133 resultados para Core promoter
Regulation of the vitellogenin gene B1 promoter after transfer into hepatocytes in primary cultures.
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The estrogen-dependent and tissue-specific regulation of the Xenopus laevis vitellogenin gene B1 promoter has been studied by lipid-mediated DNA transfer into Xenopus hepatocytes in primary culture. Hepatocytes achieve an efficient hormonal control of this promoter through a functional interaction between the estrogen responsive elements and a promoter proximal region upstream of the TATA box, which is characterized by a high density of binding sites for the transcription factors CTF/NF-1, C/EBP and HNF3. DNA accessibility to restriction enzymes within the chromosomal copy of the vitellogenin gene B1 promoter shows that the estrogen responsive unit and the promoter proximal region are sensitive to digestion in uninduced and estrogen-induced hepatocytes but not in erythrocyte nuclei. Together, these findings support the notion that chromatin configuration as well as the interplay of promoter elements mediate proper hormone-dependent and tissue-specific expression of the B1 vitellogenin gene.
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Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood.
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PURPOSE: Rechallenge with temozolomide (TMZ) at first progression of glioblastoma after temozolomide chemoradiotherapy (TMZ/RT→TMZ) has been studied in retrospective and single-arm prospective studies, applying temozolomide continuously or using 7/14 or 21/28 days schedules. The DIRECTOR trial sought to show superiority of the 7/14 regimen. EXPERIMENTAL DESIGN: Patients with glioblastoma at first progression after TMZ/RT→TMZ and at least two maintenance temozolomide cycles were randomized to Arm A [one week on (120 mg/m(2) per day)/one week off] or Arm B [3 weeks on (80 mg/m(2) per day)/one week off]. The primary endpoint was median time-to-treatment failure (TTF) defined as progression, premature temozolomide discontinuation for toxicity, or death from any cause. O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation was prospectively assessed by methylation-specific PCR. RESULTS: Because of withdrawal of support, the trial was prematurely closed to accrual after 105 patients. There was a similar outcome in both arms for median TTF [A: 1.8 months; 95% confidence intervals (CI), 1.8-3.2 vs. B: 2.0 months; 95% CI, 1.8-3.5] and overall survival [A: 9.8 months (95% CI, 6.7-13.0) vs. B: 10.6 months (95% CI, 8.1-11.6)]. Median TTF in patients with MGMT-methylated tumors was 3.2 months (95% CI, 1.8-7.4) versus 1.8 months (95% CI, 1.8-2) in MGMT-unmethylated glioblastoma. Progression-free survival rates at 6 months (PFS-6) were 39.7% with versus 6.9% without MGMT promoter methylation. CONCLUSIONS: Temozolomide rechallenge is a treatment option for MGMT promoter-methylated recurrent glioblastoma. Alternative strategies need to be considered for patients with progressive glioblastoma without MGMT promoter methylation.
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INTRODUCTION: Perfusion-CT (PCT) processing involves deconvolution, a mathematical operation that computes the perfusion parameters from the PCT time density curves and an arterial curve. Delay-sensitive deconvolution does not correct for arrival delay of contrast, whereas delay-insensitive deconvolution does. The goal of this study was to compare delay-sensitive and delay-insensitive deconvolution PCT in terms of delineation of the ischemic core and penumbra. METHODS: We retrospectively identified 100 patients with acute ischemic stroke who underwent admission PCT and CT angiography (CTA), a follow-up vascular study to determine recanalization status, and a follow-up noncontrast head CT (NCT) or MRI to calculate final infarct volume. PCT datasets were processed twice, once using delay-sensitive deconvolution and once using delay-insensitive deconvolution. Regions of interest (ROIs) were drawn, and cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) in these ROIs were recorded and compared. Volume and geographic distribution of ischemic core and penumbra using both deconvolution methods were also recorded and compared. RESULTS: MTT and CBF values are affected by the deconvolution method used (p < 0.05), while CBV values remain unchanged. Optimal thresholds to delineate ischemic core and penumbra are different for delay-sensitive (145 % MTT, CBV 2 ml × 100 g(-1) × min(-1)) and delay-insensitive deconvolution (135 % MTT, CBV 2 ml × 100 g(-1) × min(-1) for delay-insensitive deconvolution). When applying these different thresholds, however, the predicted ischemic core (p = 0.366) and penumbra (p = 0.405) were similar with both methods. CONCLUSION: Both delay-sensitive and delay-insensitive deconvolution methods are appropriate for PCT processing in acute ischemic stroke patients. The predicted ischemic core and penumbra are similar with both methods when using different sets of thresholds, specific for each deconvolution method.
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Sediments can be natural archives to reconstruct the history of pollutant inputs into coastal areas. This is important to improve management strategies and evaluate the success of pollution control measurements. In this work, the vertical distribution of organochlorine pesticides (DDTs, Lindane, HCB, Heptachlor, Aldrin and Mirex) was determined in a sediment core collected from the Gulf of Batabanó, Cuba, which was dated by using the (210)Pb dating method and validated with the (239,240)Pu fallout peak. Results showed significant changes in sediment accumulation during the last 40 years: recent mass accumulation rates (0.321 g cm(-2) yr(-1)) double those estimated before 1970 (0.15 g cm(-2) yr(-1)). This change matches closely land use change in the region (intense deforestation and regulation of the Colon River in the late 1970s). Among pesticides, only DDTs isomers, Lindane and HCB were detected, and ranged from 0.029 to 0.374 ng g(-1) dw for DDTs, from<0.006 to 0.05 ng g(-1) dw for Lindane and from<0.04 to 0.134 ng g(-1) dw for HCB. Heptachlor, Aldrin and Mirex were below the detection limits (∼0.003 ng g(-1)), indicating that these compounds had a limited application in the Coloma watershed. Pesticide contamination was evident since the 1970s. DDTs and HCB records showed that management strategies, namely the banning the use of organochlorine contaminants, led to a concentration decline. However, Lindane, which was restricted in 1990, can still be found in the watershed. According to NOAA guidelines, pesticides concentrations encountered in these sediments are low and probably not having an adverse effect on sediment dwelling organisms.
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L'élément génétique intégratif et conjugatif auto-transférable de 103 kb qui se trouve dans le génome de Pseudomonas knackmussii B13 (ICEc/c) confère la capacité de dégrader le 3-chlorobenzoate et le 2-aminophénol. L'élément ICE c/c peut être transféré par conjugaison de la souche B13 à diverses bêta- et gamma- protéobactéries. Seule une sous-population de 3 à 5% des cellules transfère l'élément, les cellules dites "compétentes pour le transfert". L'acquisition de la compétence pour le transfert est vraisemblablement la conséquence d'une régulation bistable, conduisant une partie des cellules au transfert de l'élément ICE c/c tandis que, dans les autres, l'élément reste quiescent et ne se transfère pas. À ce jour, les mécanismes et les acteurs moléculaires qui régulent l'activation bistable de l'élément sont restés inconnus. Mon travail de doctorat visait à identifier les éléments bistables du régulon de la compétence pour le transfert et d'analyser les fondements moléculaires de la bistabilité de l'élément ICE c/c chez P. knackmussii. Le premier chapitre introduit le thème du transfert génétique horizontal avec un accent particulier sur les éléments intégratifs et conjugatifs (ICE) et ICEcIc. L'état actuel des connaissances sur l'organisation génétique, la régulation, l'intégration et le transfert de différents modèles de ICEs est exposé en détail. En outre, je m'étends sur les phénomènes d'hétérogénéité et de bistabilité phénotyplques, qu'on peut distinguer dans une population isogénique dans des conditions de culture homogènes, et qui sont susceptibles de jouer un rôle dans le transfert de l'élément ICE c/c, dans la mesure où il ne s'active et n'est transférable que dans une très petite sous-population de cellules. Dans le chapitre 2, je présente une analyse globale des régions promotrices minimales des gènes appartenant au régulon de la compétence pour le transfert de l'élément ICE c/c. Nous avons étudié les caractéristiques d'expression des promoteurs et, s'ils s'avéraient bistables, leur activation dans le temps par comparaison avec le mutant lntB13. Pour ce faire, nous avons utilisé des fusions de promoteurs avec des gènes rapporteurs et testé l'expression bistable chez P. knackmussii par microscopie à épifluorescence. Pour six promoteurs présentant une expression bistable, nous avons employé de la microscopie temporelle pour déterminer la chronologie de leur expression par rapport à Pint et PinR. Parmi eux, nous avons identifié deux gènes exprimés précocement et trois gènes exprimés tardivement dans le processus d'acquisition de la compétence de transfert. Dans le chapitre 3, j'expose une analyse d'expression génétique pour