant tissues so far, I have chosen one of the SSGP family, SSGP-1, which are conserved among all three gall midge species, for further analysis in chapter 4. Members in family SSGP-1 are also the most abundantly expressed at the transcript level. Based on Hessian fly data, family 1 contains seven genes and are named SSGP-1A1, SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1C2, SSGP-1D1, and SSGP-1E1. To detect the presence of these proteins in the infested wheat tissues, and to identify probable targets from wheat that interact with the SSGPs in the feeding site, we have generated and purified recombinant proteins for five of the seven proteins, namely SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1D1, and SSGP-1E1 (since SSGP-1A1 and SSGP-1C2 are very similar to SSGP-1A2 and SSGP-1C1, respectively). Antibodies were produced for the recombinant proteins for western blot analyses and indirect immunostaining.
Immunostaining on dissected tissues including salivary glands, guts, and Malpighian tubules from 3-day old larvae, was conducted with antibodies against the five SSGPs, and detected a specific localization of all proteins in salivary glands except SSGP-1E1, which exhibited a weak signal in the foregut, in addition to localization in salivary glands. Western blot analyses demonstrated that these five proteins were expressed in larvae at all stages. The continuous production of these proteins suggests that they play roles in initiation and maintenance in Hessian fly infestation. Consistent with their effector functions, these five proteins were detected for the first time in infested wheat tissues based on western blot analyses.
To identify possible target proteins from host plants that interact with SSGP-1 family proteins, in vitro pull-down assays were performed. Putative interacting targets for SSGP-1A2, SSGP-1B1, and SSGP-1C1 have been identified by LC-MS/MS. These putative interaction target proteins included uncharacterized proteins, ribosomal proteins, a lipoxygenase, and a tubulin. Identification of these putative targets provided a base for further confirmation of their interaction with Hessian fly effectors in the future.
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Elucidating the molecular basis of copper stress in Erwinia amylovoraÁguila Clares, Begoña 23 May 2017 (has links)
Erwinia amylovora, a quarantine organism of the European Union (EU), is the causal agent of fire blight. This disease causes substantial economic losses in all countries where it is present and its control turns out difficult, due to the absence of effective chemical and biological treatments and the ability of persistence and dissemination of E. amylovora. Cupric treatments constitute the base of the integrated management of fire blight in the European Union countries, because the antibiotics, although have been proved useful against this disease, are forbidden in the EU for plant treatments.
This thesis, mostly performed in a P2 security lab, is aimed to dilucidate molecular mechanisms implicated in the response of E. amylovora to copper sulfate as a stress factor, considering that copper is a well known toxic element for bacterial cells over a certain threshold concentration. The global objective was first addressed with the study of a selection of genes that have been related in other bacterial models with copper stress or with stress in general. The quantification of the rpoS gene expression in presence of copper showed that, at least in long-term survival, this gene may be involved in the E. amylovora response to copper stress.
Second, a transcriptomic study was performed by microarray after subdue the bacteria to a copper shock treatment. The analysis of the microarray results showed that 44 genes were differentially expressed in presence of this metal. Each one of these genes was studied by gene ontology and, after comparing them with databases published in NCBI, they were classified in functional categories. The gene expression of twenty-five out of fourty-four differentially expressed genes was validated by real-time PCR. In the validation, copA gene was expressed more than 19-fold in presence than in absence of copper and, because of that, it was selected together with other seven genes (soxS, yjcE, ygcF, yhhQ, galF, arcB, EAM_3469), which also showed an increased expression, to generate mutants of E. amylovora. The responses of mutants to copper, and the fact that the wild phenotype was restored in the complemented mutants, has shown the role of copA, soxS, yjcE, ygcF, arcB and yhhQ genes in the E. amylovora in vitro survival against copper stress. Besides, the implication of copA gene has also been proved in planta, in copper treated shoots from pear trees. Finally, all the results obtained along this thesis have allowed to elaborate a putative model of the different genetic mechanisms that seem are involved in the interaction between E. amylovora and copper. The most important mechanism seems to be to face up reactive oxygen species (ROS) by the activation of the soxS and yjcE genes. The activity of these genes is supported by CopA protein, which pumps copper from inside the cell out to the periplasmic space. The activation of arcB gene, which allows the change from aerobic metabolism to anaerobic metabolism, would also help E. amylovora to reduce ROS.
Taking together, the results of this thesis have allowed an approximation to the genetic basis of E. amylovora response to copper stress and they constitute a start point to move forward in the knowledge of the molecular mechanisms underlying that response. / Erwinia amylovora, organismo de cuarentena en la Unión Europea (UE), es el agente causal del fuego bacteriano. Esta enfermedad produce grandes pérdidas económicas en todos los países en los que está presente y su control resulta muy difícil, debido a la carencia de tratamientos químicos y biológicos eficaces y a la persistencia y facilidad de diseminación de E. amylovora. Los tratamientos con compuestos cúpricos constituyen la base de la gestión integrada del fuego bacteriano en los países de la UE, puesto que el uso de antibióticos, aunque se ha demostrado útil contra esta enfermedad, está prohibido en la UE para el tratamiento de bacteriosis en plantas.
