Potencial do uso do ácido cafeico em formulações cosméticas: atividade antioxidante, atividade despigmentante e citotoxicidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo químico dos escapos de Paepalanthus geniculatus Kunth

Eriocaulaceae species are popularly known as sempre-vivas. This family comprising approximately 1.200 species divided into 10 genera, with high endemic levels. Paepalanthus genus has high incidence in the Espinhaço Range region and the report of biological activities. Despite the large number of previous studies with Eriocaulaceae species, Paepalanthus geniculatus has no studies about their scapes, which demonstrates the need for new research to identify their chemical and biological composition. The aim of this work was to study the chemical composition of P. geniculatus ethanolic extract seeking the isolation and identification of metabolites and evaluate the radical scavenging activity of the extract and isolated substances. P. geniculatus were collected in Serra do Cipó-MG in 2013 (Voucher: SANO 3193) and the scapes were dried, crushed and the powder was percolated with ethanol. With the etanolic extract it is carried out a fractionation by gel permeation chromatography, yielding fractions analyzed by TLC assay. The fraction J89 (45,6 mg) was purified by semipreparative HPLC-PDA, resulting in the isolation of substance S1. The fractionation was also performed by medium pressure liquid chromatography, yielding fractions analyzed by TLC assay. The fraction number 4 (92,6 mg) were purified by semipreparative HPLC-PDA resulting in the isolation of substances S2 and S3. The substances S1 and S3 were analyzed by mono and twodimensional NMR, resulting in the identification of 3,4-dihydroxybenzoic acid (protocatechuic acid) and the flavonoid 6-hydroxyquercetin-7-O-β-Dglucopyranoside. The substance S2 were identified by comparison with standards, were it was possible to determine the presence of the flavonoid 6-hydroxy-7- methoxyquercetin-3-O-β-D-glucopyranoside. The evaluation of radical scavenging activity for the extract and the isolated substances using DPPH, showed consistent activity to S1...

Potencial do uso do ácido cafeico em formulações cosméticas: atividade antioxidante, atividade despigmentante e citotoxicidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development of albendazole sulfoxide-loaded Eudragit microparticles: A potential strategy to improve the drug bioavailability

Albendazole sulfoxide (ABZSO), a broad spectrum anthelmintic drug extensively used in veterinary medicine, exhibits a low and erratic bioavailability due to its poor solubility in biological fluids. The aims of this study were the development, physicochemical characterization, and in vitro release profile evaluation of ABZSO-loaded Eudragit RS PO (R) microparticles (MPs) in order to improve the rate of dissolution and the dissolved percentage of the drug in pH 7.4. MPs were successfully obtained by the emulsification/solvent evaporation method, achieving entrapment efficiency and process yield of about 60% and mean size of 254 nm. The in vitro release profile study showed that dissolution of ABZSO followed a pseudo-second order kinetics and MPs were able to increase significantly (p < 0.05) the rate of dissolution of ABZSO compared to the micronized and non-micronized free drug, what could lead to an improvement in bioavailability and, consequently, in the antiparasitic activity. (C) 2011 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.

Poly(2,5-bibenzimidazole) Membranes: Physico-Chemical Characterization Focused on Fuel Cell Applications

Membranes of Poly(2,5-benzimidazole) (ABPBI), prepared by polycondensation in polyphosphoric acid, were characterized from the fuel cell application point of view: mechanical properties of the membranes for different acid doping levels, thermal stability, permeability for the different gases/vapors susceptible of use in the cell (hydrogen, oxygen, methanol and ethanol), electro-osmotic water drag coefficient, oxidation stability to hydroxyl radicals, phosphoric acid leaching rate and, finally, in-plane membrane conductivity. ABPBI membranes presented an excellent thermal stability, above 500 degrees C in oxygen, suitable mechanical properties for high phosphoric acid doping levels, a low methanol and ethanol limiting permeation currents, and oxygen permeability compared to Nafion membranes, and a low phosphoric acid leaching rate when exposed to water vapor. On the contrary, hydrogen permeation current was higher than that of Nafion, and the chemical stability was very limited. Membrane conductivity achieved 0.07 S cm(-1) after equilibration with a humid environment. Fuel cell tests showed reasonable good performances, with a maximum power peak of 170 mW cm(-2) for H-2/air at 170 degrees C operating under a humidified hydrogen stream, 39.9 mW cm(-2) for CH3OH/O-2 at 200 degrees C for a methanol/water weight ratio of 1: 2, and 31.5 mW cm(-2) for CH3CH2OH/O-2 at the same conditions than for methanol. (C) 2012 The Electrochemical Society. [DOI: 10.1149/2.014207jes] All rights reserved.

