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Molecular cloning and functional characterization of genes involved in the biosynthesis of polyunsaturated fatty acids in oat (Avena sativa L.)2014 April 1900 (has links)
This thesis research started with analysis of oat fatty acids by using three different transmethylation methods. Basic sodium methoxide was compared with traditional acidic methanol for the total fatty acid analysis, while diazomethane was used to analyze free fatty acids. Epoxy FAs were readily hydrolyzed to dihydroxy fatty acids under the acidic condition, which suggest an overestimation of hydroxyl fatty acids and underestimation of epoxy fatty acids in previous analyses. The sodium methoxide method proved more reliable to quantify the oat seed fatty acid composition. CDC Dancer oat seed analyzed here was comprised mostly of palmitic acid (PA), oleic acid (OA) and the polyunsaturated fatty acid (PUFA) linoleic acid (LA) in quantities of 23%, 32%, and 37% of total seed FA, respectively. As well, the seed contained small quantities of another PUFA, α-linolenic (ALA) and several unusual oxygenated fatty acids (UFAs), Δ15-hydroxy fatty acid (15HFA) and epoxy fatty acids in quantities of 0.85%, 0.68%, and 2.3%, respectively. This thesis further aimed to identify and assemble all FAD2-like genes from an oat Expressed-Sequence Tag (EST) database using FAD2 and FAD2-like proteins from other organisms as query sequences in order to clone all putative FAD2-like genes-of-interest (GOIs) from oat. From the contig assemblies of retrieved oat ESTs, four distinct, putative genes were identified. From the Δ12-desaturase (FAD2) queries, a putative FAD2-like (AsFAD2) gene was identified; the Δ15-desaturase (FAD3) queries revealed two putative oat FAD3-like (AsFAD3-1 and AsFAD3-2) genes, while an ω-3 desaturase (FAD7) query identified a fourth putative full-length FAD6-like coding sequence of two possible lengths, AsFADX and AsFADX+. The GOIs were then subcloned into a yeast expression vector and functionally characterized. AsFAD2a and AsFAD2b both demonstrated Δ12 desaturation on 18:1-9c substrate. AsFAD3-1 had no activity on any substrates present, while AsFAD3-2 exhibited weak Δ15-desaturation activity specifically on 18:2-9c,12c. Finally, AsFADX converted 18:1-9c to 18:2-9c,12c, while AsFADX+ had no activity. Then, a comparative analysis of transcript levels of these GOIs via quantitative real-time PCR (qRT-PCR) was performed across oat germinating seed, root, leaf, and developing seed. AsFAD2 transcript abundance was generally much higher than AsFAD3-1 and AsFAD3-2 in all tissues. AsFAD3-1 mRNA level was highest in developing seed tissue, slightly lower in leaf tissue, and lowest in root. AsFAD3-2 mRNA was highest in germinating seed, and lowest in leaf tissue. In summary, the data produced from this thesis could be used to enhance breeding efforts for establishing oat cultivars with healthier oil content.
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Neuropeptides et phéromones sexuelles impliqués dans le contrôle de la ponte chez la seiche Sepia Officinalis / Egg-laying regulation in the cuttlefish Sepia officinalis : neuropeptides and sex pheromonesEndress, Maxime 10 April 2018 (has links)
La seiche commune Sepia officinalis est un Mollusque Céphalopode présent sur les côtes Normandes. En Baie de Seine, elle représente la troisième espèce visée par les flottilles hauturière et côtière, ce qui en fait une ressource économique importante pour la région. Dans un contexte de protection de la ressource, l’étude et la compréhension des mécanismes liés à la reproduction représentent un objectif majeur. Cette étude est donc focalisée sur les mécanismes régulateurs de la ponte, et en particulier sur les facteurs impliqués dans l’émission des ovocytes. Des recherches antérieures effectuées au laboratoire ont mis en évidence un contrôle multifactoriel du processus de ponte, avec trois niveaux de régulation. (i) Les neuropeptides sont impliqués dans la perception des paramètres environnementaux, (ii) les facteurs ovariens assurent la régulation paracrine du tractus génital, et (iii) les phéromones sexuelles, tout en interférant avec la ponte, sont suspectées de jouer un rôle important dans le comportement des géniteurs associé à la reproduction. Cette thèse met en lumière le rôle de 2 familles de neuropeptides identifiées récemment : les CCAPs et les FLGamides (ou So-orcokinines