LUIZ FELIPE TEIXEIRA DA SILVA

LUIZ FELIPE TEIXEIRA DA SILVA

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Micro-Raman spectroscopy identification of hydroxyapatite in dental pulp stem cells
2023 - SILVA, FLAVIA R.O.; PASCOAL, DIEGO R.C.; BERECZKI, ALLAN; SIPERT, CARLA R.; BRAGA, ROBERTO R.; BELLINI, MARIA H.; SILVA, LUIS F.T.; FREITAS, ANDERSON Z.; WETTER, NIKLAUS U.
Cell differentiation using calcium phosphate nanoparticles was studied. The hydroxyapatite was internalized in human dental pulp stem cells and characterized by Raman spectroscopy. Raman spectra showed the hydroxyapatite distribution in nanoparticles nodules in the cells.
NF-ĸΒ1 knockout reduces IL6 expression under hypoxia in renal cell carcinoma
2023 - TEIXEIRA, LUIZ F.S.; BELLINI, MARIA H.
Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer, accounting for more than 90% of all renal neoplasms. Clear cell RCC (ccRCC) is the most common subtype of RCC. Most patients with ccRCC have a mutation in the von Hippel-Lindau (VHL) tumor suppressor gene, which encodes a protein that downregulates various intracellular proteins, including hypoxia-inducible factor (HIF). Many molecules have been identified to be responsible for the aggressive phenotype of ccRCC, including the transcription factor nuclear factor kappa B (NF-кB). The increase in NF-кB activity observed in RCC is correlated with an increase in angiogenesis markers, such as interleukin 6 (IL-6). In recent years, several groups have demonstrated the functional role of NF-кB1 in RCC tumorigenicity. Herein, we used the CRISPR/Cas-9 technique to obtain an NF-кB1 knockout-human renal adenocarcinoma cell line. Expression of IL-6 at the mRNA and protein levels was analyzed under normoxia and hypoxia by real time-polymerase chain reaction and multiplex assay, respectively. The CRISPR/Cas9 technique was effective in producing 786-0 knockout cells for NF-κB1 (p105/p50), as confirmed by western blot analysis. Suppression of p50 expression in 786-0 single guide RNA (sg)1, 786-0 sg2 and 786-0 sg3 cells downregulated IL-6 mRNA and protein expression under normoxia and hypoxia. The observed decrease in the differential expression of IL-6 in hypoxia/normoxia is suggestive of a change in cellular responsiveness to hypoxia with respect to IL-6.
Evaluation of angiogenic capacity of human adenocarcinoma cell line knockout for NF-ĸΒ1 protein
2022 - TEIXEIRA, LUIZ F.S.; BELLINI, MARIA H.
INTRODUCTION: Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer. The most frequent subtype of RCC is clear cell (ccRCC). Most of ccRCC patients have a mutation in the Von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene encodes a protein, the VHL, which can up-regulate a series of intracellular proteins, including the hypoxia inducible factor (HIF). The transcription factor NF-кB is increased in the ccRCC. OBJECTIVES: To evaluate the impact of the NF-кB1 gene knockout on the VEGF and IL6 expression in the human RCC cells under normoxia and hypoxia. MATERIALS AND METHODS: The CRISPR/Cas-9 technique was used to obtain 786-0 cells knockout for the NF-кB1 protein. Western Blot assay was used to selected the clones. A hypoxia-inducing humid chamber was used and its effectiveness was validated its effectiveness was certified by the analysis of HIF-2α expression levels. The quantification of VEGF and IL-6 levels was measured using Real Time-PCR and MILLIPLEX assay. DISCUSSION AND RESULTS: The VEGF gene expression in the clones was significantly lower than that presented by the control both in normoxia (786-0-sg1 99.68±0.09%, 786-0-sg2 78.55±0.85%, 786-0-sg3 91.70±0.87%) and in hypoxia (786-0-sg1 98.30±1.49%, 786-0-sg2 75.21±4.14%, 786-0-sg3 98.44±0.18%). The expression of IL-6 gene was also significant lower in normoxia (786-0-sg1 49.03±0.80%, 786-0-sg2 76.59±12.43%, 786-0-sg3 66.98±10.89%) and in hypoxia (786-0-sg1 95.85±0.36%, 786-0-sg2 96.45±0.49%, 786-0-sg3 91.08±1.42%). The MILLIPLEX results show that there was a significant reduction of both VEGF and IL-6 in the culture medium of cells knocked out in normoxia and hypoxia compared to control group. CONCLUSION: Suppression of p50 expression in the clones resulted in the reduction of VEGF and IL6 in both conditions. The reduction in the IL-6 relative expression hypoxia/normoxia demonstrates a change in cellular responsiveness to decreased levels of oxygen.
Micro-Raman spectroscopy characterization of dental pulp stem cells differentiation induced by calcium phosphate nanoparticles
2022 - 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.
Identification of appropriate housekeeping genes for gene expression studies in human renal cell carcinoma under hypoxic conditions
2022 - TEIXEIRA, LUIZ F.S.; GIGLIOTTI, RODRIGO; FERREIRA, LUANA da S.; BELLINI, MARIA H.
Background: Hypoxia pathways are deregulated in clear renal cell carcinoma (ccRCC) because of the loss of the von Hippel-Lindau tumor suppressor function. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. Methods and Results: Four different tools were used to rank the most stable genes—geNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable. There was no agreement among the results from the algorithms for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes for use in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. Conclusions: To the best of our knowledge, this is the first study to identify reliable genes that can be used for gene expression analysis in ccRCC in a hypoxic environment.
