Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

Zeolites are crystalline aluminosilicates which have highly ordered pore structures and high surface area. Also the tailorable surface properties, high ion-exchange capability, high chemical, thermal, and mechanical strength make these particles an important candidate for various application such as sensors, catalysis, dielectric materials, separation, and membrane technologies. Although zeolites have these unique properties, applications where zeolites are integrated into devices according to their application areas, are limited due to the powder form of the material. The purpose of the current study was to investigate the effect of zeolite nanoparticles on conductometric biosensor performance and cell viability measurements. Firstly, zeolite attachment on silicon surfaces was investigated by attaching silicalite and zeolite A nanoparticles onto the silicon substrates by direct attachment methodology in a closely packed monolayer form with perfect orientation and full coverage without using any chemical linker. Furthermore, the ability to pattern these zeolite crystals on silicon substrates with electron beam lithography and photolithography techniques was investigated. With the combination of electron beam lithography and direct attachment methodology, zeolite patterns were produced with feature sizes as small as a single silicalite nanoparticle thick line, that is approximately 500 nm. This approach has the ability of patterning very small features on silicon substrate, but the drawback is the long patterning time and lack of electron beam stability during long pattern formation process. Accordingly, it is almost impossible to form large patterns with electron beam lithography systems. Afterwards, to have full control on surfaces with differentiated areas on solid substrates, patterns of one type of zeolite crystals was formed on the monolayer of another type of zeolite layer with electron beam lithography for the first time. The same closed packed and highly oriented silicalite patterns were successfully formed on zeolite A monolayers and vice versa. Then photolithography technique was combined with direct attachment methodology to overcome the problem of the lack of total patterned area. With this technique, it was possible to pattern the whole silicon wafer in a couple of seconds, however the feature size of the zeolite patterns was limited with the infrastructures of the mask fabricated for photolithography studies. In this particular study, zeolite lines patterns with a minimum of 5 &micro / m thickness were prepared and the total patterned area was kept constant at 1 cm2. Similar to what was obtained by electron beam lithography study, zeolite A patterns were formed on silicalite monolayers with the minimum feature size of 5 &micro / m and vice versa. In the second part of the study, zeolite films were prepared on the transducers of conductometric biosensors using dip coating technique and named as Zeolite Coated Transducers (ZCT). Electrodes prepared using a mixture of zeolite and enzyme solution and then subjected to casting using glutaraldehyde were called Zeolite Membrane Transducers (ZMT). The operational and storage stabilities were determined to be in an acceptable range using ZCTs for conductometric urea biosensors. It was observed that using electrodes fabricated by the ZCT technique enhanced the biosensor signals up to two times and showed a rapid response after the addition of urea to the medium when it was compared with Standard Membrane Transducers (SMT). This enhancement can be explained by the lack of GA layer on top of the film, which acts as a diffusion barrier and inhibits the activity of the enzyme. On the second part of this conductometric biosensor study, effect of zeolite modification with methyl viologen (MV) and silver nanoparticles (Ag+ and Ag0), as well as the effect of changing Si/Al ratio was investigated with three different zeolite Beta particles which have Si/Al ratios of 40, 50, and 60. There were no significant effect of MV modification on ZMTs and there was no response observed with Ag+ and Ag0 modified zeolites. However, it was observed that conductometric responses increased with increasing Si/Al ratio for ZMTs. This behavior can be due to an increased hydrophobicity and/or the increasing acidic strength with the increasing Si/Al ratio within the zeolite crystals. Also ZCTs showed higher responses with respect to both SMTs and ZMTs. When compared with SMTs and ZMTs, ZCTs had higher reproducibility due to the controlled thickness of zeolite thin film by dip coating, and the controlled amount of enzyme adsorbed on this film. In the third part of the study, effect of zeolites on cell proliferation with MG63 osteoblast cells and NIH3T3 fibroblast cells were investigated. For that purpose, zeolite A, silicalite, and calcined forms of these zeolites were patterned with photolithography technique onto silicon wafers. Three different patterns prepared for this particular study, which has 0.125cm2, 0.08825cm2, and 0.04167cm2 zeolite patterned areas on 1 cm2 samples. In that way, not only the zeolite type and effect of calcination of zeolites, but also the effect of zeolite amount on MG63 osteoblast cells and NIH3T3 fibroblast cells were investigated. Silicalite coated samples were observed to have higher amount of cells than zeolite A coated samples after 24, 48, and 72 hours of incubation. This may be referred to the hydrophilic/hydrophobic properties, surface charge, and/or particle size of zeolites. Also it is observed that higher zeolite amount on samples resulted in an increase in the number of cells attached to the samples. There was also a significant increase in the number of cells upon using calcined silicalite samples. Accordingly, it can be hypothesized that zeolite pores result in an enhancement of protein adsorption and proliferation, even if this only occurs at the pore openings. On the other hand, there was no positive effect of calcining zeolite A. This result was expected since there is no structure directing agent used in synthesis procedure of zeolite A, which again supports the fact that pores might have some role in cell attachment.

Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the treatment of male erectile dysfunction (MED) in the United States early 1998, 274 adverse event reports were filed by the Food and Drug Administration (FDA) between 4 Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various neurological disturbances, including amnesia and aggressive behaviour (Milman and Arnold, 2002). These and other research findings have prompted investigations into the possible central effects of sildenafil. The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004). GPCRs signal through the phosphatidylinositol signal transduction pathway known to activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal transduction pathway is also known to involve the activation of PKs (via cyclic guanosine monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the action of antidepressants by elevating cGMP levels. It is generally assumed that excitotoxic delayed cell death is pathologically linked to an increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate antagonists, especially those that block the define NMDA-receptors, are neuroprotective, showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels. Aims: The aims of the study were to investigate any neuroprotective properties of sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function. Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug (control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX - ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ - 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used to determine mAChR function by constructing dose-response curves of methacholine or to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for cell viability. Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of methacholine in neuronal cells but did not show a significant increase in epithelial cells The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay could also not be used, due to severe cell loss. Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a neurodegenerative, and not a protective property, in neuronal cells. This is not likely to be associated with its PDE5 inhibitory action, but may possibly be linked to an increase in cGMP levels via the NO-cGMP pathway. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

<p>Basella alba (BA) and Hibiscus macranthus (HM) are used by traditional healers in Cameroon to treat male sexual fertility problems. Previous studies showed that in vivo administration of the leaf extracts of both plants caused a significant increase in rat seminal vesicle weight and spermatozoa numbers was accompanied by a significant increase in serum testosterone. The aim of this study was to establish the effects of BA and HM extracts on Sertoli cell functions. TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells (Mather, 1982). Sertoli cell play a key role in spermatogenesis by regulating and supporting germ cell development. Therefore, any alterations in Sertoli cell physiology or structure may lead to impaired spermatogenesis, germ cell loss and male infertility. Developing germ cells in the seminiferous tubule require a constant supply of lactate and pyruvate (Jutte et al, 1981 / 1982) and toxicant induced alterations in these nutrients have been shown to induce germ cell necrosis (Monsees et al., 2000). TM4 Sertoli cells were cultured in DMEM/Ham F-12 (M) for one day and exposed to<br /> 0.01, 0.1, 1, 10, 100 &mu / g/ml of BA and HM extracts, respectively, for four further days. The extracts were dissolved in 0.5 % DMSO in M, while 0.5 % and 2% DMSO in M were used as negative or positive controls, respectively, and 100mM ethanol as positive control where indicated. Results obtained from the Sertoli cells exposed to BA extracts, showed that the plant extract had no significant effect on the cell viability but induced a significant concentration-dependent increase in lactate (19-67%) and pyruvate levels (39-102%) and a concentration-dependent decrease in the protein content (9-42%). The H&amp / E histological study confirmed that the BA extract had no cytotoxic effect, as there were no changes in the morphology of the cell. Likewise, apoptotic study using DAPI showed no alteration in the nucleus when compared to the negative control. The HM plant extract significantly enhanced mitochondrial dehydrogenase activity (7fold) in the Sertoli cells but caused only slight alterations in the lactate and pyruvate levels. There was no effect seen in the protein content of the Sertoli cells. H&amp / E and DAPI staining revealed that there were neither changes in the morphology of the cells nor any alteration regarding the mitotic and apoptotic indices. Thus, the HM extract did not have a cytotoxic effect on the cells. This study demonstrated that the Basella alba methanol extract may enhance spermatogenesis as it stimulated the source of energy required for the development of germ cells without exerting a cytotoxic effect. The Hibiscus macranthus extract stimulated mitochondrial dehydrogenase activities and may thus trigger changes in Sertoli cell physiology. In summary, both plant extracts enhanced certain Sertoli cell<br /> functions and thus might explain the positive in vivo effects of the combined plant extracts on rat spermatogenesis observed by Moundipa et al. (1999).</p>