B). Ces deux familles participent au transport de l’ovocyte et à la sécrétion capsulaire en agissant sur l’activité contractile du complexe oviducte/glande de l’oviducte et des glandes nidamentaires principales. Ces neuropeptides sont par ailleurs suspectés de jouer un rôle dans la biosynthèse des protéines vitellines et des protéines capsulaires. Parallèlement, des phéromones sexuelles sont exprimées par la glande de l’oviducte et sécrétées avec les protéines capsulaires. Des travaux antérieurs ont permis de caractériser des produits d’expression issus de clivages de type prohormone convertase. Dans cette nouvelle étude, un second mode de clivage a été mis en évidence avec l’identification de produits de clivage de masse moléculaire supérieure à 20 kDa. L’un de ces produits, la phéromone beta, a été produit en système recombinant mais son activité biologique sur l’appareil génital mâle et sur l’oviducte n’a pas été démontrée. Quant aux tests comportementaux, ils n’ont pu être exploités du fait d’un nombre d’animaux testés trop restreint. Enfin, une approche transcriptomique comparative et différentielle réalisée à partir de l’organe olfactif mâle et du pavillon de l’oviducte a permis d’identifier un récepteur candidat à la liaison avec les phéromones sexuelles / The common cuttlefish (Sepia officinalis) is a Cephalopod mollusk found on the Normandy coasts. In the Bay of Seine, it is the third species targeted by offshore and coastal fleets, so it is an important economic resource for the whole region. In a context of resource protection, the study and understanding of its reproductive mechanisms is a major objective. This study is therefore focused on the regulatory mechanisms of egg-laying, and in particular on the factors involved in the release of oocytes. Previous research in the laboratory demonstrated multifactorial control of the egg-laying process, with three levels of regulation: (i) neuropeptides involved in the perception of environmental cues, (ii) ovarian factors providing paracrine regulation of the genital tract, and (iii) sexual pheromones, which are implied in egg-laying and are suspected to play a determining role in the reproductive behavior. This thesis highlights the role of two families of recently identified neuropeptides called CCAPs and FLGamides (or So-orcokinins B). These two families participate in oocyte transport and in egg capsule secretion by acting on the contractile activity of the oviduct/oviduct gland complex and of the main nidamental glands. These neuropeptides are also suspected to play a role in the biosynthesis of yolk and capsular proteins. At the same time, sex pheromones are expressed by the oviduct gland and secreted along with capsular proteins. Previous work made it possible to characterize expression products resulting from cleavages of the prohormone convertase type. In this new study, a second mode of cleavage is highlighted, evidenced by the identification of cleavage products with MWs greater than 20 kDa. One of these products, beta pheromone, was produced in a recombinant system but its biological activity on the male reproductive system and on the oviduct was not demonstrated. As for behavioral tests, they were not exploited because the number of animals tested was too small. Finally, a comparative and differential transcriptomic approach of the male olfactory organ and the oviduct pavilion evidenced a candidate receptor expected to bind to sex pheromones
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Clonagem, expressão e caracterização de uma flavina monooxigenase de Coffea arabica / Cloning, expression and characterization of flavin-containing monooxygenese from Coffea arabicaCesarino, Igor, 1984- 12 August 2018 (has links)
Orientador: Paulo Mazzafera / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T20:42:22Z (GMT). No. of bitstreams: 1
Cesarino_Igor_M.pdf: 984828 bytes, checksum: 754aac1bc4502a9d5eb3102a955408d0 (MD5)
