992 resultados para RNA extraction
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Several alternative approaches have been discussed: Levenberg-Marquardt - no satisfactory convergence speed + local minimum, Bacterial algorithm - problems with large dimensionality (speed), Clustering - no safe criterion for number of clusters + dimentionality problem.
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Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011
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Regional Innovation Systems describe the relations between actors, structures and infrastructures in a region in order to stimulate innovation and regional development. For these systems the collection and organization of information is crucial. In the present paper we investigate the possibilities to extract information from websites of companies. First we describe regional innovation systems and the information types that are necessary to create them. Then we discuss the possibilities of text mining and keyword extraction techniques to extract this information from company websites. Finally, we describe a small scale experiment in which keywords related to economic sectors and commodities are extracted from the websites of over 200 companies. This experiment shows what the main challenges are for information extraction from websites for regional innovation systems.
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Tese de doutoramento, Informática (Engenharia Informática), Universidade de Lisboa, Faculdade de Ciências, 2014
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Tese de mestrado em Bioinformática e Biologia Computacional (Bioinformática), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014
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Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2016
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Tese de doutoramento, Medicina Dentária (Periodontologia), Universidade de Lisboa, Faculdade de Medicina Dentária, 2016
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The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, a method was developed known as RT-Bst for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of PCR-amplified products. Using random primers a superior performance was observed when amplifying MS2 RNA following RT-Bst compared to RT alone, indicating that greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify 8 target regions spanning the respiratory syncytial virus (RSV) genome. Six out of 8 targets were amplified consistently by PCR subsequent to RT-Bst amplification whereas only 3 out of 8 targets could be amplified after RT alone. RSV sequences were selectively amplified using RSV specific primers from a mixed template containing an excess of MS2 RNA in a RT-Bst reaction without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence specific primers and enhances the yield of cDNA when compared to RT alone.
Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells
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The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.
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The volatiles from Coriandrum sativum L., Satureja montana L., Santolina chamaecyparissus L., and Thymus vulgaris L. were isolated by hydrodistillation (essential oil) and supercritical fluid extraction (volatile oil). Their effect on seed germination and root and shoot growth of the surviving seedlings of four crops (Zea mays L., Triticum durum L., Pisum sativum L., and Lactuca sativa L.) and two weeds (Portulaca oleracea L. and Vicia sativa L.) was investigated and compared with those of two synthetic herbicides, Agrocide and Prowl. The volatile oils of thyme and cotton lavender seemed to be promising alternatives to the synthetic herbicides because they were the least injurious to the crop species. The essential oil of winter savory, on the other hand, affected both crop and weeds and can be appropriate for uncultivated fields.
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Supercritical fluid extraction (SEE) of the volatile oil from Thymus vulgaris L. aerial flowering parts was performed under different conditions of pressure, temperature, mean particle size and CO2 flow rate and the correspondent yield and composition were compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils were analyzed by GC and GC-MS and 52 components were identified. The main volatile components obtained were p-cymene (10.0-42.6% for SFE and 28.9-34.8% for HD), gamma-terpinene (0.8-6.9% for SFE and 5.1-7.0% for HD), linalool (2.3-5.3% for SFE and 2.8-3.1% for HD), thymol (19.5-40.8% for SFE and 35.4-41.6% for HD), and carvacrol (1.4-3.1% for SFE and 2.6-3.1% for HD). The main difference was found to be the relative percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether (1.0-1.2% for HD versus t-0.4 for SFE) which can explain the higher antioxidant activity, assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone is considered a strong antioxidant compound.
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The modelling of the experimental data of the extraction of the volatile oil from six aromatic plants (coriander, fennel, savoury, winter savoury, cotton lavender and thyme) was performed using five mathematical models, based on differential mass balances. In all cases the extraction was internal diffusion controlled and the internal mass transfer coefficienty (k(s)) have been found to change with pressure, temperature and particle size. For fennel, savoury and cotton lavender, the external mass transfer and the equilibrium phase also influenced the second extraction period, since k(s) changed with the tested flow rates. In general, the axial dispersion coefficient could be neglected for the conditions studied, since Peclet numbers were high. On the other hand, the solute-matrix interaction had to be considered in order to ensure a satisfactory description of the experimental data.