Esta tesis, realizada en su mayoría en un laboratorio de seguridad biológica P2, pretende dilucidar mecanismos moleculares implicados en la respuesta de E. amylovora al sulfato de cobre como factor de estrés, ya que este metal es un elemento tóxico para las células bacterianas por encima de una determinada concentración umbral. El objetivo global se abordó, en primer lugar, con el estudio de una selección de genes que se han relacionado en otros modelos bacterianos con el estrés que produce el cobre o con el estrés en general. La cuantificación de la expresión del gen rpoS en presencia de cobre mostró que este gen puede estar implicado en la supervivencia a largo plazo de E. amylovora para combatir el estrés que produce este metal.
En una segunda aproximación, se realizó un estudio transcriptómico mediante microarray tras someter a la bacteria a un breve tratamiento de cobre. El análisis de los resultados del microarray reveló que 44 genes se expresaban de forma diferencial en presencia del metal. Cada uno de ellos se estudió mediante gene ontology y por comparación con las bases de datos publicadas en el NCBI, y así se clasificaron en categorías funcionales. Las categorías de estrés y transporte fueron las más abundantes, tanto respecto a los genes que aumentaron su expresión tras la aplicación de cobre como a los que la disminuyeron. De los 44 genes que se expresaron de forma diferencial, se validó la expresión de 25 de ellos por PCR en tiempo real. En dicha validación, el gen copA se expresó 19 veces más en presencia que en ausencia de cobre, por lo que fue seleccionado, junto con siete genes más (soxS, yjcE, ygcF, yhhQ, galF, arcB, EAM_3469), en los que el incremento en la expresión fue menos pronunciado, para generar mutantes de E. amylovora. La respuesta de los mutantes a la presencia de cobre, y la restauración de fenotipos al complementar las mutaciones generadas, han revelado el papel de los genes copA, soxS, yjcE, ygcF, arcB y yhhQ en la supervivencia in vitro de E. amylovora frente al estrés por cobre. Además, la implicación del gen copA se ha demostrado también in planta en brotes de peral tratados con cobre. Finalmente, todos los resultados obtenidos han permitido elaborar un posible modelo de los diferentes mecanismos genéticos que parecen estar implicados en la interacción de E. amylovora con el cobre. El mecanismo más importante parece ser combatir las especies reactivas del oxígeno (ERO), mediante la activación de la expresión de los genes soxS e yjcE. La actividad de estos genes está apoyada, además, por la proteína CopA, que bombea cobre desde el interior celular al espacio periplásmico. La activación del gen arcB, que permite el cambio de un metabolismo aerobio a uno anaerobio, también ayudaría a la reducción de las ERO. En definitiva, los resultados han permitido una aproximación al sustrato genético de la respuesta de E. amylovora al estrés por cobre, y constituyen un punto de partida para avanzar en el conocimiento de los mecanismos moleculares implicados en dicha respuesta. / E. amylovora, organisme de quarantena a la Unió Europea (UE), és l'agent causal del foc bacterià. Aquesta malaltia produeix grans pèrdues econòmiques a tots els països on està present, i el seu control resulta molt difícil, a causa de l' absència de productes químics i biològics eficaços i també per la capacitat de persistència i disseminació d'E. amylovora. Els tractaments amb composts cúprics constitueixen la base de la gestió integrada del foc bacterià als països europeus, ja que l'ús d'antibiòtics, tot i que s'ha demostrat eficaç per a combatre aquesta malaltía, està prohibit a la UE per al tractament de bacteriosi en plantes.
Aquesta tesi, realitzada majoritàriament a un laboratori de seguretat biològica P2, pretén dilucidar mecanismes moleculars implicats en la resposta d'E. amylovora davant del coure com a factor d'estrés, ja que el coure és un element tòxic per la cèl.lula per damunt d'una determinada concentració umbral. L'objectiu global es va abordar, en primer lloc, amb l'estudi d'una selecció de gens relacionats en altres models bacterians amb l'estrés que produeix el coure, o amb l'estrés en general. La quantificació de l'expressió del gen rpoS en presència de coure va mostrar que aquest gen pot estar implicat en la supervivència a llarg termini d'E. amylovora per a combatre l'estrés que produeix aquest metall.
En una segona aproximació, es va realitzar un estudi transcriptòmic mitjançant microarrays després de sotmetre els bacteris a un breu tractament de coure. L'anàlisi dels resultats dels microarrays va revelar que 44 gens s'expressen de forma diferencial en presència del metall. Cadascun d'ells es va estudiar mitjançant gene ontology i, per comparació amb les bases de dades publicades al NCBI, es van classificar en categories funcionals. Les categories d'estrés i transport van ser les més enriquides, tant en els gens que augmentaren la seua expressió després de l'aplicació de coure com en aquells que la van reduir. Dels 44 gens que s'expressaren de forma diferencial, es va validar l'expressió de 25 d'ells per PCR a temps real. En la validació, el gen copA es va expressar 19 vegades més en presència que en absència de coure, per aquesta raó va ser seleccionat junt amb set gens més (soxS, yjcE, ygcF, yhhQ, galF, arcB, EAM_3469), en els que l'increment de l'expressió va ser menys pronunciada, per a generar mutants d'E. amylovora. La resposta dels mutants a la presència de coure, i la restauració dels fenotips originals al complementar les mutacions generades, han revelat el paper dels gens copA, soxS, yjcE, ygcF, arcB i yhhQ en la supervivència in vitro d'E. amylovora davant a l'estrés per coure. A més a més, la implicació del gen copA s'ha demostrat també in planta, en brots de perera tractats amb coure. Finalment, tots els resultats obtinguts han permès elaborar un possible model dels diferents mecanismes genètics que semblen estar implicats en la interacció d'E. amylovora amb el coure. El mecanisme més important sembla ser combatre les especies reactives de l'oxigen (ERO), mitjançant l'activació de l'expressió dels gens soxS i yjcE. L'activitat d'aquestos gens és recolzada també per l'acció de la proteïna copA, que bombeja coure des de l'interior cel.lular a l'espai periplàsmic. L'activació del gen arcB, que permet el canvi d'un metabolisme aerobi a un metabolisme anaerobi, també ajudaria a reduir la producción de les ERO. En conclusió, els resultats han suposat una aproximació al substrat genètic de la resposta d'E. amylovora a l'estrés per coure, i constitueixen un punt de partida per avançar en el coneixement dels mecanismes moleculars implicats en aquesta resposta. / Águila Clares, B. (2017). Elucidating the molecular basis of copper stress in Erwinia amylovora [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/81658 / TESIS
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Increased virulence and processing resistance of Salmonella Enteritidis in the egg environment: Understanding the paradigm of food as a vehicle for human infectionXu, Yumin 12 September 2022 (has links)
No description available.