Previous issue date: 2009 / Resumo: Uma grande quantidade de genes que codificam flavina monooxigenases (Flavin containing monooxygenases - FMOs) é encontrada em genomas vegetais, embora poucas funções biológicas tenham sido relacionadas com esse grupo de enzimas em plantas. Um importante papel desempenhado por FMOs é a conversão de triptamina em N-hidroxil niptamina, reação catalisada pelas proteínas YUCCA de Arabidopsis thaliana e que constitui o passo limitante da via de síntese de auxina a partir de triptofano. Proteínas similares às YUCCA foram descobertas e caracterizadas em outras espécies vegetais, como OsYUCCA em arroz. FLOOZY em petúnia, ToFZY em tomate e SPIl em milho, todas comprovadamente envolvidas na produção do hormônio citado. Análises da proteína recombinante CaFM08 de Coffea arabica revelou características similares às YUCCA, sugerindo que esta proteína de café é a primeira YUCCA-like descrita para esta espécie e, inclusive, para a família Rubiaceae. CaFM08 apresenta os mesmos motivos protéicos conservados entre FMOs vegetais, e particularmente entre proteínas YUCCA-like. O padrão de expressão espacial do gene que codifica CaFM08 indica possível relação com o desenvolvimento de raízes, folhas e flores de café. Apesar de grandes semelhanças com as proteínas YUCCA, a atividade de N-hidroxilação da triptamina não foi comprovada para CaFM08 recombinante in vitro. Uma análise minuciosa a respeito da funcionalidade de CaFM08 produzida em E. coli deve ser feita antes de descartar a participação desta proteína na síntese de auxina. / Abstract: A large number of genes coding flavin-containing monooxygenases (FMOs) is found in plant genomes, although only few biological functions have been related with these enzymes in plants. An important role for FMOs is the conversion of tryptamine in N-hydroxyl triptamine, catalyzed by the YUCCA protein family in Arabidopsis thaliana. These proteins perform "the rate-limiting step in tryptophan-dependent auxin biosynthesis. Similar YUCCA proteins were discovered and characterized in other plant species, like OsYUCCA in rice, FLOOZY in petunia, ToFZY in tomato and SPIl in maize. All of them are shown to be involved in auxin synthesis. Analysis of the recombinant CaFM08 from Colfea Arabica showed features similar to YUCCA proteins, suggesting that CaFM08 is the first described YUCCA-like protein from coffee and, indeed, from the entire Rubiaceae family. CaFM08 has the same conserved motifs found in other plant FMOs and particulary conserved in YUCCA like proteins. The spatial expression pattern from the CaFM08 coding gene suggests a probable role in the development of roots, leaves and flowers. Although very similar to YUCCA proteins, the CaFM08-mediated convertion of tryptamine in N-hydroxyl tryptamine has not been confirmed in vitro. A further analysis of CaFM08 functionality should address the relation of CaFM08 to auxin production. / Mestrado / Mestre em Biologia Vegetal
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Caracterização funcional da interação entre as proteínas CTSP-1 e CTCF / Functional characterization of the interaction between the proteins CTSP-1 and CTCF.Inoue, Lilian Tiemi 06 December 2011 (has links)
Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose. / Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis.
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Genetic and Functional Characterization of RUNX2Stephens, Alexandre, N/A January 2007 (has links)
RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.
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Caracterização funcional da interação entre as proteínas CTSP-1 e CTCF / Functional characterization of the interaction between the proteins CTSP-1 and CTCF.Lilian Tiemi Inoue 06 December 2011 (has links)
Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose. / Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis.
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Functional characterization of INTS11 loss-of-function in zebrafishHerold, Aveeva 10 1900 (has links)
Le gène INTS11 est une sous-unité catalytique du complexe Integrator qui joue un rôle central dans le traitement de divers ARN naissants. Récemment, des patients présentant des mutations de perte de fonction dans le gène INTS11 ont été signalés comme ayant des problèmes neurodéveloppementaux graves, des problèmes ataxiques et des retards de développement globaux. À ce jour, aucune mutation dans INTS11 n'a été liée à des maladies humaines, et aucune preuve ne soutient leur rôle dans des problèmes neurodéveloppementaux. Par conséquent, nous avons développé un modèle INTS11 knock-out (KO) F0 CRISPRant chez le poisson-zèbre pour caractériser fonctionnellement les mutations de perte de fonction de ce gène in vivo. Nos larves INTS11-KO présentent une accumulation accrue de snARN mal traités, confirmant la perturbation de la fonction du gène. De plus, les larves INTS11-KO meurent