The putative peroxisome proliferator-activated receptor (PPAR) α ligand, conjugated linoleic acid (CLA) induced cytoplasmic lipid droplet (LD) formation in H4IIE rat hepatoma cells. Currently, the mechanism(s) by which CLA isomers affects hepatic LD formation is unclear. We have investigated the role of PPARα and fatty acid (FA) activation in the regulation of hepatic LD formation induced by CLA isomers [cis-9,trans-11 (c9,t11), trans-10,cis-12 (t10,c12)] and linoleic acid (LA) in an in vitro model of lipid accumulation. Dose response of c9,t11 and t10,c12 CLA isomers as well as LA in quiescent H4IIE cells was assessed by Oil Red O staining and subsequent quantification after 24 hours. LD formation was induced by the CLA isomers similar to LA in a dose-dependent manner. However, treatment with the acyl CoA synthetase (ACS) inhibitor, triacsin C, resulted in significantly reduced LD formation. A similar reduction in lipid accumulation was observed with the PPARα activator, Wy14643. Furthermore, CLA isomers promoted H4IIE viability at 60 µM but decreased viability at a higher dose of 180 µM. To further understand the role of PPARα in hepatic steatosis, we studied the level and phosphorylation of PPARα in livers of male lean and fa/fa Zucker rats fed either a control diet or fa/fa Zucker rats fed a CLA isomer (0.4% wt/wt c9,t11 or 0.4% wt/wt t10,c12) diet for 8 weeks. Immunoblotting results showed that only the t10,c12 CLA isomer significantly reduced phospho-PPARα S21 compared to the lean control (ln Ctl) and it was associated with a significant increase in the phosphorylation of p38 mitogen activated protein kinase (MAPK).These changes were not observed with the c9,t11 CLA isomer. Taken together, we have shown that CLA isomers directly induce LD formation in quiescent H4IIEs by activation of the lipid storage pathway which was significantly reduced by triacsin C or Wy14643. Also, we demonstrate for the first time that only the t10,c12 CLA isomer significantly reduced PPARα phosphorylation while it increased p38 MAPK phosphorylation. These results indicate that the anti-steatotic effects of the t10,c12 CLA isomer is associated with changes in PPARα phosphorylation and thereby its activity in a MAPK-independent manner.

Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the treatment of male erectile dysfunction (MED) in the United States early 1998, 274 adverse event reports were filed by the Food and Drug Administration (FDA) between 4 Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various neurological disturbances, including amnesia and aggressive behaviour (Milman and Arnold, 2002). These and other research findings have prompted investigations into the possible central effects of sildenafil. The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004). GPCRs signal through the phosphatidylinositol signal transduction pathway known to activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal transduction pathway is also known to involve the activation of PKs (via cyclic guanosine monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the action of antidepressants by elevating cGMP levels. It is generally assumed that excitotoxic delayed cell death is pathologically linked to an increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate antagonists, especially those that block the define NMDA-receptors, are neuroprotective, showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels. Aims: The aims of the study were to investigate any neuroprotective properties of sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function. Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug (control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX - ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ - 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used to determine mAChR function by constructing dose-response curves of methacholine or to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for cell viability. Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of methacholine in neuronal cells but did not show a significant increase in epithelial cells The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay could also not be used, due to severe cell loss. Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a neurodegenerative, and not a protective property, in neuronal cells. This is not likely to be associated with its PDE5 inhibitory action, but may possibly be linked to an increase in cGMP levels via the NO-cGMP pathway. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