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TARGET IDENTIFICATION THROUGH THE TRANSCRIPTOMIC CHARACTERIZATION OF PROFIBROTIC MONOCYTES/MACROPHAGES IN IDIOPATHIC PULMONARY FIBROSIS / CHARACTERIZING MONOCYTES/MACROPHAGES IN PULMONARY FIBROSISVierhout, Megan January 2020 (has links)
Idiopathic pulmonary fibrosis (IPF) is a disease of unknown pathogenesis characterized by scarring of the lung and declining respiratory function. Originating from bone marrow, circulating monocytes can be recruited into the lung tissue and polarized toward the alternatively activated (M2) profibrotic macrophage phenotype. Recent literature has shown that cluster of differentiation 14 positive (CD14+) monocytes are more abundant in IPF patient blood and are associated with disease outcome and acute exacerbation. Additionally, a 52-gene risk profile from peripheral blood mononuclear cells for outcome prediction in IPF was recently published. Here, we began by characterizing macrophages in human IPF lung tissue. We then assembled a biobank and examined transcriptomic characteristics of blood-derived circulating monocytes from IPF patients.
Various histological assessments were completed on a tissue microarray including lung biopsies from 24 IPF patients and 17 controls, to characterize M2 macrophage expression in human tissue. Whole blood samples were collected from 50 IPF patients and 12 control subjects. CD14+ monocytes were isolated and mRNA was extracted for bulk RNA sequencing. Data were analyzed for differential expression (DE), and Gene Set Enrichment Analysis (GSEA) was performed to examine enrichment of the previously published 52-gene risk profile in our dataset.
We found that M2 macrophage expression was increased in IPF lung tissue compared to controls. CD14+ monocyte levels were significantly elevated in IPF patients in our cohort compared to control participants, and was negatively correlated with forced vital capacity (FVC). DE analysis comparing IPF and control monocytes yielded a 35-gene signature, with 16 up-regulated genes and 19 down-regulated genes. When comparing the signature related to long transplant-free survival from the published dataset to our data, GSEA demonstrated that this signature is enriched in donors from our dataset, supporting concurrence between the meanings of the two datasets. Overall, these results provide insight to identify targets to modulate monocyte/macrophage function in IPF and potentially affect progressive disease. / Thesis / Master of Science (MSc) / Idiopathic pulmonary fibrosis (IPF) is a disease of unknown cause that results in excessive scarring of the lungs and progressive impairment in lung function. We believe that white blood cells called monocytes and macrophages play a key role in the development and progression of this disease. Overall, it is thought that monocytes, which circulate in the blood, enter the lung tissue and become macrophages. These macrophages then lead to the formation of scar tissue, which is characteristic to IPF. In order to better understand how these cells contribute to IPF, we studied their properties in blood and lung biopsies from IPF patients. We found significant differences between monocytes/macrophages in IPF than those in healthy controls, that may help explain disease progression. We hope that these findings will provide insight into causes of the IPF, and potential avenues for therapeutic intervention.