prématurément à 14 jours et présentent un phénotype comportemental aberrant, similaire à d'autres modèles génétiques de poisson-zèbre des troubles neurodéveloppementaux. Aussi, les larves INTS11-KO présentent une réduction de la taille du cerveau avec une réduction du contenu neuronal. Enfin, nos résultats d'immunomarquage ont révélé une réduction de la taille du cervelet chez les larves INTS11-KO. Dans l'ensemble, ces données soutiennent le rôle d'INTS11 dans le développement cérébral et sont cohérentes avec les retards neurodéveloppementaux décrits chez les patients présentant des mutations délétères dans ce gène. Notre étude montre comment des organismes modèles simples tels que le poisson-zèbre peuvent aider à caractériser l'étiologie génétique des troubles génétiques. Les résultats de nos recherches pourraient contribuer à des diagnostics plus précis et ouvrir la voie à la découverte de mécanismes pathogènes clés qui pourraient être exploités pour le développement de traitements pour les patients présentant des mutations dans INTS11. / The INTS11 gene is a catalytic subunit of the Integrator complex that plays a central role
in processing various nascent RNAs. Recently, patients with loss-of-function mutations in
the INTS11 gene have been reported to have severe neurodevelopmental issues, ataxic
problems, and global developmental delays. To date, mutations in INTS11 have not been
linked to human diseases, and no evidence supports their role in neurodevelopmental
problems. Therefore, we developed an ints11 F0 CRISPRant knock-out (KO) model in
zebrafish to functionally characterize loss-of-function mutations in this gene in vivo. Our
ints11-KO larvae exhibited an increased accumulation of unprocessed snRNAs,
confirming the disruption of the ints11 function. Moreover, ints11-KO larvae die
prematurely by 14 days of age and display an aberrant behavioural phenotype, similar to
other zebrafish genetic models of neurodevelopmental disorders. Furthermore, ints11-KO
larvae show reduced brain size with reduced neuronal content. Finally, immunostaining
results revealed a reduction in cerebellum size in our ints11-KO. Altogether, these data
support the role of INTS11 in brain development and are consistent with the
neurodevelopment delays described in patients with deleterious mutations in this gene. Our
study shows how simple model organisms like zebrafish can help characterize the genetic
etiology of genetic disorders. The results from our research could aid in more accurate
diagnoses and open the path to unveiling key pathogenic mechanisms that could be
leveraged for the development of treatment for patients with mutations in INTS11.
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Caracterização funcional e estrutural de uma fosfolipase A2 ácida isolada do veneno de Bothrops pirajai / Functional and structural characterization of acidic phospholipase A2 isolated from Bothrops pirajai snake venom.Teixeira, Sabrina Schaaf 04 March 2009 (has links)
A caracterização proteômica de venenos de serpentes contribui significativamente para o avanço das Ciências na área Biomédica. Várias destas moléculas, utilizadas como instrumentos de investigação de mecanismos celulares e moleculares, estão envolvidas em diversos processos fisiopatológicos e de intoxicação. No entanto, os venenos de serpentes ainda requerem caracterização funcional e/ou biológica adicionais. As fosfolipases A2s (PLA2s) são enzimas que induzem vários efeitos farmacológicos, e geralmente, correspondem a maior porcentagem do conteúdo protéico dos venenos de serpentes. Desta forma, o isolamento e a caracterização bioquímica, funcional e estrutural de PLA2s poderão gerar informações importantes para o melhor entendimento dos efeitos farmacológicos e de intoxicação ocasionados por estas proteínas. Este trabalho descreve o isolamento e a caracterização bioquímica, enzimática, funcional e estrutural da primeira PLA2 Asp49 ácida, denominada Bpir-I-PLA2, isolada do veneno da serpente Bothrops pirajai, espécie endêmica da região Sul do Estado da Bahia e atualmente, integrante da Lista Nacional das Espécies da Fauna Brasileira Ameaçadas de Extinção. O isolamento da Bpir-I-PLA2 foi realizada através de dois passos cromatográficos, envolvendo inicialmente cromatografia de troca iônica, seguida de HPLC de fase reversa. Quanto submetida à eletroforese em gel de poliacrilamida para proteínas ácidas, Bpir-I-PLA2 apresentou alto grau de