Intracellular membrane trafficking in Osteoclasts The role of direct Rab protein – Rac 1 interactions in the targeting of intracellular vesicles

Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.

Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hall- mark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM (the calcein acetoxymethyl-ester) on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phos-phate pathway was significantly altered. To elucidate whe-ther these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

Efeitos dos parâmetros operacionais na fotodegradação do azo corante direct red 23 na interface dióxido de titânio/água

The decolorization and degradation of direct red 23 azo dye have been investigated in aqueous suspension of titanium dioxide under artificial irradiation. The effects of some operational parameters such as azo dye concentration, catalyst loading, and solution pH were investigated at 30.0 ºC and optimized values were obtained. The first-order kinetic model was used to discuss the results. The UV-Vis spectra changes showed that the azo dye sample, collected after 6 h irradiation, was 98% decolorized while the residual total carbon was 97.9% degraded, indicating simultaneous photodecolorization and degradation.

An easy and direct method for the synthesis of 1,2,4-triazole derivatives through carboxylic acids and hydrazinophthalazine

We have developed an easy method for the synthesis of thirteen compounds derived from 1,2,4-triazoles through a carboxylic acid and hydrazinophthalazine reaction, with a 75-85% yield mediated by the use of agents such as 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride and 1-hydroxybenzotriazole. The operational simplicity of this method and the good yield of products make it valuable for the synthesis of new compounds with pharmacological activity.

Effects of Surface Properties and Wood Modification into Direct Printing on Wood

Building industry is a high volume branch which could provide prominent markets for wood based interior decoration solutions. Competition in interior decoration markets requires versatility in appearance. Versatility in wood appearance and added value could be achieved by printing grain patterns of different species or images directly onto wood. The problem when planning wood printing’s implementing into durable applications is basically how to transfer a high quality image or print sustainably onto wood, which is porous, heterogeneous, dimensionally unstable, non-white and rough. Wood preservation or treating, and modification can provide durability against degradation but also effect to the surface properties of wood which will effect on printability. Optimal adhesion is essential into print quality, as too high ink absorbance can cause spreading and too low ink absorbance cause pale prints. Different printing techniques have different requirements on materials and production. The direct printing on wood means, that intermedias are not used. Printing techniques with flexible printing plates or in fact non-impact techniques provide the best basis for wood printing. Inkjet printing of wood with different mechanical or chemical surface treatments, and wood plastic composite material gave good results that encourage further studies of the subject. Sanding the wood surface anti-parallel to the grain gave the best overall printing quality. Spreading parallel to the grain could not be avoided totally, except in cases where wood was treated hydrophobic so adhesion of the ink was not sufficient. Grain pattern of the underlying wood stays clearly visible in the printed images. Further studies should be made to fine tune the methods that already gave good results. Also effects of moisture content of wood, different inks, and long-term exposure to UV-radiation should be tested.

A comparison of electrodeposited Ti/β-PbO2 and Ti-Pt/β-PbO2 anodes in the electrochemical degradation of the direct yellow 86 dye

The electrochemical performance of electrodeposited Ti/β-PbO2 and Ti-Pt/β-PbO2 anodes was galvanostatically evaluated (batch mode, 50 mA cm-2) to degrade the Direct Yellow 86 dye (100 or 200 mg L-1 in 0.1 mol L-1 Na2SO4 + 1.5 g L-1 NaCl), investigating the effect of pH and temperature. Similar results were obtained for both electrodes and the best conditions for removal of color and chemical oxygen demand are pH 7 and 40 °C, when 90% decolorization is attained by passing a charge of only ~0.13 A h L-1 and total mineralization is achieved with expenditure of ~5 kW h m-3.

Influence of chloride-mediated oxidation on the electrochemical degradation of the direct black 22 dye using boron-doped diamond and β-PbO2 anodes

The Direct Black 22 dye was electrooxidized at 30 mA cm-2 in a flow cell using a BDD or β-PbO2 anode, varying pH (3, 7, 11), temperature (10, 25, 45 °C), and [NaCl] (0 or 1.5 g L-1). In the presence of NaCl, decolorization rates were similar for all conditions investigated, but much higher than predicted through a theoretical model assuming mass-transport control; similar behavior was observed for COD removal (at pH 7, 25 °C), independently of the anode. With no NaCl, COD removals were also higher than predicted with a theoretical model, which suggests the existence of distinct dye degradation pathways.

DIRECT INFUSION ESI-MS APPLIED IN THE DETECTION OF BYPRODUCTS DUE TO REDUCTIVE DEGRADATION OF ACETAMIPRID BY ZERO-VALENT IRON

This study investigated the reductive degradation of acetamiprid (5 mg L-1) in aqueous medium (at pH 2.0) induced by zero-valent iron (50 mg). The process was monitored using high-performance liquid chromatography (HPLC) to determine the degradation rate as a function of reaction time, and direct infusion electrospray ionization mass spectrometry (DI-ESI-MS) to search for (and potentially characterize) any possible byproducts formed during degradation. The results obtained via HPLC showed that after 60 min, the degradation of the substrate reached nearly 100% in an acidic medium, whereas the mineralization rate (as determined by total organic carbon measurements) was as low as 3%. Data obtained by DI-ESI-MS showed that byproducts were formed mainly by insertions of hydrogen atoms into the nitrile, imine, and pyridine ring moieties, in addition to the observation of chlorine substitution by hydrogen replacement (hydrodechlorination) reactions.

