Kinetic studies of alginate gelation

Comunicação oral selecionada

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ2235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC50 on ATP concentration that allows prediction of the IC50 at different ATP concentrations in enzyme and cellular assays. Comparison of the drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K/PTEN/AKT/mTOR1 network in order to understand mechanisms of drug sensitivity and resistance in different cancer cell lines. We suggest that using these models in systems biology investigation of the PI3K/AKT/mTOR1 signalling in cancer cells can bridge the gap between direct drug target action and the therapeutic response to these drugs and their combinations.

Kinetic and mechanistic studies on unconventional antioxidant activities of natural compounds

In chapter one, the autoxidation kinetics of natural oil substrates, including, triglyceric sunflower oil, olive oil, terpenic squalene, and p-cymene were calibrated through differential oximetry methods. Calibration allows their use as reference oxidizable substrates for further studies, e.g. for quantitative testing of antioxidants under biomimetic settings. Several essential oils samples, of different botanical species or different productions of same species were studied for their antioxidant activity in inhibited autoxidation kinetics. Their antioxidant activities were matched with their composition analyzed by GC-MS. In chapter two, the molecular mechanism of the synergy between the common phenolic antioxidants such as tocopherol and catechols with widespread essential component gamma-terpinene was studied through lipid oxidation kinetics. Wherein, gamma-terpinene was able to disclose the key intermediacy HOO·, which acted as a reducing agent regenerating the phenolic antioxidant. This counterintuitive role of HOO· radicals was further investigated in detail and allowed to rationalize for the first time the purported antioxidant behavior of PDA melanin nanoparticles. It will also open to a deeper understanding of the redox biology of quinones. Regarding melanin, its role is broadly important in living organisms and its control, including its inhibition, is of great importance with several relevant applications ranging from food preservation to control of human skin pigmentation. In chapter three, an oximetry method combined with the traditional UV-Vis spectroscopy was developed to study the tyrosinase inhibition kinetics, which allowed identifying Glabridin (from G. glabra, L.), as one of the most effective natural tyrosinase inhibitors.

Kinetic and mechanistic studies on peroxidation and stabilization of lipids with industrial and biological relevance

Lipid peroxidation is a complex mechanism that causes the degradation of lipid material of both industrial and biological significance. During processing, it is known that thermal stress produces oxidation and polymerization of oils. Additionally, biological lipids with both structural and bioactive roles are prone to peroxidation, which can have pathogenic effects including cancer and long-term degenerative disorders. To create innovative strategies to slow down the deterioration of lipids, it is crucial to improve our understanding of oxidation reactions and kinetics. To this purpose, Chapter II of this thesis focuses on the kinetic study of the oxidation reactions that take place during the thermal processing of bio-oils for industrial application. Through a new method it was possible to evaluate the kinetic parameters of oxidation of various lipid materials. This allowed us to distinguish between the different lipid materials based on their intrinsic properties. The effect of 18 antioxidants from the major families of natural and synthetic phenols were studied using the same methodology in order to acquire crucial data for enhancing the antioxidant activity of phenols based on structure-activity at high temperatures. Finally, it has been described how the antioxidant activity of α-tocopherol, revealed to be scarce in our conditions, can be improved in the presence of gamma-terpinene, through a synergistic action. Chapter III describes the synthesis and study of the antioxidant activity of polydopamine nanoparticles, in order to clarify the unclear mechanism of action of this material. Finally, in Chapter IV it was reported how the gamma-terpinene strongly inhibits the peroxidation of unsaturated lipids in heterogeneous model systems (micelles and liposomes) by forming hydroperoxyl radicals which diffuse outside the lipid nucleus, blocking the propagation of the chain radical. Furthermore, gamma-terpinene shows a very potent protective activity against ferroptosis being effective in the nanomolar range in the human neuroblastoma cell model.

Kinetic modeling for the synthesis of ethyl levulinate

This work is focused on studying the kinetics of esterification of levulinic acid in an isothermal batch reactor using ethanol as a reactant and as a protic polar solvent at the same time and in the presence of an acid catalyst (sulfuric acid). The choice of solvent is important as it affects the kinetics and thermodynamics of the reaction system moreover, the knowledge of the reaction kinetics plays an important role in the design of the process. This work is divided into two stages; The first stage is the experimental part in which the experimental matrix was developed by changing the process variables one at a time (temperature, molar ratio between reactants, and catalyst concentration) in order to study their influence on the kinetics; the second stage is using the obtained data from the experiments to build the modeling part in order to estimate the thermodynamics parameters.