The putative peroxisome proliferator-activated receptor (PPAR) α ligand, conjugated linoleic acid (CLA) induced cytoplasmic lipid droplet (LD) formation in H4IIE rat hepatoma cells. Currently, the mechanism(s) by which CLA isomers affects hepatic LD formation is unclear. We have investigated the role of PPARα and fatty acid (FA) activation in the regulation of hepatic LD formation induced by CLA isomers [cis-9,trans-11 (c9,t11), trans-10,cis-12 (t10,c12)] and linoleic acid (LA) in an in vitro model of lipid accumulation. Dose response of c9,t11 and t10,c12 CLA isomers as well as LA in quiescent H4IIE cells was assessed by Oil Red O staining and subsequent quantification after 24 hours. LD formation was induced by the CLA isomers similar to LA in a dose-dependent manner. However, treatment with the acyl CoA synthetase (ACS) inhibitor, triacsin C, resulted in significantly reduced LD formation. A similar reduction in lipid accumulation was observed with the PPARα activator, Wy14643. Furthermore, CLA isomers promoted H4IIE viability at 60 µM but decreased viability at a higher dose of 180 µM. To further understand the role of PPARα in hepatic steatosis, we studied the level and phosphorylation of PPARα in livers of male lean and fa/fa Zucker rats fed either a control diet or fa/fa Zucker rats fed a CLA isomer (0.4% wt/wt c9,t11 or 0.4% wt/wt t10,c12) diet for 8 weeks. Immunoblotting results showed that only the t10,c12 CLA isomer significantly reduced phospho-PPARα S21 compared to the lean control (ln Ctl) and it was associated with a significant increase in the phosphorylation of p38 mitogen activated protein kinase (MAPK).These changes were not observed with the c9,t11 CLA isomer. Taken together, we have shown that CLA isomers directly induce LD formation in quiescent H4IIEs by activation of the lipid storage pathway which was significantly reduced by triacsin C or Wy14643. Also, we demonstrate for the first time that only the t10,c12 CLA isomer significantly reduced PPARα phosphorylation while it increased p38 MAPK phosphorylation. These results indicate that the anti-steatotic effects of the t10,c12 CLA isomer is associated with changes in PPARα phosphorylation and thereby its activity in a MAPK-independent manner.

<p>Basella alba (BA) and Hibiscus macranthus (HM) are used by traditional healers in Cameroon to treat male sexual fertility problems. Previous studies showed that in vivo administration of the leaf extracts of both plants caused a significant increase in rat seminal vesicle weight and spermatozoa numbers was accompanied by a significant increase in serum testosterone. The aim of this study was to establish the effects of BA and HM extracts on Sertoli cell functions. TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells (Mather, 1982). Sertoli cell play a key role in spermatogenesis by regulating and supporting germ cell development. Therefore, any alterations in Sertoli cell physiology or structure may lead to impaired spermatogenesis, germ cell loss and male infertility. Developing germ cells in the seminiferous tubule require a constant supply of lactate and pyruvate (Jutte et al, 1981 / 1982) and toxicant induced alterations in these nutrients have been shown to induce germ cell necrosis (Monsees et al., 2000). TM4 Sertoli cells were cultured in DMEM/Ham F-12 (M) for one day and exposed to<br /> 0.01, 0.1, 1, 10, 100 &mu / g/ml of BA and HM extracts, respectively, for four further days. The extracts were dissolved in 0.5 % DMSO in M, while 0.5 % and 2% DMSO in M were used as negative or positive controls, respectively, and 100mM ethanol as positive control where indicated. Results obtained from the Sertoli cells exposed to BA extracts, showed that the plant extract had no significant effect on the cell viability but induced a significant concentration-dependent increase in lactate (19-67%) and pyruvate levels (39-102%) and a concentration-dependent decrease in the protein content (9-42%). The H&amp / E histological study confirmed that the BA extract had no cytotoxic effect, as there were no changes in the morphology of the cell. Likewise, apoptotic study using DAPI showed no alteration in the nucleus when compared to the negative control. The HM plant extract significantly enhanced mitochondrial dehydrogenase activity (7fold) in the Sertoli cells but caused only slight alterations in the lactate and pyruvate levels. There was no effect seen in the protein content of the Sertoli cells. H&amp / E and DAPI staining revealed that there were neither changes in the morphology of the cells nor any alteration regarding the mitotic and apoptotic indices. Thus, the HM extract did not have a cytotoxic effect on the cells. This study demonstrated that the Basella alba methanol extract may enhance spermatogenesis as it stimulated the source of energy required for the development of germ cells without exerting a cytotoxic effect. The Hibiscus macranthus extract stimulated mitochondrial dehydrogenase activities and may thus trigger changes in Sertoli cell physiology. In summary, both plant extracts enhanced certain Sertoli cell<br /> functions and thus might explain the positive in vivo effects of the combined plant extracts on rat spermatogenesis observed by Moundipa et al. (1999).</p>

