Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987. Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/ Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP

Nanostructured system based on hydroxyapatite and curcumin: A promising candidate for osteosarcoma therapy:A promising candidate for osteosarcoma therapy

Osteosarcoma is the most common type of bone cancer. Despite therapeutic progress, survival rates for metastatic cases or that do not respond well to chemotherapy remain in the 30% range. In this sense, the use of nanotechnology to develop targeted and more effective therapies is a promising tool in the fight against cancer. Nanostructured hydroxyapatite, due to its biocompatibility and the wide possibility of functionalization, is an interesting material to design nanoplatforms for targeted drug delivery. These platforms have the potential to enable the use of natural substances in the fight against cancer, such as curcumin. Curcumin is a polyphenol with promising properties in treating various types of cancer, including osteosarcoma. In this work, hydroxyapatite (n-HA) nanorods synthesized by the hydrothermal method were investigated as a carrier for curcumin. For this, first-principle calculations based on the Density Functional Theory (DFT) were performed, in which the modification of curcumin (CM) with the coupling agent (3-aminopropyl) triethoxysilane (APTES) was theoretically evaluated. Curcumin was incorporated in n-HA and the drug loading stability was evaluated by leaching test. Samples were characterized by a multi-techniques approach, including Fourier transform infrared spectroscopy (FTIR), UV–visible spectroscopy (UV–Vis), X-ray diffraction (XRD), X-ray fluorescence spectrometry (FRX), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), zeta potential analysis (ζ), X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The results show that n-HAs with a 90 nm average size were obtained and successful incorporation of curcumin in the nanostructure was achieved. Cell viability and the number of osteosarcoma cells were decreased by CMAP-HA treatment. Furthermore, the stability test suggests that hydroxyapatite nanoparticles present great potential for the transportation of curcumin in the bloodstream, crediting this system for biological performance evaluations aiming at the treatment of osteosarcomas. Keywords: nanostructures, curcumin, hydroxyapatite, osteosarcoma

Nanostructured lipid carriers as a novel tool to deliver sclareol: physicochemical characterisation and evaluation in human cancer cell lines

Sclareol (SC) is arousing great interest due to its cytostatic and cytotoxic activities in several cancer cell lines. However, its hydrophobicity is a limiting factor for its in vivo administration. One way to solve this problem is through nanoencapsulation. Therefore, solid lipid nanoparticles (SLN-SC) and nanostructured lipid carriers (NLC-SC) loaded with SC were produced and compared regarding their physicochemical properties. NLC-SC showed better SC encapsulation than SLN-SC and was chosen to be compared with free SC in human cancer cell lines (MDA-MB-231 and HCT-116). Free SC had slightly higher cytotoxicity than NLC-SC and produced subdiploid DNA content in both cell lines. On the other hand, NLC-SC led to subdiploid content in MDA-MB-231 cells and G2/M checkpoint arrest in HCT-116 cells. These findings suggest that SC encapsulation in NLC is a way to allow the in vivo administration of SC and might alter its biological properties

SET domain-containing protein 4 (SETD4) is a newly identified cytosolic and nuclear Lysine Methyltransferase involved in breast cancer cell proliferation

Cancer is comprised of a multitude of epigenetic abnormalities, including the global loss and regional gain of DNA methylation as well as alterations in histone methylation. Here, we characterize a new methyltransferase, SET domain-containing protein 4 (SETD4), which is involved in breast carcinogenesis. Quantitative real-time PCR (qPCR) showed elevated expression levels of SETD4 in several breast cancer cell lines. SETD4 overexpression was confirmed by western blot analysis suggesting a correlation between high expression of SETD4 and a lack of the estrogen receptor (ER) in breast cancer. In addition, cell fractionation studies and confocal immunofluorescence revealed the nuclear and non-nuclear localization of this new protein. SETD4 knockdown in breast cancer cell lines significantly suppressed their proliferation and delayed the G1/S cell cycle transition without affecting apoptosis. Furthermore, western blot analysis showed that knockdown of SETD4 decreased cyclin D1 expression, revealing the involvement of SETD4 in cell cycle regulation. These data imply that SETD4 plays a crucial role in breast carcinogenesis and could be a novel molecular target for the development of new strategies for the diagnosis and treatment of breast cancer

