Lower Extremities Magnetic Resonance Angiography With Blood Pressure Cuff Compression: Quantitative Dynamic Analysis

Purpose: To quantitatively evaluate changes induced by the application of a femoral blood-pressure cuff (BPC) on run-off magnetic resonance angiography (MRA). which is a method generally previously proposed to reduce venous contamination in the leg. Materials and Methods: This study was Health Insurance Portability and Accountability Act (HIPAA)- and Institutional Review Board (IRB)-compliant, We used time-resolved gradient-echo gadolinium (Gd)-enhanced MRA to measure BPC effects on arterial, venous, and soft-tissue enhancement. Seven healthy volunteers (six men) were studied with the BPC applied at the mid-femoral level unilaterally using a 1.5T MR system after intravenous injection of Gd-BOPTA. Different statistical tools were used such as the Wilcoxon signed rank test and a cubic smoothing spline fit. Results: We found that BPC application induces delayed venous filling (as previously described), but also induces significant decreases in arterial inflow, arterial enhancement, vascular-soft tissue contrast, and delayed peak enhancement (which have not been previously measured). Conclusion: The potential benefits from using a BPC for run-off MRA must be balanced against the potential pitfalls, elucidated by our findings.

Influence of matrix composition on polymerization stress development of experimental composites

Objective. Stress development at the tooth/restoration interface is one of the most important reasons for failure of adhesive restorations. The aim of this study was to evaluate the influence of BisGMA/TEGDMA (B/T) and UDMA/TEGDMA (U/T) ratios on polymerization stress (PS) and on the variables related to its development: degree of conversion (DC), polymerization maximum rate (Rp(max)), volumetric shrinkage (VS), elastic modulus (E), stress relaxation (SR) and viscosity of experimental composites. Method. Composites were formulated containing B/T or U/T in mol% ratios of 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3 and 8: 2, and 15 wt% of fumed silica. PS was determined with a universal testing machine. VS was measured with a linometer. E and SR were obtained in three-point bending. DC and Rp(max) were determined by real time NIR spectroscopy and viscosity was measured in viscometer. Data were submitted to one-way ANOVA, Tukey test (alpha = 0.05%) and regression analyses. Results. PS, VS, E and DC decreased and viscosity and Rp(max) increased with base monomer content in both series. PS showed strong correlation with VS, DC and viscosity. PS, VS and DC were higher and viscosity was lower for UDMA-based materials. Significance. Reduced viscosity, kinetics parameters and molecular characteristics led UDMA-based composites to elevated conversion and relatively lower PS at lower TEGDMA contents, compared to B/T composites. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Chemical reaction network of flavylium ions in heterogeneous media

Dissertação apresentada para a obtenção do Grau de Doutor em Química, especialidade em Química-Física, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Color and Luminescence stability of selected dental materials in vitro

To study luminescence, reflectance, and color stability of dental composites and ceramics. Materials and Methods: IPS e.max, IPS Classic, Gradia, and Sinfony materials were tested, both unpolished (as-cast) and polished specimens. Coffee, tea, red wine, and distilled water (control) were used as staining drinks. Disk-shaped specimens were soaked in the staining drinks for up to 5 days. Color was measured by a colorimeter. Fluorescence was recorded using a spectrofluorometer, in the front-face geometry. Time-resolved fluorescence spectra were recorded using a laser nanosecond spectrofluorometer. Results: The exposure of the examined dental materials to staining drinks caused changes in color of the composites and ceramics, with the polished specimens exhibiting significantly lower color changes as compared to unpolished specimens. Composites exhibited lower color stability as compared to ceramic materials. Water also caused perceptible color changes in most materials. The materials tested demonstrated significantly different initial luminescence intensities. Upon exposure to staining drinks, luminescence became weaker by up to 40%, dependent on the drink and the material. Time-resolved luminescence spectra exhibited some red shift of the emission band at longer times, with the lifetimes in the range of tens of nanoseconds. Conclusions: Unpolished specimens with a more developed surface have lower color stability. Specimens stored in water develop some changes in their visual appearance. The presently proposed methods are effective in evaluating the luminescence of dental materials. Luminescence needs to be tested in addition to color, as the two characteristics are uncorrelated. It is important to further improve the color and luminescence stability of dental materials.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

Aminobenzocoumarinylmehtyl esters as photoactive precursors for the release of butyric acid

The evaluation of the photorelease of a carboxylic acid drug, using butyric acid as a representative model, was carried out by using 7-amino-4-chloromethyl-2-oxo-2Hnaphtho[1,2-b] pyran, an aminobenzocoumarin, and its mono- and di-methylated or ethylated derivatives. This study was intended to improve the release of butyric acid from benzocoumarins by the addition of an amino group to the heterocycle by applying the knowledge of second-generation coumarinylmethyl-based photoremovable protecting groups. Photolysis studies were performed on the resultant ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80:20 solutions as solvent. The data obtained showed that these new fluorescent aminobenzocoumarins are superior to all the previously tested benzocoumarins with the same or different ring fusions. As well as the photolysis, the photophysics of the compounds were characterised by both steady state and time-resolved methods.

