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This study aims to evaluate the effects of tea of copaíba extract (Copaifera luetzelburgii), in differentconcentrations, added to the Tris-egg yolk extender, the functionality of the plasma membrane sperm ofcryopreserved goats. Were used four goats breeds, adults, male, six of each breed, aged between 1 and 6 years,a pool of ejaculates were formed to analyze. The animals were randomly divided into four experimentaltreatments: TI without adding of tea of copaíba extract (control); TII, added 0,1 mg of tea of copaíba extract;TIII, 0,2 mg; and TIV, 0,5 mg. The samples were frozen and stored, after 30 days were thawed for analysis byhisposmotic test (HOST). Adding tea of copaíba extract showed no significant difference between treatments forHOST, so the use of tea of copaíba extract the seminal dilution was not effective in preserving the functionalityplasma membrane sperm of goats post-thawing.

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This study aimed to evaluate the effect of insulin on the membrane integrity evaluated by the eosin testand nigrosine in ovine semen. Seven sheep were used, harvest were made with the aid of eletroejaculator. Theyolk-citrate extender was used for dilution of the ejaculate (25x106 sperm / mL) using quail's egg yolk. Eachejaculate was divided into four equal fractions, corresponding to treatments: control group (Gcont) using onlydilutive citrat gem; Group 50 (G50) plus dilutive 50UI/ml insulin type N; Group 100 (G100) plus dilutive 100IU/Ml Insulin type N; Group 200 (G200) plus dilutive 200 IU/Ml Insulin type N. After dilution, the semenremained in water - bath at 45°C to perform the eosin test and nigrosine for evaluation by time (0, 20, 40 and 60minutes) for membrane integrity. Thus, the use of insulin does not preserve the integrity of the membranesduring the incubation period of 45C, with a gradual decrease due to stress caused by time and temperature atwhich the semen was subjected.

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This study evaluate the effect of melatonin supplementation to TRIS-GEMA diluter in plasmatic andmitochondria membrane integrity of semen ram cryopreserved. Was used seven ejaculates of six ram,whichwere pooled in pool, according to the experimental groups: Control; T1 - 1x10-7 mol / L and T2 - 1x10-9 mol / Lmelatonin. Posteriorly the diluting the samples were packaged in straws of 0.25 ml and cryopreserved. Afterthawing at 37°C for 30 seconds, the samples were analyzed for Test thermoresistance slow (TTR) andacrosomal membrane integrity. For statistical analysis we used the Duncan test (α = 0.05) for meancomparison. Treatments 1 and 2 differed statistically control for plasma membrane integrity at times 60, 120and 180min. It was concluded that supplementation 1x10-7mol / L and 1x10-9mol / L of melatonin increased theintegrity of the plasma membrane to 60,120 and 180 minutes.

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This study aimed to evaluate the effect of insulin on the membrane integrity evaluated by the eosin testand nigrosine in ovine semen. Seven sheep were used, harvest were made with the aid of eletroejaculator. Theyolk-citrate extender was used for dilution of the ejaculate (25x106 sperm / mL) using quail's egg yolk. Eachejaculate was divided into four equal fractions, corresponding to treatments: control group (Gcont) using onlydilutive citrat gem; Group 50 (G50) plus dilutive 50UI/ml insulin type N; Group 100 (G100) plus dilutive 100IU/Ml Insulin type N; Group 200 (G200) plus dilutive 200 IU/Ml Insulin type N. After dilution, the semenremained in water - bath at 45°C to perform the eosin test and nigrosine for evaluation by time (0, 20, 40 and 60minutes) for membrane integrity. Thus, the use of insulin does not preserve the integrity of the membranesduring the incubation period of 45C, with a gradual decrease due to stress caused by time and temperature atwhich the semen was subjected.(AU)

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This study evaluate the effect of melatonin supplementation to TRIS-GEMA diluter in plasmatic andmitochondria membrane integrity of semen ram cryopreserved. Was used seven ejaculates of six ram,whichwere pooled in pool, according to the experimental groups: Control; T1 - 1x10-7 mol / L and T2 - 1x10-9 mol / Lmelatonin. Posteriorly the diluting the samples were packaged in straws of 0.25 ml and cryopreserved. Afterthawing at 37°C for 30 seconds, the samples were analyzed for Test thermoresistance slow (TTR) andacrosomal membrane integrity. For statistical analysis we used the Duncan test (α = 0.05) for meancomparison. Treatments 1 and 2 differed statistically control for plasma membrane integrity at times 60, 120and 180min. It was concluded that supplementation 1x10-7mol / L and 1x10-9mol / L of melatonin increased theintegrity of the plasma membrane to 60,120 and 180 minutes.(AU)