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Réponse transcriptomique des tissus cérébraux sains et tumoraux à la radiothérapie par microfaisceaux synchrotron / differential response of healthy and tumoral tissu after microbeam radiation therapyBouchet, Audrey 31 October 2012 (has links)
La radiothérapie par microfaisceaux (MRT) synchrotron est une méthode de radiothérapie alternative pour les tumeurs cérébrales, qui présente l'avantage unique de pouvoir déposer de très hautes doses d'irradiation (plusieurs 100aines de Gy) au niveau de la masse tumorale. En effet, le fractionnement spatial des rayons X en microfaisceaux parallèles de quelques dizaines de micromètres s'est montré efficace dans le traitement des tumeurs cérébrales du rongeur tout en préservant le tissu cérébral péritumoral. Pour autant, son mode d'action sur le plan biologique n'est qu'en partie connu. Si l'effet différentiel de cette irradiation sur les vaisseaux sains et tumoraux a pu être démontré ces dernières années, il ne peut expliquer à lui seul l'efficacité de la MRT. Dans ce travail, nous avons établi une description de la réponse transcriptomique précoce des tissus sains et tumoraux (gliosarcome 9L) à la MRT et les fonctions biologiques et voies de signalisation associées. Ces résultats constituent une base de données interrogeable à partir d'hypothèses précises. Cette base a ainsi permis d'identifier des transcrits impliqués dans la réponse de la tumeur à la MRT et dont l'inhibition n'interfèrerait pas avec la réparation des tissus sains : nous avons proposé 3 cibles potentielles qui permettraient d'augmenter l'index thérapeutique de la MRT. (i) L'inhibition radio-induite d'un groupe de 13 gènes (Plk1, Cdc20, Ccnb1, Pttg1, Bub1, Dlgap5, Cenpf, Kif20a, Traf4af1, Depdc1b, Mxd3, Cenpe et Cenpf), participerait au contrôle tumoral précoce après MRT par la perturbation de la division cellulaire et pourrait être amplifié pour prolonger l'inhibition de la croissance tumorale. (ii) La mise à profit de l'activation du promoteur de Clecsf6 au sein des tumeurs irradiées permettrait la surexpression locale, via les monocytes modifiés et infiltrés, de protéine d'intérêt thérapeutique. (iii) Areg (codant pour l'Amphiréguline) est surexprimé au sein du tissu tumoral après MRT et son implication connue dans la chimio/radiorésistance nous conduit à considérer que son inhibition pourrait être une stratégie de renforcement des effets de la MRT. Par ailleurs, nous avons montré que la MRT engendrait de meilleurs résultats sur le contrôle tumoral et la survie animale qu'une irradiation synchrotron en champ plein (avec une dose équivalente à la vallée MRT). Cependant, aucune différence transcriptomique ne pouvant soutenir cet effet n'a pu être mis en évidence. / Synchrotron Microbeam Radiation Therapy (MRT) is a novel form of radiosurgery of brain tumors which allows high dose deposition (few hundreds of Gy) in pathologic tissues. The spatial fractionation of the incident beam into arrays of near-parallel microbeams has shown efficiency on brain tumors implanted in rodents while sparing normal tissues. The preferential effects observed on tumor vessels could not entirely explain the efficiency of MRT and other biological mechanisms might be involved in tumor control. In this work, we described the early whole transcriptomic responses of normal and tumoral (9L gliosarcoma) tissues to MRT and the associated biofunctions and pathways. This provides a questionable data base which can be used by the whole MRT community. This base allows to identify transcripts involved in tumor response to MRT and which inhibition would have no consequence in healthy tissue repair. We identified 3 relevant targets which might increase the therapeutic index of MRT. (i) The radio-induced inhibition of a cluster of 13 genes (Plk1, Cdc20, Ccnb1, Pttg1, Bub1, Dlgap5, Cenpf, Kif20a, Traf4af1, Depdc1b, Mxd3, Cenpe and Cenpf) may be involved in tumor control after MRT through the deregulation of cell division and could be amplified to continue the tumor growth inhibition. (ii) We might benefit from the activation of the Clecsf6 promoter in irradiated tumors by delivering, via modified and injected monocytes, some therapeutic proteins. (iii) Finally, Areg (encoding for Amphiregulin) is overexpressed in tumors after MRT and its involvement described in chimio/radioresistance enable to consider that its inhibition might help in tumor control after irradiation. We also showed that MRT induces a greater tumor control and survival rates compared with similar broad beam irradiations but no differences in transcriptomic responses have been highlighted.
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Recherche de facteurs génétiques contrôlant la résistance de lignées de souris consanguines à une infection expérimentale par Yersinia pestis, l’agent de la peste. / Identification of genetic factors involved in the resistance of inbred strains of mice to an experimental infection with Yersinia pestis, the plague agent.Chevallier, Lucie 05 December 2012 (has links)
Yersinia pestis, l'agent de la peste, est une bactérie à Gram-négatif classée comme agent pathogène ré-émergent et potentielle arme de bioterrorisme. De plus, l'apparition d'une souche multi-résistance de cette bactérie souligne la nécessité de mieux comprendre comment cette bactérie hyper-virulente interagit avec son hôte. Afin d'identifier des facteurs génétiques de vulnérabilité à la peste, notre laboratoire travaille sur la réponse de souris résistantes versus sensibles à Y. pestis. Notre stratégie pour identifier les facteurs génétiques impliqués dans la résistance/sensibilité à la peste combine une approche de cartographie de QTL (Quantitative Trait Locus) et d'analyse d'expression génique. Nous avons précédemment décrit la lignée SEG/Pas, issue de Mus spretus, comme la première résistante à une souche virulente de Y. pestis, alors que la plupart des lignées murines de laboratoire, telle que la lignée C57BL/6J, sont extrêmement sensible à la bactérie. Des croisements entre SEG/Pas et C57BL/6J nous ont permis d'identifier trois QTL impliqués dans la résistance à Y. pestis, localisés sur les chromosomes 3, 4 et 6. Deux des QTL (situés sur les chromosomes 4 et 6) ont pu être confirmés par l'analyse de lignées congéniques. Plus de 40 % des