Lanthanide-based β-tricalcium phosphate upconversion nanoparticles as an effective theranostic nonviral vectors for image-guided gene therapy
2022 - SILVA, FLAVIA R.O.; LIMA, NELSON B.; BELLINI, MARIA H.; TEIXEIRA, LUIZ F.S.; DU, ERIC Y.; JAMSHIDI, NILOUFAR; GOODING, JUSTIN; MARTIN, ADAM D.; MACMILLAN, ALEXANDER; MARQUIS, CHRISTOPHER P.; THORDARSON, PALL
Lanthanide-based beta-tricalcium phosphate (β-TCP) upconversion nanoparticles are exploited as a non-viral vector for imaging guided-gene therapy by virtue of their unique optical properties and multi-modality imaging ability, high transfection efficiency, high biocompatibility, dispersibility, simplicity of synthesis and surface modification. Ytterbium and thulium-doped β-TCP nanoparticles (βTCPYbTm) are synthesized via co-precipitation method, coated with polyethylenimine (PEI) and functionalized with a nuclear-targeting peptide (TAT). Further, in vitro studies revealed that the nanotheranostic carriers are able to transfect cells with the plasmid eGFP at a high efficiency, with approximately 60% of total cells producing the fluorescent green protein. The optimized protocol developed comprises the most efficient βTCPYbTm/PEI configuration, the amount and the order of assembly of βTCPYbTm:PEI, TAT, plasmid DNA and the culturing conditions. With having excellent dispersibility and high chemical affinity toward nucleic acid, calcium ions released from βTCPYbTm:PEI nanoparticles can participate in delivering nucleic acids and other therapeutic molecules, overcoming the nuclear barriers and improving the transfection efficacy. Equally important, the feasibility of the upconversion multifunctional nanovector to serve as an effective contrast agent for imaging modality, capable of converting low-energy light to higher-energy photons via a multi-photons mechanism, endowing greater unique luminescent properties, was successfully demonstrated.
In vitro and in vivo response of PSMA-617 radiolabeled with CA and NCA lutetium-177
2022 - BOAS, CRISTIAN A.W.V.; SILVA, JEFFERSON de J.; DIAS, LUIS A.P.; FREIRE, MARIA R.B.; BALIEIRO, LUIZA M.; SANTOS, CAROLINA S.F. dos; VIVALDINI, BIANCA F.; BENEDETTO, RAQUEL; VIEIRA, DANIEL P.; PASSOS, PRISCILA de Q.S.; MARUMO, MARIA H.; TEIXEIRA, LUIS F.S.; ARAUJO, ELAINE B. de
The PSMA-targeted radionuclide therapy has been explored since 2015 with radioisotope lutetium-177, whose β− emission range is adequate for micrometastases treatment. This radioisotope is obtained by two different production routes that directly affect the specific activity of lutetium-177 (non-carrier added and carrier added) and, consequently, the specific activity of radiopharmaceuticals, like 177Lu-PSMA-617. The influence of the specific activity of lutetium-177 on the properties of the radiopharmaceutical PSMA-617 was evaluated through pre-clinical studies. The in vitro study pointed to a lower constant of dissociation with non-carrier added lutetium-177 due to the difference in the specific activity. However, competition and internalization assays resulted in similar results for both lutetium-177. Based on these pre-clinical experiments, the total in vitro tumor cell binding and tumor uptake in vivo were similar, with no influence of the specific activity of the 177Lu-PSMA-617. Regardless the specific activity did not directly affect tumor uptake, the tumor/non-target organs ratios were higher for the radiopharmaceutical labeled with carrier added lutetium-177, which had the lowest specific activity.
Avaliação da capacidade angiogênica da linhagem celular de adenocarcinoma renal humano knockout para a proteína NF-kB1
2021 - SILVA, LUIZ F.T. da
O 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.
Study of the automated synthesis of the radiopharmaceutical [18F]fluoroestradiol
2021 - 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.
Silencing of nuclear factor kappa b 1 gene expression inhibits colony formation, cell migration and invasion via the downregulation of interleukin 1 beta and matrix metallopeptidase 9 in renal cell carcinoma
2020 - TEIXEIRA, LUIZ F.S.; PERON, JEAN P.S.; BELLINI, MARIA H.
Renal cell carcinoma (RCC) is a highly deadly urological tumor due to its high metastatic incidence and its notorious chemoresistance. The nuclear transcription factor kappa B (NF-κB) family has been associated with apoptosis resistance and cellular invasion in RCC. The purpose of this study was to evaluate the impact of NF-κB1 gene silencing on the colony formation, cell migration and invasion abilities of the RCC cell line. Renca–mock and Renca-shRNA-NF-κB1 cells were used in this work. NF-κB1 downregulation was assessed by western blotting. The mRNA expression levels of interleukin-1 beta (IL-1β) and MMP-9 were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). The IL-1β levels in the culture media were determined by a commercial ELISA kit. The MMP-9 protein expression and gelatinolytic activity were evaluated by western blotting and zymography, respectively, and the migration and invasion abilities were analysed. The expression levels of p105 and p50 in Renca-shRNA-NF-κBmoc1 cells were significantly reduced compared with those in the Renca–mock cells. The colony numbers of shRNA-NF-кB1 cells were lower than the colony numbers of the Renca– mock cells. NF-κB1 knockdown inhibited the cell migration and invasion of Renca-shRNA-NF-κB1 cells. These cells also exhibited reduced levels of IL-1β. The MMP-9 expression and activity levels were significantly reduced in Renca-shRNANF- κB1 cells. Taken together, these results indicate that the downregulation of NF-κB1 suppresses the tumourigenicity of RCC by reducing MMP-9 expression and activity; thus, NF-κB1 could be a molecular target for RCC treatment.