l'un des groupes de gènes dont la transcription est la plus élevée dans la région conservée de ICE c/c, les gènes orf81655-orf68241 contenus dans une région de 14 kb. Nous montrons d'abord que cet opéron fait partie du même régulon bistable que intB13 et inrR et analysons les caractéristiques génétiques qui conduisent à une transcription élevée. Nous étudions les fonctions biologiques de ce groupe de gènes par des délétlons ciblées et montrons que certaines d'entre elles empêchent le transfert de l'élément. Nous approfondissons la caractérlsatlon de I'orf8l655 en construisant une fusion transcrlptionnelle avec le gène codant pour la protéine fluorescente verte (egfp) (en utilisant le système minl-Tn5). L'expression de Vorf81655 dans des cellules individuelles est comparée au signal mesuré par hybridation in situ en fluorescence (FISH) sur le ARN messager du gène. En utilisant FISH, des délétlons du promoteur et de l'analyse directe de transcription, nous avons localisé la région promotrice du groupe de gènes. En outre, nous avons utilisé des mutations dirigées pour comprendre la bistabilité de cette région promotrice, caractérisée par une transcription très élevée et une traduction lente de l'ARN messager. Dans le chapitre 4, nous nous efforçons de comprendre comment la bistabilité est générée au sein du régulon te de l'élément ICE c/c. Pour ce faire, nous avons tenté de reconstituer une expression bistable, dans un hôte qui ne présente pas de bistabilité naturellement, à partir d'éléments génétiques individuels. L'hôte choisi est Pseudomonas putida dans lequel nous avons introduit une copie unique de Pint, PinR ou PaipA fusionnés à la egfp, construits qui permettent d'observer l'apparition de bistabilité. Nous avons ensuite construit différents assemblages de composants génétiques de l'élément ICE c/c, en nous concentrant sur la région parA-inrR. En effet, nous avons pu démontrer qu'une expression bistable apparaît dans P. putida grâce à ces éléments en l'absence de l'élément ICE c/c complet. À noter que la plupart des construits génétiques activent PaipA ou P|,,R, mais qu'un seul recrée la bistabilité de Pint, ce qui suggère que la région parA-inrR permet à la fois d'engendrer la bistabilité et d'opérer la transition entre les promoteurs précoces et les promoteurs tardifs du régulon de la bistabilité. Dans le chapitre 5, nous concluons sur une discussion de la pertinence de nos résultats et sur de futures perspectives de recherche. -- The 103-kb self-transmissible integrative and conjugative element (ICE) of Pseudomonas knackmussii B13 (ICEc/c) confers the capacity to degrade 3- chlorobenzoate and 2-aminophenol. ICEc/c can be conjugated from strain B13 to a variety of Beta- and Gammaproteobacteria. Interestingly, ICE c/c transfer is observed in a subpopulatlon of cells (3-5%) only, the so-called 'transfer competent' cells. The formation of transfer competence (tc) is thought to be the consequence of a 'bistable' decision, which forces those cells to follow the developmental path which leads to ICEc/c transfer, whereas in others ICE c/c remains silent and does not transfer. So far, the mechanisms and molecular partners generating this bistable transfer activation in cells of P. knackmussii B13 remain mostly unidentified. This thesis aimed at understanding the extent of the tc bistability regulon and to dissect the molecular basis of bistabillty formation of ICEc/c in P. knackmussii. The first chapter is a general Introduction on horizontal gene transfer (HGT) with particular emphasis on ICEs and ICE c/c. The emphasis is made on the current knowledge about the HGT gene organization, regulation and specific integration and transfer aspects of the different ICEs models. Furthermore, I focus on the phenomena of phenotypic heterogeneity and bistability (the property of two distinguishable phenotypes existing within an isogenic population under homogeneous conditions), which may play a particular role in ICEc/c behaviour, since ICE activation and transfer only occurs in a very small subpopulation of cells. In Chapter Two, I focus on a global analysis of the different core promoters that might belong to the ICEc/c tc pathway regulon. We studied both expression patterns of ICEc/c promoters and, once being identified as "bistable", their temporal activation compared to that of intB13. In order to do this, we used promoter reporter fusions and tested blstability expression in