femelles bi-congéniques hétérozygotes pour ces deux QTL ont survécu à l'infection, alors que tous les témoins C57BL/6J ont succombé. La dissection de ces deux QTL par l'analyse de lignées sous-congéniques, nous a permis d'affiner l'architecture génétique de la résistance à la peste chez SEG/Pas. Nous avons conclu qu'un minimum de quatre facteurs génétiques, au sein de ces deux QTL, sont nécessaires pour augmenter la résistance à Y. pestis chez la Souris. Cependant, la production de plusieurs lignées congéniques portant le QTL situé sur le chromosome 3, dont une lignée triple congénique, ne nous a pas permis de confirmer l'existence de ce QTL. En parallèle de l'analyse génétique, nous avons déterminé que les macrophages de SEG/Pas et de C57BL/6J présentaient des caractéristiques différentes après exposition à Y. pestis. Une analyse différentielle du profil transcriptionnel des macrophages de ces deux lignées a été réalisée à l'aide de puces à ADN. Nos résultats montrent une forte activation de la production cytokinique dans les macrophages de SEG/Pas en réponse à la bactérie, activation qui n'est pas observée dans la lignée C57BL/6J. Ces résultats suggèrent que les souris SEG/Pas sont capables de mettre en place une réponse immune innée plus forte ou peut-être plus précoce que C57BL/6J. Nous avons ensuite étudié par qRT-PCR l'expression en cinétique de 44 gènes dans des macrophages de SEG/Pas, C57BL/6J et des bi-congéniques portant les QTL sur les chromosomes 4 et 6. Cette étude nous a permis de confirmer que les souris SEG/Pas sont capables se mettre en place une forte réponse inflammatoire lors de l'infection. Cependant, aucune différence significative n'a été observée entre la lignée bicongénique et la lignée parentale C57BL/6J. D'autres expériences seront nécessaires afin de mieux comprendre les mécanismes biologiques impliqués dans la résistance intermédiaire de cette lignée. La dissection génétique associée à l'analyse de l'expression génique de ces lignées résistante et sensible permet d'augmenter notre compréhension de la réponse de l'hôte à Y. pestis. / Yersinia pestis, the agent of plague, is a deadly gram-negative bacterium classified as a re-emerging pathogen and class A biological weapon. The appearance of a multi-resistant strain highlights the need to better understand how this pathogen kills its host. To identify genetic factors of host susceptibility to plague, our laboratory is investigating the response of resistant versus susceptible mice to Y. pestis. Our strategy to decipher genetic determinants involved in resistance to plague combines Quantitative Trait Loci (QTL) mapping with gene expression analysis. We previously described the Mus spretus-derived SEG/Pas strain as the first to resist fully virulent Y. pestis, while most inbred strains, such as C57BL/6, are highly susceptible. Crosses between these two strains identified three QTLs (located on chromosome 3, 4 and 6) contributing to resistance. Two of the QTLs (on chromosome 4 and 6) were confirmed through creation of congenic mice. Up to 40% of the congenic mice heterozygous at these two QTLs, on a C57BL/6J background, survived the infection while all C57BL/6J mice died. Further dissection of these two QTLs, through the use of subcongenic strains, enabled us to refine the genetic architecture of resistance to plague in SEG/Pas mice. We concluded that a minimum of four genetic factors, within these two QTLs, are required to increased resistance to Y. pestis in mice. Despite production of numerous congenic strains, including triple congenic mice, we were not able to confirm the existence of the third QTL identified on chromosome 3. In parallel to genetic studies, we determined that SEG/Pas and C57BL/6J macrophages exhibit distinct characteristics upon in vitro exposure to Y. pestis. The underlying molecular differences were investigated by using microarrays. Our results show strong activation of cytokines in SEG/Pas macrophages in response to Y. pestis, which is not found in C57BL/6J macrophages. These results suggest that SEG/Pas mice are able to better activate innate immune response to Y. pestis than C57BL/6J mice.We further studied the expression of 44 genes in a kinetic study on macrophages in vitro of SEG/Pas, C57BL/6J and bicongenic mice (carrying QTLs on chromosome 4 and 6). This study confirmed that SEG/Pas mice are able to build a stronger inflammatory response at early time of infection. Nevertheless no significant differences were observed in the bicongenic strain compared to C57BL/6J. Further studies will be required to understand the mechanisms involved in the intermediate resistance of this strain. This combination of genetic dissection and gene expression analysis of resistant and susceptible mouse strains will enhance our ability to better understand the host response to plague.
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TRANSCRIPTOMIC ANALYSES OF <em>CATHATRANTHUS ROSEUS</em> HAIRY ROOTS OVEREXPRESSING CRMYC2 AND ORCA3 AND ROLES OF CROSS-FAMILY TRANSCRIPTION FACTOR INTERACTION IN TERPENOID INDOLE ALKALOID BIOSYNTHESISSui, Xueyi 01 January 2017 (has links)
Catharanthus roseus (Madagascar periwinkle), is a well-known medicinal plant that produces a vast array of terpenoid indole alkaloids (TIAs), including two anticancer compounds vinblastine and vincristine. Industrial scale production of TIAs is hampered by the difficulties of total chemical synthesis of these compounds and the fragmented knowledge on TIA pathway. Transcriptional regulation of the TIA biosynthetic pathway has not been thoroughly investigated in Catharanthus and only a few structural genes have been identified as the targets of two master regulators: the basic helix-loop-helix (bHLH) transcription factor (TF) CrMYC2 and APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), ORCA3. Next generation sequencing (NGS) has been used as a tool to isolate novel genes encoding enzymes and regulators of TIA pathway in Catharanthus. In this dissertation, I have performed the transcriptomic analysis of transgenic Catharanthus hairy roots ectopically expressing a dominant repressive form of CrMYC2 or ORCA3 in order to understand their potential impact on the TIA transcriptional regulatory network and to identify and characterize novel target(s) of these two key TFs.