Influence of the relative amounts of crystalline and amorphous phases on the mechanical properties of polyamide-6 nanocomposites

The relative amounts of amorphous and crystalline ?- and a-phases in polyamide-6 nanocomposites, estimated from the deconvolution of X-ray diffraction peaks using Gaussian functions, correlates with their mechanical, thermomechanical, and barrier properties. The incorporation of organoclay platelets (Cloisite 15A and 30B) induced the crystallization of the polymer in the ? form at expense of the amorphous phase, such that 12 wt % of Cloisite is enough to enhance the mechanical and the thermomechanical properties. However, higher nanofiller loads were necessary to achieve good barrier effects, because this property is mainly dependent on the tortuous path permeation mechanism of the gas molecules through the nanocomposite films. (C) 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

In Vitro Characterization of Chitosan Gels for Buccal Delivery of Celecoxib: Influence of a Penetration Enhancer

Celecoxib (Cx) shows high efficacy in the treatment of osteoarthritis and rheumatoid arthritis as a result of its high specificity for COX-2, without gastrolesivity or interference with platelet function at therapeutic concentrations. Besides of anti-inflammatory effects, Cx also has a potential role for oral cancer chemoprevention. For these conditions, oral administration in long-term treatment is a concern due to its systemic side effects. However, local application at the site of injury (e.g., buccal inflammation conditions or chemoprevention of oral cancer) is a promising way to reduce its toxicity. In this study, the in vitro characterization of mucoadhesive chitosan (CHT) gels associated to AzoneA (R) was assessed to explore the potential buccal mucosal administration of Cx in this tissue. Rheological properties of gels were analyzed by a rheometer with cone-plate geometry. In vitro Cx release and permeability studies used artificial membranes and pig cheek mucosa, respectively. Mucoadhesion were measured with a universal test machine. CHT gels (3.0%) containing 2.0% or 3.0% Az showed more appropriate characteristics compared to the others: pH values, rheology, higher amount of Cx retained in the mucosa, and minimal permeation through mucosa, besides the highest mucoadhesion values, ideal for buccal application. Moreover, the flux (J) and amounts of drug released decreased with increased CHT and Az concentrations. CHT gels (3.0%) associated with 2.0% or 3.0% Az may be considered potential delivery systems for buccal administration of Cx.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

Interaction of gramicidin with DPPC/DODAB bilayer fragments

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids. (C) 2012 Elsevier B.V. All rights reserved.

Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles

Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50 degrees C. This effect becomes negligible at temperatures higher that ca. 60 degrees C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis. Published by Elsevier B.V.

p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma

CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis.

Movement of NH3 through the human urea transporter B: a new gas channel

Aquaporins and Rh proteins can function as gas (CO2 and NH3) channels. The present study explores the urea, H2O, CO2, and NH3 permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [14C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (∆pHS) caused by exposing oocytes to 5% CO2/33 mM HCO3- (pHS increase) or 0.5 mM NH3/NH4+ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H2O-injected control oocytes. UT-B expression had no effect on the CO2-induced ∆pHS but doubled the NH3-induced ∆pHS. Phloretin reduced UT-B-dependent urea uptake (Jurea * ) by 45%, Pf * by 50%, and (- ∆pHS * )NH3 by 70%. p-Chloromercuribenzene sulfonate reduced Jurea * by 25%, Pf * by 30%, and (∆pHS * )NH3 by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H2O and NH3 and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H2O and NH3 permeability. Our data confirm that UT-B has significant H2O permeability and for the first time demonstrate significant NH3 permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH3 and H2O pass through the three monomeric urea channels in UT-B.

