Análise do efeito in vitro de acridonas na replicação do Vírus da Hepatite C

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influência de Lactobacillus rhamnosus na patogenicidade e na expressão de genes de virulência de Candida albicans: estudo in vitro e in vivo

Pós-graduação em Biopatologia Bucal - ICT

Efeitos de reguladores do metabolismo lipídico no desenvolvimento in vitro de embriões bovinos e na sobrevivência à vitrificação

Pós-graduação em Medicina Veterinária - FCAV

Principais mecanismos envolvidos na maturação oocitária em bovinos: da oogenese a maturação in vitro

Despite the efforts made to improve the production of bovine embryos in vitro, their efficiency is still low, since only 30-40% of developed blastocysts are obtained from oocytes after in vitro maturation (IVM), fertilization and cultured embryos. Assisted reproductive technologies have a limiting impact due a lack of oocytes capable to fertilization.The comprehension of mechanism involved in oocyte maturation are crucial to establish a culture system that allows a larger number production of good quality embryos. The study of the early stages of oocyte and follicle development in vivo is important for a better understanding of the molecular pathways that regulate oogenesis, folliculogenesis and oocyte maturation. Thus the physiological biochemical and molecular mechanisms involved in maturation may contribute to the increased efficiency of in vitro embryo production. Therefore, the aim of this literature review is to understand the basic mechanisms that underlie oocyte maturation in cattle, since oocyte and follicle cells in vivo formation to its use in the in vitro environment.

In vitro production of bovine embryos using Sigma antioxidant supplement®, α-tocopherol and L-ascorbic acid

This study tested the effect of Sigma antioxidant supplement®, α-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) in the culture medium of bovine embryos. In experiment 1, in vitro produced bovine zygotes were cultured in Human Tubal Fluid (HTF): Eagle’s Basic Medium (BME) with: Group 1 – 50 µm vitamin C; Group 2 – 200 µm vitamin E; Group 3 – 25 µm vitamin C and 100 µm vitamin E; Group 4 – 1 µl/ml Sigma antioxidant supplement®; and the Control group – HTF:BME only. In experiment 2, embryos were cultured in high or low oxygen tension with HTF:BME + Sigma antioxidant supplement® or in HTF:BME alone (Control). The data were analyzed using ANOVA followed by Tukey’s test. The results of experiment 1 showed a negative effect (P < 0.05) of vitamin E on blastocyst production in Group 2 (19.7 ± 0.1%). This effect was reduced in Group 3 by the addition of vitamin C (26.1 ± 0.2%). The use of vitamin C alone (34.9 ± 0.3%) or the Sigma antioxidant supplement® (33.3 ± 0.7%) did not increase (P > 0.05) the number of blastocysts produced compared with the control group (30.1 ± 0.5%). During experiment 2, there was no effect (P > 0.05) from the culture medium or the O2 concentrations used, indicating that the reduction of the O2 concentration did not improve blastocyst production.

Efeito do fator de crescimento epidermal (EGF) na maturação in vitro de oócitos caninos

Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP)

Uso de resíduos madeireiros da Amazônia brasileira no cultivo in vitro de Lentinus strigosus

Lentinus strigosus (Schwein.) Fr. is an exploitable edible mushroom occurring in the Brazilian Amazon, being part of a huge diversity of edible mushrooms which are little grown. The use of regional waste is recommended to reduce production costs of any kind of edible mushroom. Thus, the mycelial growth of L. strigosus in culture media based on regional wood waste extract by using substrates based on Protium puncticulatum, Cariniana micrantha and Caryocar glabum sawdust, supplemented with 20% of wheat bran (Triticum aestivum), corn bran (Zea sp.) or rice bran (Oryza sp.) was observed. Eucalyptus (Eucaliptus sp.) sawdust was used for comparison with the other wood wastes because it is commonly used in the cultivation of edible fungi. The experimental design employed was totally randomized, in 4 x 3 factorial scheme (sawdust x bran), adding up 12 treatments with 5 repetitions, being that each repetition corresponded to a Petri dish, totalizing 60 dishes, incubated at 35 ºC. The diameter of the colony was daily evaluated until the fungus reached the borders of the Petri dish in one of the treatments. After that period, the media based on P. puncticulatum sawdust obtained thebest results of mycelial growth, showing potential to be used as an alternative residuein a future production of L. strigosus in the state of Amazonas.

Resíduos de bananeira como substrato base para o cultivo in vitro de Coprinus comatus

Coprinus comatus is an edible and lignolitic fungus which has presented great potential for commercial use due to its easy development in the different residues, such as banana tree leave. Thus, the mycelial growth of Coprinus comatus in culture media based on leaves of Thap-Maeo, Prata-Anã, Pelipita and Caipira banana tree cultivars, supplemented with 20% of wheat, soy and rice brans, was evaluated. 7 mm-wide discs of CCO 01/01 strain of C. comatus were inoculated in the middle of Petri dishes containing culture medium, inside a laminar flow chamber. Next, the dishes were arranged totally at random inside an incubator at 25 ºC. The daily measurements of the mycelial growth began after 24 hours, until one of the treatments reached the borders of the Petri dish. According to the results obtained, we verified that there was not effect of the kind of supplementation for culture media based on Thap-Maeo, Prata-Anã and Pelipita; the best growth averages for culture media based on Caipira were provided by wheat and rice brans. Therefore, banana residues may be a viable and ecologically correct choice for the cultivation of C. comatus, especially for Thap-Maeo and Prata Anã sorts, which provided the best growth averages, regardless of the supplementation used.

Cultivo in vitro de Pleurotus ostreatus em resíduos de bananeira

The objective of this paper was to evaluate the mycelial growth of Pleurotus ostreatus (strain POS 09/100) in culture media based on different banana tree residues. The experimental design was totally randomized in 3 x 4 factorial scheme and consisted in three combinations of residues (pseudostem, leave and pseudostem + leave) and four banana tree cultivars (Thap Maeo, Prata Anã, Pelipita and Caipira), totalizing twelve treatments each with five repetitions, adding up sixty experimental units. Growth was measured every 24 hours until the mycelium of one of the treatments reached the border of the Petri dish, what occurred five days after the beginning of the experiment. The results obtained showed that all the combinations of banana tree residues were favorable to P. ostreatus mycelial growth, especially pseudostem + leaf of Pelipita, Thap maeo and Prata anã cultivars. Thus, the use of banana tree residues is viable for cultivation of P. ostreatus, and considered as an excellent alternative, besides reducing their disposal in the environment.

Avaliação dos fatores de virulência, atividade antimicrobiana e viabilidade celular de bactéria cariogênica na presença do Terpinen-4-ol: estudo in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação dos fatores de virulência, atividade antimicrobiana e viabilidade celular de bactéria cariogênica na presença do Terpinen-4-ol: estudo in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10-14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.