homogeneidade. A massa molecular relativa, determinada através de eletroforese em gel de poliacrilamida com agente desnaturante, foi de aproximadamente 28.000 para a forma de dímero e 14.500 para o monômero. A focalização isoelétrica demonstrou uma única banda para a fosfolipase A2, revelando pI aproximado de 4,9. A região N-terminal de Bpir-I-PLA2, seqüenciada através do método de degradação de Edman, apresentou elevada homologia entre outras PLA2s de venenos de serpentes. A composição de amioácidos da proteína corroborou com sua característica ácida, apresentando um elevado conteúdo de aminoácidos carregados negativamente. A enzima apresentou elevada atividade fosfolipásica e induziu edema moderado in vivo. Além disso, Bpir-I-PLA2 foi capaz de inibir a agregação plaquetária e a coagulação do plasma, e também induzir efeito hipotensor in vivo e citotóxico sobre células tumorais, bactérias, fungos e leishmanias. O tratamento da enzima com o reagente brometo de p-bromofenacila neutralizou as atividades enzimática, inibitória sobre a agregação plaquetária, anticoagulante, hipotensora e antitumoral. A moderada atividade edematogênica foi parcialmente inibida. O cDNA contendo 366 bp (BPIR-A) que codifica a proteína Bpir-I-PLA2 foi clonado e a proteína recombinante, expressa. Bpir-I-PLA2 recombinante apresentou a mesma sequência de aminoácidos, estrutura secundária, atividade fosfolipásica e efeito inibitório sobre plaquetas, observados para a proteína nativa Bpir-I-PLA2, sugerindo que a recPLA2 foi eficientemente expressa e purificada. Devido à baixa toxicidade demonstrada, a Bpir-I-PLA2 pode tornar-se uma ferramenta importante nos estudos das desordens da coagulação, como também um potencial modelo para a elaboração de novos fármacos para uso na clínica-médica. / The proteomic characterization of snake venoms significantly contributes to science advance in the biomedical area. Several venom toxins are used as instruments for the investigation of cellular and molecular mechanisms, and are involved in a number of physiopathological and intoxication processes. Nevertheless, snake venoms still require further functional and/or biological characterization. Phospholipases A2 (PLA2s) are enzymes that induce several pharmacological effects and usually correspond to the major percentage of snake venom protein contents. Being so, the isolation and characterization of PLA2s could generate important information for the better understanding of the pharmacological and toxic effects caused by these proteins. This work describes the isolation and biochemical, enzymatic and functional characterization of the first acidic Asp49 PLA2 (named Bpir-I-PLA2) from the venom of Bothrops pirajai snake, an endemic species from the south region of state of Bahia and that nowadays is part of the national list of Brazilian fauna species threatened of extinction. The isolation of Bpir-I-PLA2 was carried out by two chromatographic steps, an ion-exchange chromatography followed by reverse phase HPLC. When submitted to polyacrylamide gel electrophoresis for acidic proteins, Bpir-I-PLA2 showed high homogeneity level. The relative molecular mass, determined by polyacrylamide gel electrophoresis with denaturating agent, was of approximately 28,000 for the dimmer and 14,500 for the monomer. With high phospholipase activity and pharmacological effects, characteristic of acidic isoforms, Bpir-I-PLA2 did not present myotoxic activity and showed to be little edematogenic. However, the enzyme acts inhibiting platelet aggregation, delaying plasma clotting time and inducing hypotension, antitumoral effects, antibacteria, antifungal and parasiticide actions. The treatment of the enzyme with the reagent p-bromofenacil brometo neutralized the enzymatic activities, inhibition of aggregation platelet, anticoagulant, hypotensive and antitumoral. The moderate activity edematogenic was inhibited partially. The cDNA (BPIR-A) that codes the protein Bpir-I-PLA2 was cloned and the protein recombinant, expressed. Bpir-I-PLA2 recombinant presented the same sequence of amino acids, structures secondary, phospholipase activity and inhibitory effects on platelet of the native protein, suggesting that recPLA2 was efficiently express and isolated. Due to its low toxicity, Bpir-I-PLA2 could become an important tool in the study of the disorders of the clotting as well as a potential model for the elaboration of new drugs for use in the clinic-doctor.