Computational analyses on the structure-function relation in ion channels

Ion channels are protein molecules, embedded in the lipid bilayer of the cell membranes. They act as powerful sensing elements switching chemicalphysical stimuli into ion-fluxes. At a glance, ion channels are water-filled pores, which can open and close in response to different stimuli (gating), and one once open select the permeating ion species (selectivity). They play a crucial role in several physiological functions, like nerve transmission, muscular contraction, and secretion. Besides, ion channels can be used in technological applications for different purpose (sensing of organic molecules, DNA sequencing). As a result, there is remarkable interest in understanding the molecular determinants of the channel functioning. Nowadays, both the functional and the structural characteristics of ion channels can be experimentally solved. The purpose of this thesis was to investigate the structure-function relation in ion channels, by computational techniques. Most of the analyses focused on the mechanisms of ion conduction, and the numerical methodologies to compute the channel conductance. The standard techniques for atomistic simulation of complex molecular systems (Molecular Dynamics) cannot be routinely used to calculate ion fluxes in membrane channels, because of the high computational resources needed. The main step forward of the PhD research activity was the development of a computational algorithm for the calculation of ion fluxes in protein channels. The algorithm - based on the electrodiffusion theory - is computational inexpensive, and was used for an extensive analysis on the molecular determinants of the channel conductance. The first record of ion-fluxes through a single protein channel dates back to 1976, and since then measuring the single channel conductance has become a standard experimental procedure. Chapter 1 introduces ion channels, and the experimental techniques used to measure the channel currents. The abundance of functional data (channel currents) does not match with an equal abundance of structural data. The bacterial potassium channel KcsA was the first selective ion channels to be experimentally solved (1998), and after KcsA the structures of four different potassium channels were revealed. These experimental data inspired a new era in ion channel modeling. Once the atomic structures of channels are known, it is possible to define mathematical models based on physical descriptions of the molecular systems. These physically based models can provide an atomic description of ion channel functioning, and predict the effect of structural changes. Chapter 2 introduces the computation methods used throughout the thesis to model ion channels functioning at the atomic level. In Chapter 3 and Chapter 4 the ion conduction through potassium channels is analyzed, by an approach based on the Poisson-Nernst-Planck electrodiffusion theory. In the electrodiffusion theory ion conduction is modeled by the drift-diffusion equations, thus describing the ion distributions by continuum functions. The numerical solver of the Poisson- Nernst-Planck equations was tested in the KcsA potassium channel (Chapter 3), and then used to analyze how the atomic structure of the intracellular vestibule of potassium channels affects the conductance (Chapter 4). As a major result, a correlation between the channel conductance and the potassium concentration in the intracellular vestibule emerged. The atomic structure of the channel modulates the potassium concentration in the vestibule, thus its conductance. This mechanism explains the phenotype of the BK potassium channels, a sub-family of potassium channels with high single channel conductance. The functional role of the intracellular vestibule is also the subject of Chapter 5, where the affinity of the potassium channels hEag1 (involved in tumour-cell proliferation) and hErg (important in the cardiac cycle) for several pharmaceutical drugs was compared. Both experimental measurements and molecular modeling were used in order to identify differences in the blocking mechanism of the two channels, which could be exploited in the synthesis of selective blockers. The experimental data pointed out the different role of residue mutations in the blockage of hEag1 and hErg, and the molecular modeling provided a possible explanation based on different binding sites in the intracellular vestibule. Modeling ion channels at the molecular levels relates the functioning of a channel to its atomic structure (Chapters 3-5), and can also be useful to predict the structure of ion channels (Chapter 6-7). In Chapter 6 the structure of the KcsA potassium channel depleted from potassium ions is analyzed by molecular dynamics simulations. Recently, a surprisingly high osmotic permeability of the KcsA channel was experimentally measured. All the available crystallographic structure of KcsA refers to a channel occupied by potassium ions. To conduct water molecules potassium ions must be expelled from KcsA. The structure of the potassium-depleted KcsA channel and the mechanism of water permeation are still unknown, and have been investigated by numerical simulations. Molecular dynamics of KcsA identified a possible atomic structure of the potassium-depleted KcsA channel, and a mechanism for water permeation. The depletion from potassium ions is an extreme situation for potassium channels, unlikely in physiological conditions. However, the simulation of such an extreme condition could help to identify the structural conformations, so the functional states, accessible to potassium ion channels. The last chapter of the thesis deals with the atomic structure of the !- Hemolysin channel. !-Hemolysin is the major determinant of the Staphylococcus Aureus toxicity, and is also the prototype channel for a possible usage in technological applications. The atomic structure of !- Hemolysin was revealed by X-Ray crystallography, but several experimental evidences suggest the presence of an alternative atomic structure. This alternative structure was predicted, combining experimental measurements of single channel currents and numerical simulations. This thesis is organized in two parts, in the first part an overview on ion channels and on the numerical methods adopted throughout the thesis is provided, while the second part describes the research projects tackled in the course of the PhD programme. The aim of the research activity was to relate the functional characteristics of ion channels to their atomic structure. In presenting the different research projects, the role of numerical simulations to analyze the structure-function relation in ion channels is highlighted.