MYC2 acts as regulatory hub involved in diverse aspects of plant growth, development, and specialized metabolite biosynthesis by coordinating the crosstalk among different phytohormone signals. CrMYC2 was initially identified in Catharanthus as a regulator of ORCA3. CrMYC2 transactivates ORCA3 by binding to the T/G-box in jasmonate-responsive element (JRE) of ORCA3 promoter. RNA interference (RNAi) mediated knockdown of CrMYC2 strongly reduced TIA accumulation in Catharanthus cell suspension culture. However, the potential influence of CrMYC2 on the expression of other regulatory and structural genes in the TIA pathway remains poorly understood. Transcriptomic analyses revealed that CrMYC2 plays an essential role in JA-induced gene expression and the differentially expressed genes are involved in diverse aspects of growth and development as well as abiotic and biotic stress responses in Catharanthus. Additionally, the expression of genes related to auxin, ethylene, and abscisic acid signaling cascades were affected in hairy roots with modified CrMYC2 expression, suggesting this TF mediates cross-talk between JA and other phytohormones. Surprisingly, overexpression of CrMYC2 resulted in repressed expression of TIA pathway genes in transgenic hairy roots. Expressions of key activators of indole and iridoid pathway were downregulated whereas expression of repressors were upregulated in CrMYC2 hairy roots.
Activators (i.e. CrMYC2 and ORCA3) and repressors (i.e. G-box binding factors; GBFs) have been isolated and characterized for their role in regulation of TIA pathway. However, the interconnection between those regulators and the underlying molecular mechanism has not been throughly studied. I identified (i) the interaction of CrMYC2 with CrGBFs and (ii) how this cross-family transcription factor interactions fine-tunes TIA biosynthesis in Catharanthus. The expression profiles of CrMYC2 and CrGBFs were highly correlated in different tissues and in response JA. Moreover, CrMYC2 interacted with CrGBF1 and CrGBF2 in both yeast and plant cells. CrGBF1 and CrGBF2 could form homo- and hetero-dimer which bound T/G-box elements of TIA pathway gene promoters. In plant cells, CrGBF1 antagonizes the activity of CrMYC2 on target promoters in a dosage dependent-manner. Similarly, CrMYC2 can overcome CrGBF1-mediated repression of target promoters in a dosage dependent manner.
ORCA3 is another major regulator of TIA biosynthesis in Catharanthus. The transcriptomic analysis of ORCA3 transgenic hairy roots revealed (i) the effect of ORCA3 on newly identified TIA pathway biosynthetic enzymes; (ii) identify the potential effect of ORCA3 on three biological processes: abiotic stress response, plant secondary metabolic process, and response to hormonal stimulus; and (iii) the identification of potential regulator(s) of TIA biosynthesis using ORCA3 based co-expression analysis.
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Étude du rôle des micro-ARN cellulaires au cours de l’infection par le VIH-1Bellini, Nicolas 12 1900 (has links)
Avec plus de 39 millions de personnes infectées à travers le monde, le virus de l’immunodéficience humaine de type-1 (VIH-1) est un problème majeur de santé publique. Bien que la thérapie antirétrovirale contrôle la réplication virale, améliore la santé et prolongent la vie des personnes vivant avec le VIH-1, elle ne permet pas d’éradiquer complètement le virus. En effet, celui-ci établit des phases de latence en intégrant son génome dans l’ADN cellulaire des cellules cibles, entrainant la formation de ce que l’on appelle le réservoir latent du virus. Ce réservoir se situe principalement dans les lymphocytes T CD4+, bien qu’on le retrouve également dans les cellules myéloïdes, et il constitue le principal obstacle à l’éradication du virus. Il est donc impératif de mieux comprendre les facteurs de l’hôte qui régissent non seulement la susceptibilité de ces cellules à l’infection et qui contribuent également à la persistance du VIH-1. Nous postulons que les micro-ARN (miARN), des petits ARN produits par la cellule et qui régulent l’expression génique, jouent un rôle au cours de l’infection par le VIH-1.
Dans un premier temps, nous nous sommes concentrés sur le rôle des miARN dans l’entrée virale. À l’aide d’un séquençage de nouvelle génération des miARN dans les macrophages, nous avons déterminé que le miARN-103, ainsi que son paralogue le miARN-107, ciblent l’ARNm du corécepteur CCR5 utilisé par le VIH-1. Nous montrons que l’induction de l’expression des miARN-103/107 est régulée par le facteur de transcription p53 et rend les macrophages réfractaires à l’entrée du VIH-1. Nous observons que le niveau d’expression des miARN-103/107 est enrichi dans les macrophages résidant dans les tissus intestinaux de donneurs sains et les macrophages alvéolaires des personnes sous thérapie antirétrovirale, ce qui contribue vraisemblablement à leur résistance à l’infection par le VIH-1.