Photoactivation of butyric acid from 6-aminobenzocoumarin cages

A new benzocoumarin bearing an amino group is proposed as a photocleavable protecting group for carboxylic acids. The novel heterocycle, 6-amino-4-chloromethyl-2-oxo-2H-naphtho[1,2-b]pyran was used in the preparation of ester conjugates of butyric acid, and of the corresponding mono- and di-methylated or ethylated derivatives. The photolability of the ester conjugates was studied by irradiation at selected wavelengths in methanol/HEPES buffer (80:20) solutions, and the release of butyric acid was followed with HPLC/UV and 1H NMR monitoring. Release of the carboxylic acid was faster for the monoalkylated derivatives (approximately within 20 min), at the longer wavelengths of irradiation (350 and 419 nm). The photophysics of the heterocyclic conjugates was also evaluated by both steady state and time-resolved methods.

Effect of surface plasmon resonance in TiO2/Au thin films on the fluorescence of self-assembled CdTe QDs structure

The exceptional properties of localised surface plasmons (LSPs), such as local field enhancement and confinement effects, resonant behavior, make them ideal candidates to control the emission of luminescent nanoparticles. In the present work, we investigated the LSP effect on the steady-state and time-resolved emission properties of quantum dots (QDs) by organizing the dots into self-assembled dendrite structures deposited on plasmonic nanostructures. Self-assembled structures consisting of water-soluble CdTe mono-size QDs, were developed on the surface of co-sputtered TiO2 thin films doped with Au nanoparticles (NPs) annealed at different temperatures. Their steady-state fluorescence properties were probed by scanning the spatially resolved emission spectra and the energy transfer processes were investigated by the fluorescence lifetime imaging (FLIM) microscopy. Our results indicate that a resonant coupling between excitons confined in QDs and LSPs in Au NPs located beneath the self-assembled structure indeed takes place and results in (i) a shift of the ground state luminescence towards higher energies and onset of emission from excited states in QDs, and (ii) a decrease of the ground state exciton lifetime (fluorescence quenching).

Synaptobrevin N-terminally bound to syntaxin-SNAP-25 defines the primed vesicle state in regulated exocytosis.

Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)-dependent exocytosis pathway at an intermediate "cocked" state, from which fusion can be triggered by Ca(2+). It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin-SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca(2+)-triggered C-terminal assembly and membrane fusion.

Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes.

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions. The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification. The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

Analysis of GPCR properties of Frizzled receptors and establishment of the "in vitro" platform for screening of Frizzled agonists/antagonists as potential therapeutic agents

Morphogens of the Wnt protein family are the secreted lipoglycoprotein ligands which initiate several pathways heavily involved in the coordination of various developmental stages of organisms in the majority of animal species. Deregulation of these pathways in the adult leads to formation and sustaining of multiple types of cancer. The latter notion is reinforced by the fact that the very discovery of the first Wnt ligand was due to its role as the causative factor of carcinogenic transformation (Nusse and Varmus, 1982). Nowadays our knowledge on Wnt signaling has "moved with the times" and these pathways were identified to be often crucial for tumor formation, its interactions with the microenvironment, and promotion of the metastases (Huang and Du, 2008; Zerlin et al., 2008; Jessen, 2009). Thus the relevance of the pathway as the target for drug development has further increased in the light of modern paradigms of the complex cancer treatments which target also spreading and growth- promoting factors of tumors by specific and highly efficient substances (Pavet et al., 2010). Presently the field of the Wnt-targeting drug research is almost solely dominated by assays based on transcriptional activation induced by the signaling. This approach resulted in development of a number of promising substances (Lee et al., 2011). Despite its effectiveness, the method nevertheless suffers from several drawbacks. Among the major ones is the fact that this approach is prone to identify compounds targeting rather downstream effectors of the pathway, which are indiscriminately used by all the subtypes of the Wnt signaling. Additionally, proteins which are involved in several signaling cascades and not just the Wnt pathway turn out as targets of the new compounds. These issues increase risks of side effects due to off-target interactions and blockade of the pathway in healthy cells. In the present work we put forward a novel biochemical approach for drug development on the Wnt pathway. It targets Frizzleds (Fzs) - a family of 7-transmbembrane proteins which serve as receptors for Wnt ligands. They offer unique properties for the development of highly specific and effective drugs as they control all branches of the Wnt signaling. Recent advances in the understanding of the roles of heterotrimeric G proteins downstream from Fzs (Katanaev et al., 2005; Liu et al., 2005; Jernigan et al., 2010) suggest application of enzymatic properties of these effectors to monitor the receptor-mediated events. We have applied this knowledge in practice and established a specific and efficient method based on utilization of a novel high-throughput format of the GTP-binding assay to follow the activation of Fzs. This type of assay is a robust and well-established technology for the research and screenings on the GPCRs (Harrison and Traynor, 2003). The conventional method of detection involves the radioactively labeled non-hydrolysable GTP analog [35S]GTPyS. Its application in the large-scale screenings is however problematic which promoted development of the novel non-radioactive GTP analog GTP-Eu. The new molecule employs phenomenon of the time-resolved fluorescence to provide sensitivity comparable to the conventional radioactive substance. Initially GTP-Eu was tested only in one of many possible types of GTP-binding assays (Frang et al., 2003). In the present work we expand these limits by demonstrating the general comparability of the novel label with the radioactive method in various types of assays. We provide a biochemical characterization of GTP-Eu interactions with heterotrimeric and small GTPases and a comparative analysis of the behavior of the new label in the assays involving heterotrimeric G protein effectors. These developments in the GTP-binding assay were then applied to monitor G protein activation by the Fz receptors. The data obtained in mammalian cultured cell lines provides for the first time an unambiguous biochemical proof for direct coupling of Fzs with G proteins. The specificity of this interaction has been confirmed by the experiments with the antagonists of Fz and by the pertussis toxin-mediated deactivation. Additionally we have identified the specificity of Wnt3a towards several members of the Fz family and analyzed the properties of human Fz-1 which was found to be the receptor coupled to the Gi/o family of G proteins. Another process playing significant role in the functioning of every GPCR is endocytosis. This phenomenon can also be employed for drug screenings on GPCRs (Bickle, 2010). In the present work we have demonstrated that Drosophila Fz receptors are involved in an unusual for many GPCRs manifestation of the receptor-mediated internalization. Through combination of biochemical approaches and studies on Drosophila as the model organism we have shown that direct interactions of the Fzs and the α-subunit of the heterotrimeric G protein Go with the small GTPase Rab5 regulate internalization of the receptor in early endosomes. We provide data uncovering the decisive role of this self-promoted endocytosis in formation of a proper signaling output in the canonical as well as planar cell polarity (PCP) pathways regulated by Fz. The results of this work thus establish a platform for the high-throughput screening to identify substances active in the cancer-related Wnt pathways. This methodology has been adjusted and applied to provide the important insights in Fz functioning and will be instrumental for further investigations on the Wnt-mediated pathways.