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A discussion of the most interesting results obtained in our laboratories, during the supercritical CO(2) extraction of bioactive compounds from microalgae and volatile oils from aromatic plants, was carried out. Concerning the microalgae, the studies on Botryococcus braunii and Chlorella vulgaris were selected. Hydrocarbons from the first microalgae, which are mainly linear alkadienes (C(23)-C(31)) with an odd number of carbon atoms, were selectively extracted at 313 K increasing the pressure up to 30.0 MPa. These hydrocarbons are easily extracted at this pressure, since they are located outside the cellular walls. The extraction of carotenoids, mainly canthaxanthin and astaxanthin, from C. vulgaris is more difficult. The extraction yield of these components at 313 K and 35.0 MPa increased with the degree of crushing of the microalga, since they are not extracellular. On the other hand, for the extraction of volatile oils from aromatic plants, studies on Mentha pulegium and Satureja montana L were chosen. For the first aromatic plant, the composition of the volatile and essential oils was similar, the main components being the pulegone and menthone. However, this volatile oil contained small amounts of waxes, which content decreased with decreasing particle size of the plant matrix. For S. montana L it was also observed that both oils have a similar composition, the main components being carvacrol and thymol. The main difference is the relative amount of thymoquinone, which content can be 15 times higher in volatile oil. This oxygenated monoterpene has important biological activities. Moreover, experimental studies on anticholinesterase activity of supercritical extracts of S. montana were also carried out. The supercritical nonvolatile fraction, which presented the highest content of the protocatechuic, vanilic, chlorogenic and (+)-catechin acids, is the most promising inhibitor of the enzyme butyrylcholinesterase. In contrast, the Soxhlet acetone extract did not affect the activity of this enzyme at the concentrations tested. (C) 2011 Elsevier B.V. All rights reserved.
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Soil vapor extraction (SVE) is an efficient, well-known and widely applied soil remediation technology. However, under certain conditions it cannot achieve the defined cleanup goals, requiring further treatment, for example, through bioremediation (BR). The sequential application of these technologies is presented as a valid option but is not yet entirely studied. This work presents the study of the remediation of ethylbenzene (EB)-contaminated soils, with different soil water and natural organic matter (NOMC) contents, using sequential SVE and BR. The obtained results allow the conclusion that: (1) SVE was sufficient to reach the cleanup goals in 63% of the experiments (all the soils with NOMC below 4%), (2) higher NOMCs led to longer SVE remediation times, (3) BR showed to be a possible and cost-effective option when EB concentrations were lower than 335 mg kgsoil −1, and (4) concentrations of EB above 438 mg kgsoil −1 showed to be inhibitory for microbial activity.
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An accurate and sensitive method for determination of 18 polycyclic aromatic hydrocarbons (PAHs) (16 PAHs considered by USEPA as priority pollutants, dibenzo[a,l]pyrene and benzo[j]fluoranthene) in fish samples was validated. Analysis was performed by microwave-assisted extraction and liquid chromatography with photodiode array and fluorescence detection. Response surface methodology was used to find the optimal extraction parameters. Validation of the overall methodology was performed by spiking assays at four levels and using SRM 2977. Quantification limits ranging from 0.15–27.16 ng/g wet weight were obtained. The established method was applied in edible tissues of three commonly consumed and commercially valuable fish species (sardine, chub mackerel and horse mackerel) originated from Atlantic Ocean. Variable levels of naphthalene (1.03–2.95 ng/g wet weight), fluorene (0.34–1.09 ng/g wet weight) and phenanthrene (0.34–3.54 ng/g wet weight) were detected in the analysed samples. None of the samples contained detectable amounts of benzo[a]pyrene, the marker used for evaluating the occurrence and carcinogenic effects of PAHs in food.