Made available in DSpace on 2015-05-14T12:56:03Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2062730 bytes, checksum: f8aa4f1dba574b15daf66619df74d887 (MD5) Previous issue date: 2013-12-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Candida albicans is a yeast that lives harmlessly on various parts of the human body, including the oral cavity, however, under certain circumstances, may cause superficial infections of the mucous membranes, such as denture stomatitis, in which a biofilm of Candida is formed on the acrylic surface of dentures. Thymol is a phenolic terpene found in several plant species, which has antimicrobial activity against oral microorganisms such as Candida albicans, since it can significantly interfere with fungal biofilm formation and inhibit the metabolic activity of these microorganisms by direct action on cell membrane. This study evaluated through fluorescence technique the cell viability of Candida albicans biofilms under the antifungal activity of thymol. It was used strains of Candida albicans (ATCC® 11006 ). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the antifungal drugs (miconazole and thymol) were determined by microdilution tests in Sabouraud dextrose broth. The drugs were prepared in dimethylsulfoxide (DMSO) and the inoculum was standardized to correspond to 0.5 of McFarland s scale (106 UFC/mL). Biofilms of Candida albicans were grown, from Sabouraud broth supplemented with 10% dextrose, on the surface of acrylic resin disks in parallel flow chambers and, after 12 hours of biofilm formation in a continuous flow system, it was exposure to the antifungal agents, being evaluated periods of 5, 15 and 30 minutes. For counting of colony-forming units, the fungal solution was sequentially diluted and plated on Sabouraud dextrose agar. Cell viability was quantified by fluorescence by mixing SYTO 9 and Propidium Iodide dyes. Data were evaluated by analysis of variance (ANOVA), Tukey's test and t-test at 5% probability. Biofilms treated with thymol showed, on the three incubation times evaluated, low percentage numbers of viable cells detected by fluorescence technique, and the average of the three incubation times between miconazole and thymol were not statistically different (p&#707; 0.05), demonstrating that both drugs possess equivalent efficiency, taking into account their respective minimum fungicidal concentrations. It was possible to prove that both methodologies used to quantify fungal cells showed to be strongly correlated. / Candida albicans trata-se de um fungo comensal que habita inofensivamente nichos de várias partes do corpo humano, incluindo a cavidade oral, no entanto, sob certas circunstâncias, pode causar infecções superficiais das mucosas, como a estomatite protética, na qual um biofilme de Candida é formado sobre a superfície acrílica das próteses dentárias. O timol é um terpeno fenólico encontrado em diversas espécies vegetais e que possui atividade antimicrobiana frente a microrganismos orais, tal como Candida albicans, pois é capaz de interferir de forma significativa com a formação de biofilmes fúngicos, uma vez que inibe a atividade metabólica destes microrganismos por ação direta na membrana celular. Este estudo objetivou avaliar através de técnica de fluorescência a viabilidade celular de biofilmes de Candida albicans frente à ação antifúngica do timol. Foram utilizadas cepas de Candida albicans (ATCC® 11006 ). A concentração inibitória mínima (CIM) e a concentração fungicida mínima (CFM) dos agentes antifúngicos (timol e miconazol) foram determinadas através de testes de microdiluição em caldo Sabouraud dextrose, sendo as drogas preparadas em dimetilsulfóxido (DMSO) e o inóculo padronizado para corresponder a 0,5 da escala de McFarland (106 UFC/mL). Os biofilmes de Candida albicans foram cultivados sobre a superfície de discos de resina acrílica, nas células paralelas de fluxo, a partir de caldo Sabouraud suplementado com 10% de dextrose, e, após 12 horas de formação do biofilme no sistema de fluxo contínuo, foi realizada a exposição aos agentes antifúngicos, sendo avaliados períodos de 5, 15 e 30 minutos. Para contagem das unidades formadoras de colônia, a solução fúngica foi sequencialmente diluída e semeada em ágar Sabouraud dextrose. A viabilidade celular foi quantificada por fluorescência através da mistura dos corantes SYTO 9 e Iodeto de Propídio. Os valores médios foram submetidos à análise de variância (ANOVA) e ao teste T em nível de 5% de probabilidade. Os biofilmes tratados com o timol apresentaram, nos três tempos de exposição avaliados, baixos números percentuais de células viáveis detectadas através da técnica de fluorescência, e os valores médios dos três tempos de exposição entre miconazol e timol não diferiram estatisticamente (p&#707;0,05), demonstrando que ambas as drogas possuem eficiência equivalente, levando-se em consideração suas respectivas concentrações fungicidas mínimas. Foi possível comprovar ainda que ambas as metodologias utilizadas para quantificação das células fúngicas apresentaram-se fortemente correlacionadas.