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Analyse moléculaire de l’interaction entre peupliers et Melampsora spp. par des approches génomiques et fonctionnelles / Molecular analysis of the poplar-Melampsora spp. interaction using genomics and functional approachesLorrain, Cécile 28 March 2018 (has links)
La maladie de la rouille foliaire du peuplier causée par des champignons du genre Melampsora (Pucciniales, Basidiomycètes) affecte largement les peupleraies en France. Ces champignons possèdent des cycles de vie complexes et infectent deux hôtes différents. La sécrétion de molécules appelées effecteurs est nécessaire lors du processus d'infection par le champignon afin de manipuler les processus de l’hôte et de contourner son immunité. La compréhension de leur rôle est centrale en phytopathologie moléculaire. Au cours de cette thèse, l’analyse du transcriptome de Melampsora larici-populina (Mlp) au cours de son cycle sexué lors de l'infection des deux hôtes, le peuplier et le mélèze, révèle la présence d'une majorité de gènes exprimés communément chez les deux hôtes et d'une fraction exprimée spécifiquement chez chaque hôte, notamment des gènes codant des effecteurs candidats. Des cribles fonctionnels réalisés sur un répertoire d’effecteurs candidats de Mlp ont révélé deux candidats d’intérêt. L’effecteur MLP124017 interagit avec des protéines de la famille TOPLESS-RELATED PROTEINS et présente une structure similaire à des protéines NUCLEAR TRANSPORT FACTOR 2 LIKE. L'effecteur MLPCTP1 est localisé dans les chloroplastes en système hétérologue tabac et chez le peuplier. Les fonctions de ces effecteurs restent à élucider mais ces travaux ouvrent de nouvelles perspectives quant à la diversité et au rôle des effecteurs chez les Pucciniales. L'analyse préliminaire du génome de M. allii-populina montre des répertoires comparables en gènes et en effecteurs candidats par rapport à Mlp ainsi qu'une expansion de la taille du génome due à l’invasion par des éléments transposables / The poplar rust disease is caused by fungi belonging to the Melampsora genus (Pucciniales, Basiodiomycota) that cause important damages in poplar plantations in France. These fungi achieve their complex life cycles on two different host plants. The secretion of molecules called effectors that alter cell processes and impair immunity are required to set a successful infection. A central theme of molecular phytopathology is to understand how these molecules function in the host cell. In this PhD thesis, the transcriptome analysis of M. larici-populina (Mlp) during its sexual cycle while infecting its two host plants, poplar and larch, revealed a majority of genes commonly expressed on both hosts and a fraction specifically expressed on each host, including genes encoding candidate effectors. Effectoromic screens developed on a panel of Mlp candidate effectors revealed two candidates of interest. The candidate effector MLP124017 interacts with proteins of the TOPLESS-RELATED PROTEINS family and presents a structure similar to NUCLEAR TRANSPORT FACTOR 2 LIKE proteins. The MLPCTP1 effector is translocated inside chloroplasts of the heterologous plant tobacco and poplar. The functions of these two effectors remain to be determined but the functional characterization initiated in this thesis opens new perspectives in term of diversity and roles of effectors in Pucciniales. The preliminary analysis of the M. allii-populina genome shows similar repertoires of genes and candidate effector genes compared with Mlp as well as an increased genome size due to transposable elements invasion
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Caracterização funcional e estrutural de uma fosfolipase A2 ácida isolada do veneno de Bothrops pirajai / Functional and structural characterization of acidic phospholipase A2 isolated from Bothrops pirajai snake venom.Sabrina Schaaf Teixeira 04 March 2009 (has links)
A caracterização proteômica de venenos de serpentes contribui significativamente para o avanço das Ciências na área Biomédica. Várias destas moléculas, utilizadas como instrumentos de investigação de mecanismos celulares e moleculares, estão envolvidas em diversos processos fisiopatológicos e de intoxicação. No entanto, os venenos de serpentes ainda requerem caracterização funcional e/ou biológica adicionais. As fosfolipases A2s (PLA2s) são enzimas que induzem vários efeitos farmacológicos, e geralmente, correspondem a maior porcentagem do conteúdo protéico dos venenos de serpentes. Desta forma, o isolamento e a caracterização bioquímica, funcional e estrutural de PLA2s poderão gerar informações importantes para o melhor entendimento dos efeitos farmacológicos e de intoxicação ocasionados por estas proteínas. Este trabalho descreve o isolamento e a caracterização bioquímica, enzimática, funcional e estrutural da primeira PLA2 Asp49 ácida, denominada Bpir-I-PLA2, isolada do veneno da serpente Bothrops