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This study aims to evaluate the effects of tea of copaíba extract (Copaifera luetzelburgii), in differentconcentrations, added to the Tris-egg yolk extender, the functionality of the plasma membrane sperm ofcryopreserved goats. Were used four goats breeds, adults, male, six of each breed, aged between 1 and 6 years,a pool of ejaculates were formed to analyze. The animals were randomly divided into four experimentaltreatments: TI without adding of tea of copaíba extract (control); TII, added 0,1 mg of tea of copaíba extract;TIII, 0,2 mg; and TIV, 0,5 mg. The samples were frozen and stored, after 30 days were thawed for analysis byhisposmotic test (HOST). Adding tea of copaíba extract showed no significant difference between treatments forHOST, so the use of tea of copaíba extract the seminal dilution was not effective in preserving the functionalityplasma membrane sperm of goats post-thawing.(AU)

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Membranas celulares (MCs; Cell Sheets), constituídas por células-tronco (CTs), são autodestacáveis da placa de cultivo, e sem subcultivos geram grande quantidade de células que podem ser transplantadas de maneira mais próxima da fisiologia celular, mantendo-se as ligaçُões celulares e a matriz extracelular produzidas em cultura. O Ácido ascórbico ou vitamina C (VC) tem efeito indutor da formação destas MCs, aumentando a longevidade e tempo de indiferenciação das CTs. A similaridade observada entre respostas biológicas da VC em MCs e aquelas da Laserfototerapia (LFT) sobre células e tecidos, nos levou à hipótese de que estas terapias poderiam se complementar melhorando o prognóstico de futura aplicação clínica dessas MCs em regenerações tecidos de interesse odontológico. Para testar essa hipótese, LFT e VC foram aplicadas associadas ou não na indução de MCs de células-tronco da polpa dentária humana (hDPSCs). Para tanto, hDPSCs descongeladas, que expressaram níveis típicos de marcadores de superfície de células-tronco mesenquimais, foram plaqueadas em placas de 6 poços (5x104 células por poço). Vinte e quatro horas depois do plaqueamento as culturas foram submetidas aos tratamentos dos grupos experimentais: Controle: hDPSCs em P3 cultivadas com meio clonogênico; Senescente: hDPSCs em P27 cultivadas com meio clonogênico; VC: P3 cultivadas com meio clonogênico acrescido de VC (20µg/ml); Laser: P3 cultivadas com meio clonogênico e submetido à LFT (contato e pontual - 5 pontos / poço, 660 nm, 20 mW, 0,028 cm², 0,71 W/cm², 7 segundos, 5 J/cm², 0,14 J por ponto, 48 horas de intervalo) e Laser+VC: P3 cultivadas com meio clonogênico acrescido de VC e submetido

à LFT. Em 24 horas, 7 e 13 dias as hDPSCs dos diferentes grupos experimentais foram observadas macro e microscopicamente, e atividade da enzima telomerase foi avaliada por PCR-TRAP, complementado por ELISA. Para a avaliação da expressão de genes relacionados à natureza e indiferenciação (Mitofilina e Oct 4) e à longevidade (fase catalíca da enzima telomerase - hTERT); bem como à senescência das células do grupo senescente (­­ß-galactosidase), as hDPSCs de todos os grupos experimentais foram submetidas ao RT-qPCR As hDPSCs foram capazes de formar MCs somente nos grupos VC e Laser+VC (100%), entre 10 e 13 dias. As MCs do grupo Laser+VC apresentaram maior facilidade na manipulação. Atividade de Telomerase nas hDPSCs foi observada somente em 24 horas (Controle e LFT) e em 7 dias (VC e Laser+VC). Os marcadores de indiferenciação (Oct 4) e mesenquimal (mitofilina), bem como a hTERT foram expressos nas hDPSCs de todos os grupos experimentais. O Oct4 e o hTERT, em 7 dias, apresentaram expressões significativamente maiores nos grupos VC e Laser+VC em comparação com os demais (p < 0,0001, p = 0,0009, respectivamente). A expressão da mitofilina foi significativamente maior no grupo Laser+VC, em 7 dias (p =0,033). A técnica de obtenção de MCs de hDPSCs por essa metodologia foi considerada adequada para ser testada em procedimentos regenerativos. A LFT quando associada à VC não interferiu na formação das MCs, nem na manutenção da longevidade e indiferenciação das hDPSCs. Adicionalmente, a LFT melhorou a manipulação das MCs. Assim sendo, a associação de VC e LFT na indução de MCs parece promissora para futura utilização de MCs na odontologia regenerativa.