Optimization of the power cable for low voltage direct current system

The demand for electricity is constantly growing in contemporary world and, in the same time, quality and reliability requirements are becoming more rigid. In addition, renewable sources of energy have been widely introduced for power generation, and they create specific challenges for the network. Consequently, new solution for distribution system is required, and Low Voltage Direct Current (LVDC) system is the proposed one. This thesis focuses on the investigation of specific cable features for low voltage direct current (LVDC) distribution system. The LVDC system is public ±750 VDC distribution system, which is currently being developed at Lappeen-ranta University of Technology. The aspects, considered in the thesis, are reliable and economic power transmission in distribution networks and possible power line communication in the LVDC cable.

A direct potentiometric titration study of the dissociation of humic acid with selectively blocked functional groups

A direct potentiometric titration method was applied to commercial and soil humic acids in order to determine their carboxyl and phenol group concentrations and apparent and intrinsic pK. In that context, acid-base properties of humic acids are interpreted by selective blocking of carboxylic and phenolic groups by esterification and acetylation. Differences in underivatized and derivatized HA's acid-base properties are ascribed to carboxyl and phenol groups influence on total humic acidity. Potentiometric data were treated with the modified Henderson-Hasselbalch equation. Infra red results, the acidic group contents and the average values of apparent and intrinsic pK for underivatized and derivatized HAs confirmed the selectivity of esterification derivatization method. After blocking of the functional groups, the values of acidic group contents decreased, while the value of apparent pK increased after derivatization. Phenol groups cannot be specifically identified by the acetylation method, due to low selectivity of the acetylation method.

Direct determination of calcium in milk by atomic absorption spectrometry using flow-injection analysis

A flow-injection system with sample and reagent addition by the synchronous merging zones approach for calcium determination in milk by flame AAS is proposed. Main parameters were optimized using a factorial design with central point. The optimum conditions were 2.5% (m/v) for La concentration, 8 mL min-1 for the carrier flow-rate, 20 cm for coiled reactor and 250 ìL for sample volume. Different sample preparation procedures were evaluated such as dilution in water or acid and microwave-assisted decomposition using concentrated or diluted acids. The optimized flow system was applied to determine Ca in eleven commercial milk samples and two standard reference materials diluted in water. Similar calcium levels were encountered comparing the results obtained by the proposed method (dilution in water) with those obtained using microwave-oven digestion. Results obtained in two standard reference materials were in agreement at 95% confidence level with those certified. Recoveries of spiked samples were in the 93% - 116% range. Relative standard deviation (n = 12) was < 5.4% and the sample throughput was 150 measurements per hour, corresponding to a consumption of 250 µL of sample and 6.25 mg La per determination.

Effects of Foreign Direct Investment inflows on growth in Central and Eastern European transition countries

The aim of this master’s thesis is to analyze the effects of Foreign Direct Investments on growth in selected Central and Eastern European transition countries. The theoretical part of this thesis, introduces growth theories and how FDI is covered in those theories. In addition, the results from previous studies, which have studied FDI’s effect on growth, are presented in this master’s thesis. This work introduces also the economical progress during the transition period in selected countries. In the empirical part’s regression model, it will be searched for the direct effect of FDI on growth with panel data collected from nine transition countries.

Role of oxidation-reduction cycle of iron in direct leaching of zinc concentrate

Direct leaching is an alternative to conventional roast-leach-electrowin (RLE) zinc production method. The basic reaction of direct leach method is the oxidation of sphalerite concentrate in acidic liquid by ferric iron. The reaction mechanism and kinetics, mass transfer and current modifications of zinc concentrate direct leaching process are considered. Particular attention is paid to the oxidation-reduction cycle of iron and its role in direct leaching of zinc concentrate, since it can be one of the limiting factors of the leaching process under certain conditions. The oxidation-reduction cycle of iron was experimentally studied with goal of gaining new knowledge for developing the direct leaching of zinc concentrate. In order to obtain this aim, ferrous iron oxidation experiments were carried out. Affect of such parameters as temperature, pressure, sulfuric acid concentration, ferrous iron and copper concentrations was studied. Based on the experimental results, mathematical model of the ferrous iron oxidation rate was developed. According to results obtained during the study, the reaction rate orders for ferrous iron concentration, oxygen concentration and copper concentration are 0.777, 0.652 and 0.0951 respectively. Values predicted by model were in good concordance with the experimental results. The reliability of estimated parameters was evaluated by MCMC analysis which showed good parameters reliability.

Direct and simultaneous spectrophotometric determination of Ni (II) and Co (II) using diethanoldithiocarbamate as complexing agent

A direct spectrophotometric method for simultaneous determination of Co(II) and Ni(II), with diethanoldithiocarbamate (DEDC) as complexing agent, is proposed using the maximum absorption at 360 and 638 nm (Co(II)/DEDC) and 390 nm (Ni/DEDC). Adjusting the best metal/ligand ratio, supporting eletrolite, pH, and time of analysis, linear analytical curves from 1.0 10-6-4.0 10-4 for Co(II) in the presence of Ni 1.0 10-6-1.0 10-4 mol L-1 were observed. No further treatment or calculation processes have been necessary. Recoveries in different mixing ratios were of 99%. Interference of Fe(III), Cu(II), Zn(II) and Cd(II), and anions as NO3-, Cl-, ClO4-, citrate and phosphate has been evaluated. The method was applied to natural waters spiked with the cations.