pirajai, espécie endêmica da região Sul do Estado da Bahia e atualmente, integrante da Lista Nacional das Espécies da Fauna Brasileira Ameaçadas de Extinção. O isolamento da Bpir-I-PLA2 foi realizada através de dois passos cromatográficos, envolvendo inicialmente cromatografia de troca iônica, seguida de HPLC de fase reversa. Quanto submetida à eletroforese em gel de poliacrilamida para proteínas ácidas, Bpir-I-PLA2 apresentou alto grau de homogeneidade. A massa molecular relativa, determinada através de eletroforese em gel de poliacrilamida com agente desnaturante, foi de aproximadamente 28.000 para a forma de dímero e 14.500 para o monômero. A focalização isoelétrica demonstrou uma única banda para a fosfolipase A2, revelando pI aproximado de 4,9. A região N-terminal de Bpir-I-PLA2, seqüenciada através do método de degradação de Edman, apresentou elevada homologia entre outras PLA2s de venenos de serpentes. A composição de amioácidos da proteína corroborou com sua característica ácida, apresentando um elevado conteúdo de aminoácidos carregados negativamente. A enzima apresentou elevada atividade fosfolipásica e induziu edema moderado in vivo. Além disso, Bpir-I-PLA2 foi capaz de inibir a agregação plaquetária e a coagulação do plasma, e também induzir efeito hipotensor in vivo e citotóxico sobre células tumorais, bactérias, fungos e leishmanias. O tratamento da enzima com o reagente brometo de p-bromofenacila neutralizou as atividades enzimática, inibitória sobre a agregação plaquetária, anticoagulante, hipotensora e antitumoral. A moderada atividade edematogênica foi parcialmente inibida. O cDNA contendo 366 bp (BPIR-A) que codifica a proteína Bpir-I-PLA2 foi clonado e a proteína recombinante, expressa. Bpir-I-PLA2 recombinante apresentou a mesma sequência de aminoácidos, estrutura secundária, atividade fosfolipásica e efeito inibitório sobre plaquetas, observados para a proteína nativa Bpir-I-PLA2, sugerindo que a recPLA2 foi eficientemente expressa e purificada. Devido à baixa toxicidade demonstrada, a Bpir-I-PLA2 pode tornar-se uma ferramenta importante nos estudos das desordens da coagulação, como também um potencial modelo para a elaboração de novos fármacos para uso na clínica-médica. / The proteomic characterization of snake venoms significantly contributes to science advance in the biomedical area. Several venom toxins are used as instruments for the investigation of cellular and molecular mechanisms, and are involved in a number of physiopathological and intoxication processes. Nevertheless, snake venoms still require further functional and/or biological characterization. Phospholipases A2 (PLA2s) are enzymes that induce several pharmacological effects and usually correspond to the major percentage of snake venom protein contents. Being so, the isolation and characterization of PLA2s could generate important information for the better understanding of the pharmacological and toxic effects caused by these proteins. This work describes the isolation and biochemical, enzymatic and functional characterization of the first acidic Asp49 PLA2 (named Bpir-I-PLA2) from the venom of Bothrops pirajai snake, an endemic species from the south region of state of Bahia and that nowadays is part of the national list of Brazilian fauna species threatened of extinction. The isolation of Bpir-I-PLA2 was carried out by two chromatographic steps, an ion-exchange chromatography followed by reverse phase HPLC. When submitted to polyacrylamide gel electrophoresis for acidic proteins, Bpir-I-PLA2 showed high homogeneity level. The relative molecular mass, determined by polyacrylamide gel electrophoresis with denaturating agent, was of approximately 28,000 for the dimmer and 14,500 for the monomer. With high phospholipase activity and pharmacological effects, characteristic of acidic isoforms, Bpir-I-PLA2 did not present myotoxic activity and showed to be little edematogenic. However, the enzyme acts inhibiting platelet aggregation, delaying plasma clotting time and inducing hypotension, antitumoral effects, antibacteria, antifungal and parasiticide actions. The treatment of the enzyme with the reagent p-bromofenacil brometo neutralized the enzymatic activities, inhibition of aggregation platelet, anticoagulant, hypotensive and antitumoral. The moderate activity edematogenic was inhibited partially. The cDNA (BPIR-A) that codes the protein Bpir-I-PLA2 was cloned and the protein recombinant, expressed. Bpir-I-PLA2 recombinant presented the same sequence of amino acids, structures secondary, phospholipase activity and inhibitory effects on platelet of the native protein, suggesting that recPLA2 was efficiently express and isolated. Due to its low toxicity, Bpir-I-PLA2 could become an important tool in the study of the disorders of the clotting as well as a potential model for the elaboration of new drugs for use in the clinic-doctor.
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