Étant donné que les lymphocytes T CD4+ constituent le principal réservoir du VIH-1, nous avons ensuite étendu l’étude du miARN-103 dans ces cellules. Récemment, il a été montré que la latence virale serait la conséquence de l’infection de lymphocytes T CD4+ dans une fenêtre très étroite suite à leur activation. Ces cellules qui
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transitionnent vers un phénotype mémoire posséderaient des propriétés uniques, comme une augmentation temporaire de l’expression du corécepteur viral CCR5, ainsi qu’une capacité réduite de transcrire l’ADN proviral intégré. Nos résultats montrent que le miARN-103, qui cible également le CCR5 dans les lymphocytes T CD4+, participe à la modulation du CCR5 selon l’état d’activation de la cellule et contribue indirectement à l’établissement de la latence virale dans ces cellules. De plus, nos résultats montrent que les lymphocytes T CD4+ issus d’individus qui contrôlent l’infection par le VIH-1 expriment des niveaux réduits d’ARNm de CCR5 (lorsque comparés à ceux d’individus non-contrôleurs). Cet état étant associé à une tendance à la hausse du miARN-103, suggère que le miARN-103 pourrait participer au contrôle de l’expression de CCR5 in vivo.
Dans un deuxième temps, nous avons réalisé un séquençage de nouvelle génération de l’ARN des lymphocytes T CD4+ infectés. À la suite de cette analyse, nous avons déterminé que le miARN-26a participe à la régulation de CD59, une protéine cellulaire incorporée dans les virions jouant un rôle clé dans l’activation du complément en inhibant la formation du complexe d’attaque membranaire. Au cours de l’infection, ce miARN est diminué dans les cellules productivement infectées. Cette diminution est associée à une augmentation de CD59 dans les cellules infectées et à la surface des virions produits par ces cellules, favorisant leur échappement à la lyse médiée par le complément. Nous montrons que l’introduction d’un analogue du miARN-26a dans les cellules rend les virions produits par ces dites cellules plus sensibles à la lyse médiée par le complément.
En conclusion, les observations et analyses réalisées dans le cadre de cette thèse nous aident à mieux comprendre le rôle des miARN dans l’infection et la persistance du VIH-1. Ces résultats aident à notre compréhension des facteurs de l’hôte qui régissent la susceptibilité de ces cellules à l’infection ainsi que la persistance virale, et pourraient aider au développement de stratégies thérapeutiques. / With more than 39 million people infected in the world, human immunodeficiency virus type 1 (HIV-1) is a major public health problem. Although antiretroviral therapy controls viral replication, improves the health, and prolongs the lives of people living with HIV-1, it does not completely eradicate the virus. Indeed, the virus has established latency phases by integrating its genome into the cellular DNA of target cells, leading to the formation of what is called the latent reservoir of the virus. This reservoir is located mainly in CD4+ T cells, although it is also found in myeloid cells, and it has become the main obstacle to eradication of the virus. It is therefore imperative to better understand the host factors that not only govern the susceptibility of these cells to infection but also contribute to HIV-1 persistence. We postulate that microRNAs (miRNAs), which are small RNAs produced by the cell involved in regulation of gene expression, have a role during HIV-1 infection.
First, we focused on the role of miRNAs in viral entry. Using next generation miRNA sequencing in macrophages, we determined that miRNA-103, as well as its paralogue miRNA-107, target the mRNA of CCR5, the HIV-1 coreceptor. We show that the induction of miRNA-103/107 expression is regulated by the transcription factor p53 and makes macrophages more resistant to HIV-1 entry. We observe that the expression level of miRNAs-103/107 is enriched in resident macrophages in intestinal tissues of healthy donors and alveolar macrophages of people on antiretroviral therapy, which likely contributes to their resistance to infection by HIV-1.
Given that CD4+ T cells constitute the main reservoir of HIV-1, we then extended the study of miRNA-103 in these cells. Recently, it has been shown that viral latency is the consequence of the infection of CD4+ T cells within a very narrow window following their activation. These transitioning to memory T cells are thought to possess unique properties, such as a temporary increase in the expression of the viral coreceptor CCR5, as well as a reduced capacity to transcribe integrated proviral DNA. Our results show that miRNA-103, which also targets CCR5 in CD4+ T cells, participates in the modulation of CCR5 depending on the activation state of the cell
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and indirectly contributes to the establishment of viral latency in these cells. Furthermore, our results show that CD4+ T cells from individuals who control HIV-1 infection express reduced levels of CCR5 mRNA (when compared to those from non- controller individuals), this being associated to a upward trend in miRNA-103 expression, suggesting that miRNA-103 may participate in the control of CCR5 expression in vivo.
Secondly, we carried out next generation RNA sequencing of HIV-1 infected CD4+ T cells. Their resulting analyses determined that miRNA-26a participates in the regulation of CD59, a cellular protein that is incorporated into virions and has a key role in complement activation by inhibiting the formation of the membrane attack complex. During infection, this miRNA is decreased in productively infected cells. This decrease is associated with an increase in CD59 in infected cells, as well as on the surface of virions produced by these cells, favoring their escape from complement- mediated lysis. We show that introduction of miRNA-26a mimics in cells makes the virions produced by these cells more sensitive to complement-mediated lysis.
In conclusion, the observations and analyses developed in this thesis will help us better understand the role of miRNAs in HIV-1 infection and persistence. These results help in the understanding of host factors that govern the susceptibility of these cells to infection as well as viral persistence and could help in the development of therapeutic strategies.