Funções do cálcio nuclear e citosólico na sinalização celular

Exportado OPUSMade available in DSpace on 2019-08-14T10:59:51Z (GMT). No. of bitstreams: 1 tese_gomesda_final_desbloqueada.pdf: 3680052 bytes, checksum: 4cc46db84cc4ad59eaeea55449a2b747 (MD5) Previous issue date: 18O cálcio (Ca2+ ) citoplasmático e nucleoplasmático podem ser regulados independentemente. A relativa contribuição do Ca2+ citoplasmático e nucleoplasmático em processos biológicos tais como transcrição gênica, apoptose e proliferação celular ainda é matéria de especulação. Com a finalidade de explorar esta questão, utilizamos o aumento da expressão de parvalbumina (PV) e calretinina (CR) como ferramentas moleculares para tamponar seletivamente Ca2+ tanto no nucleoplasma quanto no citoplasma. Nossos dados mostram que tanto a PV quanto a CR foram eficazes em tamponar os sinais de Ca2+ em cada um destes compartimentos subcelulares. Para maximizar o número de células que expressam a PV, estas construções foram entregues às células SkHep1 por adenovírus (ad) permitindo assim, investigar funções celulares mediadas pelo Ca2+ nuclear e citosólico. A construção ad-PV-NES (Nuclear Exclusion Signal) foi eficaz no bloqueio da fosforilação da proteina Erk 1 e da expressão da proteína p38. Em contraste, a construção ad-PV-NLS (Nuclear Localization Signal) foi capaz de bloquear a fosforilação da ciclina dependente de cinase 1 (CDK1). Para estudar os mecanismos responsáveis pela geração de InsP3 nuclear, utilizamos construções que tamponam InsP3 seletivamente no núcleo ou no citosol, denominadas de Sponge NLS e NES, respectivamente. As células transfectadas com InsP3 Sponge NLS mostraram-se eficazes em bloquear o aumento de Ca2+ induzido pelo fator de crescimento hepático (HGF), mas não por arginina vasopressina (AVP). Em contraste, a construção InsP3 Sponge NES bloqueou o aumento de Ca2+ , induzido por AVP, mas não por HGF. Demostramos que o receptor de HGF, c-met, pode translocar para o núcleo de células SkHep1 após estimulação com HGF. Além disso, observamos que células SkHep1 possuem fosfolipase C (PLCy ) no núcleo. Em conjunto, estes resultados sugerem que através de PLCy, HGF pode gerar sinais de InsP3 e consequentemente Ca nuclear. Assim, sinais de Ca2+ no núcleo ou no citoplasma têm efeitos distintos na expressão e ativação de cinases e fatores de transcrição. Estas observações sobre o tráfego e função de c-met revelam um mecanismo pelo qual os sinais de Ca2+ podem ser compartimentalizados para obter efeitos celulares distintos.Nucleoplasmic and cytoplasmic calcium (Ca2+ ) can be regulated independently. The relative contribution of nucleoplasmic and cytoplasmic Ca2+ to biological processes such as gene transcription, cell growth, apoptosis and cell cycle control remain to be -binding proteins fully defined. In order to address this question, we target the Ca2+ parvalbumin (PV) or calretinin (CR), to either the nucleus or cytoplasm as a molecular tool to selectively buffer either nucleoplasmic or cytoplasmic Ca . These data showed that expression of targeted PV or CR was sufficient to buffer Ca2+ signals in these respective sub-cellular compartments. The PV constructs were delivered by adenovirus . The construct ad-PV-NES (ad) to explorer the functions of nuclear and cytosolic Ca2+ (Nuclear Exclusion Signal) blocked the phosphorylation of Erk1 and expression of p38. In contrast, the construct ad-PV-NLS (Nuclear Localization Signal) blocked the phosphorylation of cyclin dependent cinase 1 (CDK1). We also investigated the mechanisms responsible for InsP3 generation within the nucleus. In order to address this question, we employed an InsP binding protein (InsP3 Sponge) as a molecular tool to . SkHep1 cells expressing selectively buffer either nucleoplasmic or cytoplasmic InsInsP3 Sponge in the nucleus blocked the Ca2+ response induced by Hepatocyte Growth response in the cells was not affected by stimulation with factor (HGF), while the Ca2+ arginine vasopressin (AVP). In contrast, expression of InsP3 Sponge in the cytoplasm blocked the Ca2+ response induced by AVP, but the Ca2+ response induced by HGF was not affected. In addition, stimulation with HGF induced translocation of c-met to the nucleus and let to the presence of phospholipase C (PLCy) as well within the nucleus. and Ca2+ signals, In summary, these results show that HGF can generate nuclear InsP3and that this likely results from translocation of c-met and PLCy to the nucleus. Since signals in the nucleus and cytoplasm have distinct effects on expression and Ca2+ activation of cinases and transcription factors, these observations about c-met trafficking and function reveal one mechanism by which Ca2+ signals can be compartmentalized to obtain these distinct cellular effects

Stem Cell Extracellular Vesicles in Skin Repair

Stem cell extracellular vesicles (EVs) have been widely studied because of their excellent therapeutic potential. EVs from different types of stem cell can improve vascularization as well as aid in the treatment of cancer and neurodegenerative diseases. The skin is a complex organ that is susceptible to various types of injury. Strategies designed to restore epithelial tissues’ integrity with stem cell EVs have shown promising results. Different populations of stem cell EVs are able to control inflammation, accelerate skin cell migration and proliferation, control wound scarring, improve angiogenesis, and even ameliorate signs of skin aging. However, large-scale production of such stem cell EVs for human therapy is still a challenge. This review focuses on recent studies that explore the potential of stem cell EVs in skin wound healing and skin rejuvenation, as well as challenges of their use in therapy

Intra-bone marrow injection of mesenchymal stem cells improves the femur bone mass of osteoporotic female rats

The effect of intra-bone injection of differentiated rat bone marrow mesenchymal stem cells (BMMSCs) into the femur of osteoporotic female rats was studied. Osteoporosis was induced in Wistar female rats by bilateral ovariectomy. Then, 0.75 million BMMSCs isolated from healthy rats were injected into the femurs of osteoporotic rats. Histomorphometric analysis and histology clearly revealed improvements in the treated group as compared to untreated group. In 2 months, the femurs of treated rats, unlike untreated rats, showed trabecular bone percentage almost similar to the femurs from control healthy rats. To confirm the origin of newly formed bone, the experiment was repeated with BMMSCs isolated from green fluorescent protein transgenic rats. Confocal microscopy demonstrated green fluorescent protein-positive cells at the surface of trabecular bone of the treated rats. We investigated in vitro osteogenic differentiation of BMMSCs isolated from osteoporotic rats by studying alkaline phosphatase activity, collagen synthesis, and the ability to form mineralized nodules. Osteoporotic BMMSCs showed less differentiation capabilities as compared to those isolated from healthy rats. The results clearly demonstrated the importance of BMMSCs in osteoporosis and that the disease can be treated by injection of BMMSCs.Fundacao de Amparo a Pesquisa de Minas Gerais (Fapemig)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq