Navegando por Palavras-chave "espectrometria de massas"
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- ItemSomente MetadadadosCaracterização da presença de AKT no núcleo de células de melanoma e identificação de proteínas associadas por espectrometria de massas(Universidade Federal de São Paulo, 2016-08-16) Coa, Larissa Leggieri [UNIFESP]; Machado Junior, Joel [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The serine threonine kinase AKT/PKB is a critical regulator of various essential physiological cellular processes including cell proliferation, survival, motility and size, metabolism and differentiation. Deregulation of this pathway has been implicated in many diseases including cancers. Despite AKT action is acknowledged to function mainly in the cytoplasm, where most of its known substrates are located, AKT has been reported to translocate to the nucleus of various cell types. Therefore, AKT functions may be achieved through distinct subcellular compartments, providing spatial specificity in the mediation of biological effects. However, very little is known about the mechanism required for the nuclear import of AKT. Also, the interaction of AKT with proteins that reside in the nucleus has not been well explored as well as how its kinase activity modulates signaling in this compartment. Thus, identification of additional targets or binding proteins of nuclear AKT may provide a better understanding of it functions in the nucleus. In the present study we characterized the presence of endogenous nuclear AKT in two human melanoma cell lines harboring distinct genetic backgrounds (A2058, PTEN mutant/deleted; Mewo, PTEN wild-type). Our results showed that either phosphorylated and non-phosphorylated forms of AKT are present in melanoma cells nuclei, suggesting that phosphorylation of AKT is not required for its nuclear localization. Using co-immunoprecipitation (Co-IP) combined with crosslinking and mass spectrometry we identified a series of putative protein partners of nuclear AKT. Functional groups of proteins with known nuclear role emerged, such as RNA-binding proteins involved in transcription and pre-mRNA processing [e.g. heterogeneous nuclear ribonucleoprotein (hnRNP) and Serine/arginine-rich splicing factors (SRSF)], proteins required for ribosome biogenesis and rRNA processing (Ribosomal proteins), as well as cytoskeleton proteins (e.g. Actin, Vimentin and F-capping subunit ?). We validated ?-actin as a bona fide nuclear AKT-interacting partner. We also provided evidences that proteins such as cofilin and RNA polymerase II, known to interact and work in concert with ?-actin in the nucleus, also couple with nuclear AKT. Considering that nuclear actin has been described to have a key role regulating nuclear process such as chromatin remodeling, transcription, RNA processing and export, our findings provide new insights into the possible direct involvement of AKT in the transcriptional network of nuclear actin.
- ItemSomente MetadadadosCaracterização proteômica, peptidômica e transcriptômica dos venenos de aranhas do gênero acanthoscurria(Universidade Federal de São Paulo (UNIFESP), 2015-04-09) Abreu, Thiago Ferreira de [UNIFESP]; Tashima, Alexandre Keiji Tashima [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Spiders are one of the most successful animals on the planet, currently recording 45,330 species divided into 3957 genus and separated in two suborders: Mesothelae and Opistothelae. The most studied spiders belong to the second suborder, which are organized in two groups based on chelicerae position (Araneomorph and Mygalomorphae) and are studied, among other reasons, because of their medical and biological relevance. The chelicerae inoculates a toxic secretion produced by the venom gland for subduying preys and protection against predators. The venom presents a range of components with specific functions and synergical effects. Recent studies of spider venoms demonstrated the presence of proteins and peptides with antimicrobial activity against fungi, bacteria and protozoa, ion channel modulation and selective activity against vertebrate and invertebrate receptors, indicating the biotechnological potential for these molecules. The study of the spider venom molecular arsenal is also relevant to the understanding of phylogeny and evolutionary success and to determine variations in composition, which may depend on individual variability, sexual dimorfism and diet. Nonetheless, there are few studies about these