LUIZ FELIPE TEIXEIRA DA SILVA
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Resumo IPEN-doc 29318 Micro-Raman spectroscopy characterization of dental pulp stem cells differentiation induced by calcium phosphate nanoparticles2022 - SILVA, FLAVIA R.O.; PASCOAL, DIEGO R.C.; BERECZKI, ALLAN; SIPERT, CARLA R.; BRAGA, ROBERTO R.; BELLINI, MARIA H.; SILVA, LUIS F.T. da; FREITAS, ANDERSON Z.; WETTER, NIKLAUS U.Calcium phosphates are chemical compounds used in medicine for tissue engineering. This work analyzes the process of cell differentiation by nanoparticles in dental pulp stem cells for tissues regeneration and the development of new therapeutic methods. The most widely used synthetic calcium phosphate based bioceramic is hydroxyapatite [HA, Ca10(PO4)6(OH)2]. Micro-Raman spectroscopy assays are a powerful tool for measuring and characterizing calcium phosphates nanoparticles internalized by cells because of its capability to detect the chemical bonds of nanoparticles and collagen simultaneously and evidencing their interaction within the cell-nanoparticle system. Microscope images (Fig.1a) and Raman spectra (Fig.1b) were obtained for HA-incorporated stem cells where it was possible to observe the formed nodules of calcium phosphate and the matrix in the incorporated samples. HA and collagen peaks were detected in the samples, showing that the nanoparticles induced osteogenic differentiation of the stem cells. The spectra of the nodules showed HA characteristic peaks, while matrix spectra displayed characteristic collagen peaks. Studies have been carried out for the development of new and modified calcium phosphate nanoparticles that should further improve biostimulation.Dissertação IPEN-doc 28472 Avaliação da capacidade angiogênica da linhagem celular de adenocarcinoma renal humano knockout para a proteína NF-kB12021 - SILVA, LUIZ F.T. daO carcinoma de células renais (CCR) é o câncer epitelial renal adulto mais comum, sendo responsável por mais de 90% de todas as neoplasias renais. O subtipo mais frequente de CCR é o de células claras (CCRcc). A maioria dos pacientes com CCRcc possui mutação no gene supressor tumoral de Von Hippel-Lindau (VHL). O gene VHL codifica uma proteína, a VHL, que é capaz de regular negativamente uma série de proteínas intracelulares, dentre elas o fator induzível por hipóxia (HIF). Muitas moléculas têm sido apontadas como responsáveis pelo fenótipo agressivo desse tumor, uma delas é o fator de transcrição NF-kB, que é o nome coletivo para os fatores de transcrição da família Rel. Em mamíferos são conhecidos cinco membros desta família: RelA (p65), RelB, c-Rel, NF-kB1 (p105/p50) e o NF-kB2 (p100/p52). No CCR, o aumento da atividade do NF-kB correlacionava-se com o aumento de marcadores de angiogênese tais como o fator de crescimento endotelial vascular (VEGF) e Interleucina 6 (IL-6). Nos últimos anos vários grupos têm demonstrado o papel funcional do NF-kB1 na tumorigenicidade do CCR. Nesse projeto utilizamos a técnica CRISPR/Cas-9 para obtenção de uma linhagem celular de adenocarcinoma renal humano knockout para a proteína NF-kB1. Foi realizada a verificação do gene endógeno mais estável para condições de normóxia e hipóxia. Foi feita a quantificação dos níveis de VEGF e IL-6 em condição de normóxia e hipóxia. A técnica CRISPR/Cas9 foi eficaz para obtenção de células 786-0 nocaute para NF-kB1 (p105/p50). Dentre os oito genes endógenos analisados, o TRFC foi o mais estável e, consequentemente, o mais adequado para estudos com as células 786-0 em hipóxia. A supressão da expressão da p50 nos clones 786-0 sg1, 786-0 sg2 e 786-0 sg3 resultou na redução de VEGF e IL6 tanto em normóxia quando em hipóxia. A redução do diferencial hipóxia/normóxia demonstra uma alteração na responsividade celular à hipóxia no que diz respeito à Il-6. A redução dos níveis de IL-6, pode justificar a redução da MMP-9 e migração celular observados por nosso grupo.Artigo IPEN-doc 28416 Study of the automated synthesis of the radiopharmaceutical [18F]fluoroestradiol2021 - BALIEIRO, L.M.; OLIVEIRA, H.B.; TEIXEIRA, L.F.S.; BELLINI, M.H.; MATSUDA, M.M.N.; ARAUJO, E.B.Breast cancer is the second leading cause of cancer death among women worldwide, with an incidence increase of 25 % per year. Approximately 75 % of breast cancer cells express estrogen receptors. The 16α-[18F]fluoro-17β-estradiol, [18F]FES, is a radiopharmaceutical that binds to estrogen receptors applied in PET-CT molecular images for non-invasive diagnosis of primary and metastatic breast cancer. The objective of this work was to study