P. knackmussii using epifluorescence microscopy. For the 6 promoters that showed bistable expression, we used time-lapse microscopy to study the timing of promoter expression in comparison to that of P,,,t or PlnR. We could establish two "early" and 3 "late" phase promoters in the process of transfer competence. In Chapter Three, I focused my attention on analysis of gene expression of one of the most highly transcribed gene clusters in the conserved core region of ICEc/c, a 14-kb gene cluster formed by the genes orf81655-orf68241. First we showed that this operon is part of the same bistability 'regulon' as intB13 and inrR, and analysed the genetic features that lead to high transcription. We studied the potential biological function of this cluster for ICE c/c by making specific gene deletions, showing that some interrupt ICEc/c transfer. We further analysed the orfdl655 promoter by constructing transcriptional egfp fusion reporter strains using the miniTn5 delivery system. Expression of the orf81655 promoter in single cells was compared to signals measured by Fluorescence In Situ Hybridization (FISH) on orfSl655 mRNA. We localized the promoter region of the gene cluster using FISH, promoter deletions, and by direct transcript analysis. We further used site-directed mutagenesis to understand the bistability character of the promoter region and the extremely high transcription but low translation from this mRNA. In Chapter Four, we set out to understand how bistability is generated in the tc pathway of ICEc/c. For this we tried rebuilding bistable expression from ICEc/c individual gene components in a host, which normally does not display bistability. As host we used P. putida without ICEc/c but with a single copy Pint-, PlnR- or PalpA- egfp fusion that enabled us to verify bistability formation. Subsequently, we built different assemblages of ICEc/c gene components, focusing on the parA-inrR region. Indeed, we found that bistable expression can be build from those components in P. putida without ICEc/c. Interestingly, most genetic constructs activated PaipA or PlnR, but only one resulted in bistable activation of PinT. This suggests that the parA-inrR region acts as a bistability "generator", but also as a bistability "relay" from early to late promoters in the tc pathway hierarchy. In the final fifth chapter, we conclude with a discussion of the relevance of the present thesis and the resulting perspectives for future studies.
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Chromosome 22q11.2 deletion syndrome (22q11DS) is a genetic disease known to lead to cerebral structural alterations, which we study using the framework of the macroscopic white-matter connectome. We create weighted connectomes of 44 patients with 22q11DS and 44 healthy controls using diffusion tensor magnetic resonance imaging, and perform a weighted graph theoretical analysis. After confirming global network integration deficits in 22q11DS (previously identified using binary connectomes), we identify the spatial distribution of regions responsible for global deficits. Next, we further characterize the dysconnectivity of the deficient regions in terms of sub-network properties, and investigate their relevance with respect to clinical profiles. We define the subset of regions with decreased nodal integration (evaluated using the closeness centrality measure) as the affected core (A-core) of the 22q11DS structural connectome. A-core regions are broadly bilaterally symmetric and consist of numerous network hubs - chiefly parietal and frontal cortical, as well as subcortical regions. Using a simulated lesion approach, we demonstrate that these core regions and their connections are particularly important to efficient network communication. Moreover, these regions are generally densely connected, but less so in 22q11DS. These specific disturbances are associated to a rerouting of shortest network paths that circumvent the A-core in 22q11DS, "de-centralizing" the network. Finally, the efficiency and mean connectivity strength of an orbito-frontal/cingulate circuit, included in the affected regions, correlate negatively with the extent of negative symptoms in 22q11DS patients, revealing the clinical relevance of present findings. The identified A-core overlaps numerous regions previously identified as affected in 22q11DS as well as in schizophrenia, which approximately 30-40% of 22q11DS patients develop.