Towards the kinetic resolution of mechanically planar chiral rotaxanes

In this work, we reported the synthesis and characterization of two [2]rotaxanes endowed with a central ammonium group and two triazolium recognition stations on either side, acting as complexation sites for a dibenzo-24-crown-8 ether macrocycle. These mechanically interlocked architectures were obtained through the interlocking of a functionalized achiral macrocycle with Cs symmetry (where the symmetry element is a mirror plane corresponding to plane of the ring) and a C∞v symmetric axle (where a mirror plane and a C∞ principal axis are aligned along the axle length). We took advantage of the reversible acid/base triggered molecular shuttling of the ring between two lateral triazolium units to switch the rotaxanes between prochiral and mechanically planar chiral forms, which exists as two rapidly-interconverting co-conformers. We exploited the reactivity of the central amino group to attach an optically pure chiral substituent, with the goal of demonstrating the enantiomeric nature of the co-conformers and to obtain a non-zero diastereomeric excess in the resulting diastereomeric products through a dynamic kinetic resolution. To this end, two enantiopure reagents were chosen that could perform clean and fast reaction with amines: a sulfonyl chloride and an acyl chloride. Only the acyl chloride successfully produced an amide in high yield with the deprotonated rotaxane. The group added to the central amine station acted as a stopper against the shuttling of the macrocycle along the axis, thus preventing the fast interconversion of the two mechanically planar enantiomers. We analysed the results through static and dynamic NMR spectroscopic techniques by varying temperature and solvent used. Indeed, the presence of diastereomers was recorded alongside the configurational isomers resulting from the slow rotation of the CN-CO bond of the amide moiety, thus paving the way for a dynamic kinetic resolution.

Effects Of High Pressure Homogenization On The Activity, Stability, Kinetics And Three-dimensional Conformation Of A Glucose Oxidase Produced By Aspergillus Niger.

High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5-6.0 and a remarkable activity increase (30-300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.

Improved Selective Cholesterol Adsorption By Molecularly Imprinted Poly(methacrylic Acid)/silica (pmaa-sio₂) Hybrid Material Synthesized With Different Molar Ratios.

The present paper describes the synthesis of molecularly imprinted polymer - poly(methacrylic acid)/silica and reports its performance feasibility with desired adsorption capacity and selectivity for cholesterol extraction. Two imprinted hybrid materials were synthesized at different methacrylic acid (MAA)/tetraethoxysilane (TEOS) molar ratios (6:1 and 1:5) and characterized by FT-IR, TGA, SEM and textural data. Cholesterol adsorption on hybrid materials took place preferably in apolar solvent medium, especially in chloroform. From the kinetic data, the equilibrium time was reached quickly, being 12 and 20 min for the polymers synthesized at MAA/TEOS molar ratio of 6:1 and 1:5, respectively. The pseudo-second-order model provided the best fit for cholesterol adsorption on polymers, confirming the chemical nature of the adsorption process, while the dual-site Langmuir-Freundlich equation presented the best fit to the experimental data, suggesting the existence of two kinds of adsorption sites on both polymers. The maximum adsorption capacities obtained for the polymers synthesized at MAA/TEOS molar ratios of 6:1 and 1:5 were found to be 214.8 and 166.4 mg g(-1), respectively. The results from isotherm data also indicated higher adsorption capacity for both imprinted polymers regarding to corresponding non-imprinted polymers. Nevertheless, taking into account the retention parameters and selectivity of cholesterol in the presence of structurally analogue compounds (5-α-cholestane and 7-dehydrocholesterol), it was observed that the polymer synthesized at the MAA/TEOS molar ratio of 6:1 was much more selective for cholesterol than the one prepared at the ratio of 1:5, thus suggesting that selective binding sites ascribed to the carboxyl group from MAA play a central role in the imprinting effect created on MIP.

Electrochemical Oxidation Of Landfill Leachate In A Flow Reactor: Optimization Using Response Surface Methodology.

Response surface methodology based on Box-Behnken (BBD) design was successfully applied to the optimization in the operating conditions of the electrochemical oxidation of sanitary landfill leachate aimed for making this method feasible for scale up. Landfill leachate was treated in continuous batch-recirculation system, where a dimensional stable anode (DSA(©)) coated with Ti/TiO2 and RuO2 film oxide were used. The effects of three variables, current density (milliampere per square centimeter), time of treatment (minutes), and supporting electrolyte dosage (moles per liter) upon the total organic carbon removal were evaluated. Optimized conditions were obtained for the highest desirability at 244.11 mA/cm(2), 41.78 min, and 0.07 mol/L of NaCl and 242.84 mA/cm(2), 37.07 min, and 0.07 mol/L of Na2SO4. Under the optimal conditions, 54.99 % of chemical oxygen demand (COD) and 71.07 ammonia nitrogen (NH3-N) removal was achieved with NaCl and 45.50 of COD and 62.13 NH3-N with Na2SO4. A new kinetic model predicted obtained from the relation between BBD and the kinetic model was suggested.

Removal Of Glyphosate Herbicide From Water Using Biopolymer Membranes.