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Purification, caractérisation, espression hétérologue et applications des enzymes lipolytiques de Carica papaya / Purification, characterization, heterologous expression and applications of Carica papaya lipolytic enzymes / Purificación, caracterización, expresión heteróloga y aplicaciones de las enzimas lipolíticas de Carica papayaRivera Espinosa, Ivanna 28 November 2013 (has links)
Parmi les activités catalytiques les plus intéressantes présentes dans le latex de Carica papaya on trouve l'activité lipolytique (EC 3.1.1.X). En effet les enzymes lipolytiques peuvent catalyser des réactions d’hydrolyse sur divers substrats mais également, dans des conditions thermodynamiques favorables, des réactions de synthèse. De plus, elles sont actives en présence de solvants organiques, ce qui fait de ce type d’enzymes des biocatalyseurs spécialement attractifs pour des applications industrielles. Enfin, cette activité lipolytique est associée à la matrice insoluble du latex, et constitue donc une activité immobilisée. Cependant et pour cette même raison, il n'a pas été possible jusqu'à présent d'élucider la ou les séquences protéiques dans le latex responsables de cette activité lipolytique. En conséquence, cette thèse est dédiée à l'étude de l'activité lipolytique du latex de C. papaya. Pour cela, la purification partielle du latex de C. papaya a été réalisée. Ainsi, les propriétés catalytiques des fractions partiellement purifiées ont pu être déterminées pour l'hydrolyse de triglycérides, la résolution de mélanges racémique d'octyl ester de l'acide 2-bromo-phenylacétique, la synthèse de biopolymères et des lipides structurés. Parallèlement à cela, une recherche dans le génome de C. papaya des séquences codant les motifs conservés dans les protéines lipolytiques, ainsi que des essais d'expression in vivo dans les feuilles de la plante en conditions de stress a été effectuée, ce qui a permis d’isoler pour la première fois une lipase, CpLip2, exprimée in vivo chez C. papaya. Cette protéine, ainsi que 2 autres protéines déjà identifiées dans le latex de C. papaya, CpEst et CpLip1, ont été produites sous forme fonctionnelle en employant respectivement Nicothiana sp., Yarrowia lipolytica et Pichia pastoris comme systèmes d'expression. Finalement, certaines des propriétés biochimiques et catalytiques de ces protéines recombinantes ont pu être déterminées / Lipolytic activity (EC 3.1.1.X) is one of the most interesting catalytic activities from Carica papaya latex. Indeed lipolytic enzymes can catalyze not only hydrolysis but also various synthesis reactions due to their activity in organic solvents, which make them especially attractive for industrial applications. Nevertheless, most of the lipolytic activity present in latex is tightly associated to the insoluble fraction of the latex and up to now it has not been possible to determine which enzymes are responsible for the lipolytic activity of C. papaya latex. This PhD work is dedicated to the study of the lipolytic activity present in Carica papaya. Partial purification of lipolytic activity from C. papaya latex was carried out to evaluate the catalytic properties of the partially purified fractions in the hydrolysis of triglycerides, the resolution of racemic mixtures of 2-bromo-phenylacetic octyl ester, biopolymers and structured lipids synthesis. Bioinformatic analysis was also realized on C. papaya genome by searching conserved motifs for lipolytic proteins. Finally, assays of in vivo transcriptomic in the leaves of stressed C. papaya allowed the isolation of a new lipase produced in vivo (CpLip2, gene 1973.2). This protein, as well as two previously identified lipolytic enzymes from C. papaya, CpEst (gen 25.180) and CpLip1 (gen 55.143) were functionally expressed using Nicotiana sp.,Yarrowia lipolytica and Pichia pastoris as expression hosts, respectively. Finally, some of the biochemical and catalytic properties of these produced recombinant proteins were evaluated / Una de las actividades catalíticas más interesantes presentes en el látex de Carica papaya es la actividad lipolítica (3.1.1.X).Las enzimas lipolíticas pueden catalizar reacciones de hidrólisis sobre varios sustratos e igualmente, en condiciones termodinámicas favorables, pueden catalizar reacciones de síntesis. Adicionalmente, son activas en presencia de solventes orgánicos, por lo que son biocatalizadores atractivos para aplicaciones industriales. Sin embargo, la mayor parte de la actividad lipolítica del látex se encuentra asociada a la matriz insoluble del látex, formando un biocatalizador auto-inmovilizado.Por esta razón, hasta la fecha no ha sido posible elucidar qué enzima o enzimas son responsables de la actividad lipolítica observada en el látex de C. papaya. En consecuencia, la presente investigación está dedicada al estudio de la actividad lipolítica en Carica papaya. Para ello, se realizaron estudios de purificación parcial del látex de Carica papaya, así como la evaluación de las propiedades catalíticas de las fracciones parcialmente purificadas en la hidrólisis de triglicéridos, resolución de mezclas racémicas de octil éster del ácido 2-bromo-fenilacético y síntesis de biopolímeros y lípidos estructurados. Paralelamente, se realizó un análisis genómico mediante la búsqueda con motivos conservados que codifican para diversas proteínas lipolíticas, así como ensayos de transcriptómica para buscar la expresión in vivo de secuencias seleccionadas en las hojas de la planta, que permitieron encontrar por primera vez una expresión in vivo de una lipasa en C. papaya (CpLip2). Esta proteína y otras dos previamente identificadas en el látex de papaya (CpEst y CpLip1), fueron expresadas de forma funcional empleando respectivamente Nicotiana sp., Yarrowia lipolytica y Pichia pastoris como sistemas de expresión. Finalmente algunas de las propiedades bioquímicas y catalíticas de las proteínas expresadas fueron determinadas
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