Polarization strategies to improve the emission of a Si-based light source emitting at 1.55 um

We present a electroluminescence (EL) study of the Si-rich silicon oxide (SRSO) LEDs with and without Er3+ ions under different polarization schemes: direct current (DC) and pulsed voltage (PV). The power efficiency of the devices and their main optical limitations are presented. We show that under PV polarization scheme, the devices achieve one order of magnitude superior performance in comparison with DC. Time-resolved measurements have shown that this enhancement is met only for active layers in which annealing temperature is high enough (>1000 ◦C) for silicon nanocrystal (Si-nc) formation. Modeling of the system with rate equations has been done and excitation cross-sections for both Si-nc and Er3+ ions have been extracted.

Film-free laser forward printing of transparent and weakly absorbing liquids

A laser-based technique for printing transparent and weakly absorbing liquids is developed. Its principle of operation relies in the tight focusing of short laser pulses inside the liquid and close to its free surface, in such a way that the laser radiation is absorbed in a tiny volume around the beam waist, with practically no absorption in any other location along the beam path. If the absorbed energy overcomes the optical breakdown threshold, a cavitation bubble is generated, and its expansion results in the propulsion of a small fraction of liquid which can be collected on a substrate, leading to the printing of a microdroplet for each laser pulse. The technique does not require the preparation of the liquid in thin film form, and its forward mode of operation imposes no restriction concerning the optical properties of the substrate. These characteristics make it well suited for printing a wide variety of materials of interest in diverse applications. We demonstrate that the film-free laser forward printing technique is capable of printing microdroplets with good resolution, reproducibility and control, and analyze the influence of the main process parameter, laser pulse energy. The mechanisms of liquid printing are also investigated: time-resolved imaging provides a clear picture of the dynamics of liquid transfer which allows understanding the main features observed in the printed droplets.

Detecting the oxidative reactivity of nanoparticles: a new protocol for reducing artifacts

Understanding the oxidative reactivity of nanoparticles (NPs; <100 nm) could substantially contribute to explaining their toxicity. We attempted to refine the use of 2′7-dichlorodihydrofluorescein (DCFH) to characterize NP generation of reactive oxygen species (ROS). Several fluorescent probes have been applied to testing oxidative reactivity, but despite DCFH being one of the most popular for the detection of ROS, when it has been applied to NPs there have been an unexplainably wide variability in results. Without a uniform methodology, validating even robust results is impossible. This study, therefore, identified sources of conflicting results and investigated ways of reducing occurrence of artificial results. Existing techniques were tested and combined (using their most desirable features) to form a more reliable method for the measurement of NP reactivity in aqueous dispersions. We also investigated suitable sample ranges necessary to determine generation of ROS. Specifically, ultrafiltration and time-resolved scan absorbance spectra were used to study possible optical interference when using high sample concentrations. Robust results were achieved at a 5 µM DCFH working solution with 0.5 unit/mL horseradish peroxidase (HRP) dissolved in ethanol. Sonication in DCFH-HRP working solution provided more stable data with a relatively clean background. Optimal particle concentration depends on the type of NP and in general was in the µg/mL range. Major reasons for previously reported conflicting results due to interference were different experimental approaches and NP sample concentrations. The protocol presented here could form the basis of a standardized method for applying DCFH to detect generation of ROS by NPs.