Enormous amounts of pesticides are manufactured and used worldwide, some of which reach soils and aquatic systems. Glyphosate is a non-selective herbicide that is effective against all types of weeds and has been used for many years. It can therefore be found as a contaminant in water, and procedures are required for its removal. This work investigates the use of biopolymeric membranes prepared with chitosan (CS), alginate (AG), and a chitosan/alginate combination (CS/AG) for the adsorption of glyphosate present in water samples. The adsorption of glyphosate by the different membranes was investigated using the pseudo-first order and pseudo-second order kinetic models, as well as the Langmuir and Freundlich isotherm models. The membranes were characterized regarding membrane solubility, swelling, mechanical, chemical and morphological properties. The results of kinetics experiments showed that adsorption equilibrium was reached within 4 h and that the CS membrane presented the best adsorption (10.88 mg of glyphosate/g of membrane), followed by the CS/AG bilayer (8.70 mg of glyphosate/g of membrane). The AG membrane did not show any adsorption capacity for this herbicide. The pseudo-second order model provided good fits to the glyphosate adsorption data on CS and CS/AG membranes, with high correlation coefficient values. Glyphosate adsorption by the membranes could be fitted by the Freundlich isotherm model. There was a high affinity between glyphosate and the CS membrane and moderate affinity in the case of the CS/AG membrane. Physico-chemical characterization of the membranes showed low values of solubility in water, indicating that the membranes are stable and not soluble in water. The SEM and AFM analysis showed evidence of the presence of glyphosate on CS membranes and on chitosan face on CS/AG membranes. The results showed that the glyphosate herbicide can be adsorbed by chitosan membranes and the proposed membrane-based methodology was successfully used to treat a water sample contaminated with glyphosate. Biopolymer membranes therefore potentially offer a versatile method to eliminate agricultural chemicals from water supplies.

Breaking Oil-in-water Emulsions Stabilized By Yeast.

Several biotechnological processes can show an undesirable formation of emulsions making difficult phase separation and product recovery. The breakup of oil-in-water emulsions stabilized by yeast was studied using different physical and chemical methods. These emulsions were composed by deionized water, hexadecane and commercial yeast (Saccharomyces cerevisiae). The stability of the emulsions was evaluated varying the yeast concentration from 7.47 to 22.11% (w/w) and the phases obtained after gravity separation were evaluated on chemical composition, droplet size distribution, rheological behavior and optical microscopy. The cream phase showed kinetic stability attributed to mechanisms as electrostatic repulsion between the droplets, a possible Pickering-type stabilization and the viscoelastic properties of the concentrated emulsion. Oil recovery from cream phase was performed using gravity separation, centrifugation, heating and addition of demulsifier agents (alcohols and magnetic nanoparticles). Long centrifugation time and high centrifugal forces (2h/150,000×g) were necessary to obtain a complete oil recovery. The heat treatment (60°C) was not enough to promote a satisfactory oil separation. Addition of alcohols followed by centrifugation enhanced oil recovery: butanol addition allowed almost complete phase separation of the emulsion while ethanol addition resulted in 84% of oil recovery. Implementation of this method, however, would require additional steps for solvent separation. Addition of charged magnetic nanoparticles was effective by interacting electrostatically with the interface, resulting in emulsion destabilization under a magnetic field. This method reached almost 96% of oil recovery and it was potentially advantageous since no additional steps might be necessary for further purifying the recovered oil.

Characterisation Of The Membrane Transport Of Pilocarpine In Cell Suspension Cultures Of Pilocarpus Microphyllus.

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.

Avaliação da biodegradabilidade anaeróbia de resíduos da bovinocultura e da suinocultura

The biodegradability of animal wastes production was evaluated through a simplified methodology that allowed the verification of the applicability of anaerobic processes. The experiments were performed in bath reactors, with granular sludge of three origins: UASB reactor treating dairy effluent, UASB reactor treating swine effluent and UASB reactor treating effluent of slaughterhouse of poultry. The experiments (1) - dairy effluent and poultry slaughterhouse non-adapted sludge; (2) -swine effluent and poultry slaughterhouse non-adapted sludge; (3) - dairy effluent and poultry slaughterhouse adapted sludge; (4) - swine effluent and poultry slaughterhouse adapted sludge; (5) - dairy effluent and dairy sludge, and (6) - swine effluent and swine sludge were performed in Incubator Shaker, at a temperature of 35 °C, under agitation at a 150 rpm, for 5 minutes, every 1 hour. A substrat:biomass relationship of 0.5 was used. Kinetic models of Monod, Zero Order, First and Second Order were tested and it was verified that the First Order model provided the best adjustment. The apparent First Order kinetic parameter (k1) was estimated for the experiments 1; 2; 3; 4; 5, and 6, as 2.51 x 10-2; 2.49 x 10-2; 1.90 x 10-2; 3.09 x 10-2; 2.54 x 10-2; 4.09 x 10-2 h-1, respectively.

Respostas agudas de parâmetros biomecânicos à utilização de diferentes tamanhos de palmar no nado crawl

Universidade Estadual de Campinas . Faculdade de Educação Física

Proposta e verificação da validade de testes de limiar anaerobico para natação do nado crawl

Universidade Estadual de Campinas . Faculdade de Educação Física