animals. In this study, considering the biotechnological potential and the biological relevance of the toxins in spider venoms, a quantitative proteomic study of venoms from male and female spiders belonging to Acanthoscurria gomesiana and Acanthoscurria juruenicola species was developed, besides transcriptomics of A. juruenicola venom glands, and antimicrobial assays of purified peptides. Transcriptomic analysis of female A. juruenicola venom glands resulted in 88,335 protein sequences obtained by translation of its respective cDNAs. Digestion of venoms with trypsin and thermolysin followed by mass spectrometry analysis by data-dependent acquisition (DDA) and data-independent acquisition (DIA) approaches resulted in the identification of 353 proteins in A. juruenicola venoms and 188 proteins in A. gomesiana venoms by automated de novo sequencing and database searches. Absolute quantification of proteins present in A. juruenicola spider venoms by DIA showed a prevalence of toxins, such as cysteine-rich venom protease (CRISPs), theraphotoxins, metalloendopeptidases, lipases, hyaluronidases, tyrosine-phosphatase and metallocarboxypeptidases. One of A. juruenicola theraphotoxins showed complete homology with the peptide already described from Acanthoscurria paulensis venom (U1-theraphotoxin-Ap1a) and a new theraphotoxin from A. gomesiana, Ag1a, was fully characterized. Crude venom fractionation by gel filtration chromatography in order to purify peptides (1-7 kDa) was accomplished, enabling the isolation and characterization of A. gomesiana and A. juruenicola theraphotoxins. Antimicrobial assays against a yeast (Candida albicans) and three bacteria, two Gram negatives (Pseudomonas aeruginosa and Escherichia coli) and one Gram positive (Micrococcus luteus) was accomplished with these peptides and the minimum inhibitory concentrations (MIC) were determined.
- ItemSomente MetadadadosCaracterização proteômica, peptidômica e transcriptômica dos venenos de aranhas do gênero acanthoscurria(Universidade Federal de São Paulo (UNIFESP), 2015-04-09) Abreu, Thiago Ferreira de [UNIFESP]; Tashima, Alexandre Keiji Tashima [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Spiders are one of the most successful animals on the planet, currently recording 45,330 species divided into 3957 genus and separated in two suborders: Mesothelae and Opistothelae. The most studied spiders belong to the second suborder, which are organiz
- ItemSomente MetadadadosDeterminação do perfil lipídico metabolômico plasmático, por espectrometria de massas, de pacientes com neoplasias mieloproliferativas crônicas, síndromes mielodisplásicas e leucemia mieloide aguda(Universidade Federal de São Paulo (UNIFESP), 2015-03-25) Oliveira, Adriana Ramos de [UNIFESP]; Chauffaille, Maria de Lourdes Lopes Ferrari Chauffaille [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: There are thousands of individual lipid species in the cells interacting in different compartments in the cell membrane. The functional consequences of this diversity are not yet fully understood and new technological tools are being developed with the aim of a more comprehensive investigation. Over the past two decades, mass spectrometry (MS) has emerged as the main method used in lipidomics analysis, which allows the structural characterization and quantification of complex lipids and their metabolites. Due to the importance of this field we have considered the use of the lipidomic innovative platform to identify differences in the plasma lipid metabolomic profile of hematological patients with Myeloid Neoplasms. Purpose: Determine the lipid metabolomic profile comparative of blood plasma samples from healthy individuals and patients with Myeloproliferative Neoplasms, Myelodysplastic Syndromes and Acute Myeloid Leukemia and evaluate the existence of possible biomarkers. Methods: Untargeted Shotgun MS/MS Analysis was performed from plasma samples from 153 participants were analyzed being, 90 of the Control Group, 43 Myeloproliferative Neoplasms, 11 Myelodysplastic Syndromes and 9 Acute Myeloid Leukemias. Data were acquired using the AB-Sciex Analyst TF, processed using the AB-Sciex LipidView? and the web-based analytical pipeline MetaboAnalyst 2.0. Results: Untargeted analysis identified in negative and positive-modes a total of 658 features at 2 ppm resolution. PCA and PLS-DA analysis revealed clear discrimination among groups, in particular for AML patients. Main lipid groups differentially expressed were: Monoacylglycerols (MAG), Glucosylceramide E (GlcdE), Ethyl Esters (EE), Lysophosphatidic acid (LPA), Sulfoquinovosil diacylglycerols (SQDG), Monoglycerols (MG), Methyl Ethanolamines (ME), Lysophosphatidylcholines (LPC), Dimethyl Phosfatidyletanilamines (DMPE), Monometylphosphatidiletanolamines (MMPE), Ceramide-1-phosphate (CerP), Glicerophosphoglycerols (GP), Lysomonomethyl-Phosphatidyl ethanolamines (LMMPE), Phosphatidic Acids (PA), Ergosterols (ERG), Glycerophosphoserine (PS), Diacylglycerols (DAG), Hexocylceramides (HexCer) and Lanosterol (Lan). ROC Curve Analysis revealed Total LMMPE as the strongest discriminating marker between Controls from Patients with MDS or AML (Sensitivity= 0.95 (0.824-1); Specificity= 0.8941 (0.847-0953); Positive Likelihood Ratio= 8.972 and Negative Likelihood Ratio =0.05592 and T Test= 7.576E-12). In addition these lipids were also able to differentiate MDS and AML from NMP (Sensitivity= 0.9118 (0.824-1), Specificity= 0.95 (0.85-1); Positive Likelihood Ratio= 18.2 and Negative Likelihood Ratio= 0.05592). Conclusions: The Myeloproliferative Neoplasms from the point of view of global plasma lipidomics are accompanied by several modifications. In particular the Lysomonomethyl-Phosphatidyl ethanolamines seems to play important differentiating roles among them.
- ItemSomente MetadadadosA influência do índice de massa corporal na fisiologia do folículo ovariano em ciclos de reprodução assistida(Universidade Federal de São Paulo (UNIFESP), 2013-04-03) Porciuncula, Patricia Marafon [UNIFESP]; Bertolla, Ricardo Pimenta Bertolla [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Objective: To observe the influence of Body Mass Index (BMI) on the follicular metabolism of women submitted to controlled ovarian hyperstimulation. Methods: A prospective study was carried out including 129 follicular fluid samples from women submitted to controlled ovarian hyperstimulation and classified, according to BMI, in three groups according to the World Health Organization: eutrophic (20 to <25kg/m2), overweight (25 to <30kg/m2), and obese (>30kg/m2). Follicular fluid samples were obtained by ultrasound-guided transvaginal ovarian aspiration following controlled ovarian hyperstimulation. Immediately after collection, the follicular fluid samples were centrifuged to remove cellular debris and frozen until analysis. For quantification of the steroid hormones estradiol, progesterone, testosterone, androstenedione, and cortisol, tandem mass spectrometry was utilized (ID-LC-MS/MS). For proteomic analysis, three pools were formed (one for each group, normalized to total protein in each sample), and proteins were quantified and identified utilizing the shotgun proteomics approach nanoUPLC-nanoESI-MSE, resulting in ?Identified? and ?Quantified and Identified? proteins. Proteins observed in only one replicate were excluded from the study. For steroid profiling, groups were compared using one-way ANOVA followed by a Least Significant Differences posthoc test, or using a Kruskal-Wallis followed by a Mann-Whitney test. For the proteomics experiment a Kruskal-Wallis test was used, followed by a Mann-Whitney test. An alpha of 5% was adopted in the study. Protein lists were utilized for functional enrichment analysis. Results: Regarding the steroid profile, differences were observed between the BMI groups. The obesity group presented lower progesterone levels and higher androstenedione levels when compared to the other two groups. Also, estradiol, testosterone, and cortisol were higher in both overweight and obese groups when compared to eutrophic controls. Regarding proteomics, 284 proteins were identified, of which 152 were quantified. Major enriched functions were related to immune response, inflammation and coagulation. Moreover, seven proteins were hyperexpressed only in the obese patients. Finally, 20 proteins were exclusively expressed in the obesity group, 10 in the overweight group, and 19 in the eutrophic group. Conclusion: Follicular fluid metabolism is altered due to obesity in patients submitted to controlled ovarian hyperstimulation. The follicular fluid of overweight and obese patients presents altered steroid profiles, with increased estradiol, testosterone, and cortisol, and obese patients present increased androstenedione and decreased progesterone. Also, overweightness and obesity alter the follicular fluid protein profile of patients submitted to controlled ovarian hyperstimulation, with enriched immune and inflammatory response functions.