the synthesis of the [18F]FES in the GE TRACERlab® MXFDG module, using the Chemical Kit and the ABX® disposable cassette. Moreover, to determine the process yield and the analytical parameters to be used in the routine production of this radiopharmaceutical. Automated synthesis took place in 75 minutes and included percolation of [18F]fluoride (18F-) in an anion exchange cartridge, elution of the cartridge, azeotropic drying in 3 steps, labeling of the precursor 3-methoxymethyl-16β,17β-epiestriol-O-cyclic sulfone (MMSE) and a hydrolysis step. The product was purified in the module by solid-phase extraction (SPE) cartridges. The radiochemical yield was reproductive, despite initial [18F]fluorine activity, and the results of quality control tests suggest that the radiopharmaceutical meets the acceptance criteria established in official monographs for other radiopharmaceuticals labeled with 18-fluor. In vivo biodistribution studies in healthy mice and mice bearing MCF7 tumors showed the specific uptake on breast tumor cells.Resumo IPEN-doc 24634 Downregulation of NF-ΚB1 enhances the radiosensitivity of renal cell carcinoma2017 - IKEGAMI, AMANDA; SILVA, LUIZ F.T. da; BELLINI, MARIA H.Introduction: Clear cell renal cell carcinoma (ccRCC) accounts for ∼80% of all renal cell carcinomas (RCC) and has the Von Hippel–Lindau (VHL) tumor suppressor gene mutated. The lack of VHL protein leads to a constitutionally active Hypoxia Inducible Factor (HIF) pathway that confers both chemoresistance and radioresistance for renal tumor. HIF pathway is known to interact with the transcription factor nuclear factor kappa B (NF-kB). Increased NF-κB activity is associated with the development and progression of RCC (IKEGAMI A, TEIXEIRA LF. BRAGA MS et al. The American Society for Cell Biology 2016; 26: 3948-3955). Objective: Evaluate the synergistic effect of NF-kB1 knockdown and ionizing radiation in murine renal adenocarcinoma cell line. Methods: The murine renal adenocarcinoma cell line (Renca cells) (ATCC, USA) was cultured in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin. Lentiviral shRNA vector was used to knockdown of NF-KB1 gene in Renca cells, as described previously (1). In the clonogenic cell survival assay, the cells were irradiated by 60Co source in the range from 0 to 10 Gy, using the GammaCell 220 – Irradiation Unit of Canadian-Atomic Energy Commision Ltd. (CTR-IPEN). After 10-14 days of culture, cell colonies were fixed and stained with formaldehyde 4% and rhodamine B 2% and counted. To assess cell viability, tetrazolium [3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-MTS] was performed within 24 hours after irradiation at a dose of 10Gy. The survival variables α e β were fitted according to the linear quadratic equation (SF=exp[-αD-βD2]); SF=survival fraction, D=dose of irradiation and P value was determined by F test. Multiple comparisons were assessed by One-way ANOVA followed by Bonferroni´s tests with GraphPad Prism version 6.0 software. P< 0.05 was considered statistically significant. Data are shown as the mean ± SD. Results: The Renca-shRNA-NF-kB1 cells were found to be significantly more radiosensitive than controls - Renca-WT and Renca-Mock, (P<0.001 vs Renca-Mock). The ratio α/β was increased in Renca-shRNA-NF-kB1: -0.177±0.677 compared with 7.368±1.833 and 11.960±5.240 of the Renca- WT and Renca-Mock, respectively. There was no significant difference in the survival fraction between Renca-WT and Renca-Mock groups. The lethal dose 50% (LD50) of Renca-WT was 3.33 Gy and Renca-Mock was 3.288 Gy whereas for the Renca-shRNA-NF-kB1 group it was 2.08 Gy. Corroborating these data, the Renca-shRNA-NF-kB1 showed reduction of 16.75±0.06% in the viability when compared to the Renca-Mock (P<0.001). Conclusion: The knockdown of NF-kB1 gene mediated by shRNA on Renca cells led to a decrease in the radioresistance. Therefore, this gene can be a therapeutic target for CCR treatment.Resumo IPEN-doc 22980 A regulação negativa da expressão do gene NF-κβ1 induz a diminuição da proliferação em linhagem celular de carcinoma renal2016 - IKEGAMI, A.; TEIXEIRA, L.F.S.; BELLINI, M.H.Resumo IPEN-doc 22987 A regulação negativa da expressão do gene NF-κβ1 induz o retardamento da migração e invasão e redução da clonogenicidade em linhagem celular de carcinoma renal2016 - TEIXEIRA, L.F.S.; IKEGAMI, A.; BELLINI, M.H.Resumo IPEN-doc 22955 Avaliação da capacidade clonogênica, migratória e invasiva de linhagem celular de carcinoma renal após o silenciamento do gene NF-κβ12016 - SILVA, LUIZ F.T. da; BELLINI, MARIA H.