Cell Sheets, consisting of stem cells (SCs) are self detachable from the cultivation plate, and with no subcultivation can generate large amount of cells. The cell sheets can be transplanted closer to cell physiology environment by keeping the cell connections and the extracellular matrix produced in culture. Ascorbic acid or Vitamin C (VC) has inductive effect on cell sheet formation, increasing the longevity and the stemness of the cell for long period of time. The similarity between biological responses of VC in cell sheets and those of Laserphototherapy (LPT, Laser) on cells and tissues led us to hypothesize that these therapies could improve the prognosis of future clinical application of these cell sheets in regeneration of dental tissues. To test this hypothesis, LPT and VC were applied, associated or not, to induce human dental pulp stem cells (hDPSCs). Therefore, hDPSCs, which expressed typical levels of mesenchymal stem cell surface markers, were plated in 6-well plates (5x104 cells per well). Twenty-four hours later they were subjected to the treatment of experimental groups: Control: hDPSCs in P3 cultured with regular medium; Senescent: hDPSCs in P27 cultured with regular medium; VC: P3 cultured with regular medium supplemented with VC (20 ?g/ml); Laser: P3 cultures with regular medium and submitted to LPT (punctual and contact mode-5 points / well, 660 nm, 20 mW, 0.028 cm², 0.71 W/cm²,

7 sec, 5 J/cm², 0.14 J per point, 48 hours-intervals) and Laser+VC: P3 cultured with regular medium supplemented with VC and submitted to LPT Within 24 hours, 7 and 13 days the hDPSCs of the different experimental groups were observed macroscopically and microscopically, and the telomerase enzyme activity was assessed by PCR-TRAP, complemented by ELISA. To evaluate the expression of genes related to the nature and differentiation (Mitofilina and Oct 4), longevity (catalytic phase of telomerase-hTERT enzyme), and the senescence of the senescent group cells (?-galactosidase), the hDPSCs of all experimental groups were subjected to RT-qPCR. The RT-qPCR data were compared by ANOVA complemented by the Tukey's test (p <= 0.05). The hDPSCs were able to form cell sheets only in the VC and Laser+VC groups (100%). Additionally, the cell sheets of the Laser+VC group presented easier handling...

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As alquilfosfocolinas (APC) são uma classe de fármacos derivados de fosfolipídios endógenos que apresentam potencial antitumoral. Diferentemente de outros fármacos antitumorais que agem no DNA da célula, as APCs têm como primeiro local de ação a membrana plasmática e proteínas de sinalização, como a PKC. O objetivo desse trabalho é elucidar, através de metodologias computacionais, os possíveis mecanismos de ação das APCs que provocam hemólise, inibição da PKC e interação com membranas celulares. Inicialmente, a toxicidade de um conjunto de 34 APCs foi estudada pelos métodos quimiométricos de Análise de Agrupamentos Hierárquicos (HCA) e Componentes Principais (PCA). As moléculas foram simuladas com dinâmica molecular (DM) e propriedades físico-químicas e estruturais foram calculadas para os confôrmeros de menor energia. Após aplicação de HCA e PCA, as APCs foram divididas em 3 grupos, de acordo com suas características estruturais. Os resultados sugerem que a presença de grupos catiônicos volumosos, ou anéis como adamantila e ciclohexila, aumentam a hemólise de compostos de cadeia alquílica longa. Anéis macrocíclicos como ciclopentadecila parecem ser importantes para o potencial hemolítico de compostos com cadeia alquílica curta. Com relação a compostos sem anéis e de cadeia linear, grupos catiônicos menos volumosos parecem favorecer a hemólise. Na próxima etapa do estudo, 7 derivados de APC, com diferentes grupos catiônicos, foram selecionados e ancorados no domínio C2 da PKCα. O intuito foi mapear resíduos de aminoácidos importantes para a interação dos ligantes com a enzima, e comparar com o modo de ligação do ativador endógeno fosfatidilserina (PS). Mais uma vez, HCA e PCA foram aplicados para extrair informação relevante do mapeamento. Os resultados mostraram que as cadeias laterais de Pro188, Asn189, Arg216, Trp247, Asp249 e Thr250 não permitem a aproximação adequada do ligante, o que impede que a porção fosforila se coordene com um dos átomos de cálcio. A porção catiônica da PS, em contrapartida, consegue estabelecer ligação-hidrogênio com Asn189 de forma a posicionar os oxigênios da fosforila para interagir, ao mesmo tempo, com o átomo de cálcio. Com menos pontos de coordenação, a afinidade de ligação do cálcio pela PKCα diminui e a ativação da enzima fica comprometida, interrompendo toda a cascata de sinalização que depende dela. A parte final desse trabalho se dedicou ao estudo da interação da miltefosina com diferentes bicamadas lipídicas sob o ponto de vista termodinâmico. Oito bicamadas de diferentes fosfolipídios foram simuladas por DM e a interação energética da miltefosina foi calculada por Umbrella Sampling. Os resultados mostraram que a miltefosina apresenta maior partição em bicamadas contendo colesterol, sendo a miscibilidade nesses sistemas cerca de 76 vezes maior que os valores encontrados para bicamadas sem colesterol. Além disso, verificou-se que a internalização da miltefosina é mais fácil em regiões contendo lipídeos poli-insaturados, provavelmente devido ao empacotamento mais frouxo da bicamada. Os dados sugerem que a miltefosina age principalmente em rafts lipídicos e que células contendo mais lipídicos poli-insaturados podem incorporar maior quantidade do fármaco