Neste estudo avaliamos atividades biológicas pré-clínicas de moléculas obtidas de Mangifera indica e de Valeriana glechomifolia. Mangiferina, isolada de M. indica, estimulou a proliferação celular e induziu um aumento significativo na secreção do fator de crescimento do nervo e do fator de necrose tumoral em células de glioblastoma humano U138-MG in vitro. Uma injeção sistêmica de mangiferina melhorou a consolidação da memória de longa duração (LTM) de reconhecimento de objetos (RO) e prejudicou a retenção da memória aversiva no teste da esquiva inibitória (EI) em ratos. A melhora da LTM no RO promovida pela administração sistêmica de mangiferina também foi observada com a administração intrahipocampal. Já o prejuízo da memória no teste da EI observado sistemicamente não ocorreu com a infusão no hipocampo ou amígdala. Camundongos atáxicos também apresentaram melhora na memória de RO após administração crônica de mangiferina, sem efeito na EI; um extrato comercializado de M. indica não afetou a memória no RO, mas facilitou a memória na EI. Os resultados indicam que mangiferina melhora a LTM no RO com envolvimento do hipocampo por meio de um mecanismo que pode envolver um aumento dos níveis da neurotrofina NGF e da citocina TNF-α. Valepotriatos, isolados de V. glechomifolia, demonstraram inibição da viabilidade de células tumorais U138-MG nas doses de 30 e 100μg/μl; o 8-Br- AMPc, um análogo do AMPc, atenuou à inibição dos valepotriatos na viabilidade celular, sugerindo que os valepotriatos interagem com a rota de sinalização celular do AMPc/PKA na inibição da viabilidade de células cancerosas. A administração sistêmica de valepotriatos, em camundongos 30 minutos antes dos testes, apresentou os seguintes resultados: durante a exploração no campo aberto, a dose 10mg/kg causou redução na locomoção e no comportamento exploratório e diminuição da ansiedade no teste do labirinto em cruz elevado. Não ocorreu diferença entre os tratamentos na memória de EI e na memória RO, exceto no grupo que recebeu 3mg/kg de valepotriatos que apresentou piora na LTM de RO. Os resultados indicam que os valepotriatos causaram atividades ansiolítica e sedativa sem déficits na memória de EI e RO em camundongos tratados com 10mg/kg. As atividades biológicas in vitro e in vivo encontradas nas moléculas estudadas (especialmente mangiferina e valepotriatos) geram interesse de investigações para utilizações terapêuticas na memória e no câncer. / We evaluated the biological activities of molecules obtained from Mangifera indica and Valeriana glechomifolia. Mangiferin, isolated from M. indica, stimulated cell proliferation and induced a significant increase in levels of nerve growth factor and tumor necrosis factor secreted in human glioblastoma cells U138-MG in vitro. A systemic injection of mangiferin improved long term memory (LTM) consolidation of object recognition (NOR) and impaired memory retention in aversive inhibitory avoidance test (IA) in rats. The improvement in NOR memory promoted by systemic administration of mangiferin was also observed with intrahippocampal administration. The memory impairment observed systemically in the IA did not occur with the infusion into the hippocampus or amygdala. Ataxic mice also showed improvement in NOR memory after chronic administration of mangiferin, with no effect on IA; a standardized extract of M. indica had not effect on memory in NOR, but facilitated the memory in IA. The results indicate that mangiferin improvement NOR memory involving the hippocampus through a mechanism that may involve increased levels of neurotrophins and cytokines. Valepotriates isolated from V. glechomifolia showed inhibition of the viability of U138-MG tumor cells at doses of 30 and 100μg/μl; 8-BrcAMP, an analogue of cAMP, reversed the inhibition of valepotriates on cell viability, suggesting that valepotriates interact with the cAMP/PKA signaling route in the inhibition of the viability of cancer cells. Systemic administration of valepotriates in mice, 30 minutes before tests, showed the following results: during the open-field, the dose of 10mg/kg caused a reduction in locomotion and exploratory behavior and decreased anxiety in the test of elevated plus maze. There was no difference between treatments in IA or NOR memories, except from the group receiving valepotriates at 3mg/kg, which worsened in the NOR. The results indicated that valepotriates at 10mg/kg caused anxiolytic and sedative activities without inducing memory deficits in IA and NOR. The biological activities in vitro and in vivo found with the studied molecules (notable mangiferin and valepotriates) support further research for potential therapeutic uses in cancer and in memory.