Caratterizzazione di membrane da nanofiltrazione

Nanofiltration (NF) is a pressure-driven membrane process, intermediate between reverse osmosis and ultrafiltration. Commercially available polymeric membranes have been used in a wide range of applications, such as drinking, process industry and waste water treatment. For all the applications requiring high stability and harsh washing procedures inorganic membranes are preferred due to their high chemical inertia. Typically, γ – Al2O3 as well as TiO2 and ZrO2 selective layers are used; the latter show higher chemical stability in a wide range of pH and temperatures. In this work the experimental characterization of two different type of membrane has been performed in order to investigate permeation properties, separation performance and efficiency with aqueous solutions containing strong inorganic electrolytes. The influence of salt concentration and feed pH as well as the role of concentration polarization and electrolyte type on the membrane behavior are investigated. Experimentation was performed testing a multi–layer structured NF membrane in α-Al2O3, TiO2 and ZrO2, and a polymeric membrane, in polyamide supported on polysulfone, with binary aqueous solutions containing NaCl, Na2SO4 or CaCl2; the effect of salt composition and pH in the feed side was studied both on flux and salt rejection. All the NF experimental data available for the two membranes were used to evaluate the volumetric membrane charge (X) corresponding to each operative conditions investigated, through the Donnan Steric Pore Model and Dielectric Exclusion (DSPM&DE). The results obtained allow to understand which are the main phenomena at the basis of the different behaviors observed.

Caratterizzazione di membrane inorganiche per la separazione di idrogeno da gas di reforming

The work of this thesis has been focused on the characterisation of inorganic membranes for the hydrogen purification from steam reforming gas. Composite membranes based on porous inorganic supports coated with palladium silver alloys and ceramic membranes have been analysed. A brief resume of theoretical laws governing transport of gases through dense and porous inorganic membranes and an overview on different methods to prepare inorganic membranes has been also reported. A description of the experimental apparatus used for the characterisation of gas permeability properties has been reported. The device used permits to evaluate transport properties in a wide range of temperatures (till 500°C) and pressures (till 15 bar). Data obtained from experimental campaigns reveal a good agreement with Sievert law for hydrogen transport through dense palladium based membranes while different transport mechanisms, such as Knudsen diffusion and Hagen-Poiseuille flow, have been observed for porous membranes and for palladium silver alloy ones with pinholes in the metal layer. Mixtures permeation experiments reveal also concentration polarisation phenomena and hydrogen permeability reduction due to carbon monoxide adsorption on metal surface.