Alquilfosfocolines (APCs) comprise a class of drugs with antitumor activity derived from endogenous phospholipids. Differently from other drugs whose primary site of action is the DNA, APCs act firstly in the plasma membrane and signaling proteins, such as PKC. The main objective of this work is to elucidate, via computational approaches, the possible mechanisms of actions that cause hemolysis, PKC inhibition and interaction with cellular membranes. Initially, a set of 34 APCs was studied by means of Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA). The molecules were simulated by means of molecular dynamics simulations (MD) and molecular and structural properties were calculated for the lowest-energy conformer. After HCA and PCA methodologies, the set was divided into 3 groups according to their structural features. The findings suggest that the presence of bulky cationic moieties, or the adamantyl and cyclohexil rings, increase the hemolytic potential of compounds with long alkyl chains. Macrocyclic rings, such as cyclopentadecyl, seem to be important to elevate the hemolysis of compounds with short alkyl chains. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. In the next step of this work, 7 APC derivatives were selected and docked in the C2 domain of PKCα. The aim now was to map the residues relevant for ligands interaction compared to the binding mode of the endogenous activator, phosphatidylserine (PS). HCA and PCA were again applied in order to extract relevant information from the mapping. The results showed that the lateral chains of Pro188, Asn189, Arg216, Trp247, Asp249 and Thr250 do not allow the proper approximation of the ligands, impeding the phosphoryl moiety from coordinating with one of the calcium atoms. On the other hand, the cationic moiety of PS forms hydrogen-bonding with Asn189 in order to position the oxygens to interact, at the same time, with a calcium atom. With less coordination sites, calcium binding affinity diminishes and the enzyme activation is compromised, interrupting the signaling cascade. The final part of this work was dedicated to the study of miltefosine interaction with different lipid bilayers from the thermodynamics standpoint. Eight bilayers were simulated with MD and the energetic interaction was calculated via Umbrella Sampling simulations. The findings showed that miltefosine has higher partition in bilayers containing cholesterol, with miscibility of about 76 times higher than the values referring to bilayers without cholesterol. Moreover, it was observed that the internalization of miltefosine is facilitated in regions containing polyunsaturated lipids, probably due to the looser packing. The data suggest that miltefosine acts primarily in lipid rafts, and that cells containing more polyunsaturated lipids in their membranes can incorporate higher quantities of this drug

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Por la gran relevancia que tiene el glicocalix en la relación huésped-parásito, y para adentrarse en el conocimiento de las características y función que desempeña en diferentes parasitos, en este trabajo se aisló y caracterizó químicamente el glicocalix de cinco helmintos que tienen en común que, sin dividirse en el huésped, sobreviven en él durante mucho tiempo. En el glicocalix de ninguno de los parásitos estudiados se pudo demostrar la presencia de ácido siálico; en cambio, en todos se encontraron ácidos urónicos. En el glicocalix de los cinco parásitos estudiados, el cociente de azúcares sin carga/con carga y el de azúcares con carga negativa/positiva fue mayor de 1. En el glicocalix de Cysticercus celulasae y en el de Fasciola hepatica, los cocientes de azúcares con carga -negativa/positva y de azúcares/proteínas, fueron mayores que los de Ascaris suum, Macracanthorhynchus hirudinaceus y Moniezia expansa

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