TNF-α influences the lateral dynamics of TNF receptor I in living cells

In mammalian cells, inflammation is mainly mediated by the binding of tumor necrosis factor alpha to tumor necrosis factor receptor 1. In this study, we investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy. Our single-molecule data indicates the presence of tumor necrosis factor receptor 1 with different mobilities in the plasma membrane, suggesting different molecular organizations. Cholesterol depletion led to a decrease of slow receptor species and a strong increase in the average diffusion coefficient. Moreover, as a consequence of tumor necrosis factor-alpha treatment, the mean diffusion coefficient moderately increased while its distribution narrowed. Based on our observation, we propose a refined mechanism on the structural arrangement and activation of tumor necrosis factor receptor 1 in the plasma membrane.

Caracterização de microorganismos aquáticos por processamento digital de imagens e redes neurais artificiais

A identificação e o monitoramento de microorganismos aquáticos, como bactérias e microalgas, tem sido uma tarefa árdua e morosa. Técnicas convencionais, com uso de microscópios e corantes, são complexas, exigindo um grande esforço por parte dos técnicos e pesquisadores. Uma das maiores dificuldades nos processos convencionais de identificação via microscopia é o elevado número de diferentes espécies e variantes existentes nos ambientes aquáticos, muitas com semelhança de forma e textura. O presente trabalho tem por objetivo o desenvolvimento de uma metodologia para a caracterização e classificação de microorganismos aquáticos (bactérias e microalgas), bem como a determinação de características cinemáticas, através do estudo da mobilidade de microalgas que possuem estruturas que permitem a natação (flagelos). Para caracterização e reconhecimento de padrões as metodologias empregadas foram: o processamento digital de imagens e redes neurais artificiais (RNA). Para a determinação da mobilidade dos microorganismos foram empregadas técnicas de velocimetria por processamento de imagens de partículas em movimento (Particle Tracking Velocimetry - PTV). O trabalho está dividido em duas partes: 1) caracterização e contagem de microalgas e bactérias aquáticas em amostras e 2) medição da velocidade de movimentação das microalgas em lâminas de microscópio. A primeira parte envolve a aquisição e processamento digital de imagens de microalgas, a partir de um microscópio ótico, sua caracterização e determinação da densidade de cada espécie contida em amostras. Por meio de um microscópio epifluorescente, foi possível, ainda, acompanhar o crescimento de bactérias aquáticas e efetuar a sua medição por operadores morfológicos. A segunda parte constitui-se na medição da velocidade de movimentação de microalgas, cujo parâmetro pode ser utilizado como um indicador para se avaliar o efeito de substâncias tóxicas ou fatores de estresse sobre as microalgas. O trabalho em desenvolvimento contribuirá para o projeto "Produção do Camarão Marinho Penaeus Paulensis no Sul do Brasil: Cultivo em estruturas Alternativas" em andamento na Estação Marinha de Aquacultura - EMA e para pesquisas no Laboratório de Ecologia do Fitoplâncton e de Microorganismos Marinhos do Departamento de Oceanografia da FURG. O trabalho propõe a utilização dos níveis de intensidade da imagem em padrão RGB e oito grandezas geométricas como características para reconhecimento de padrões das microalgas O conjunto proposto de características das microalgas, do ponto de vista de grandezas geométricas e da cor (nível de intensidade da imagem e transformadas Fourier e Radon), levou à geração de indicadores que permitiram o reconhecimento de padrões. As redes neurais artificiais desenvolvidas com topologia de rede multinível totalmente conectada, supervisionada, e com algoritmo de retropropagação, atingiram as metas de erro máximo estipuladas entre os neurônios de saída desejados e os obtidos, permitindo a caracterização das microalgas.

Commissioning and performance of the CMS silicon strip tracker with cosmic ray muons

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The CMS experiment at the CERN LHC

The Compact Muon Solenoid (CMS) detector is described. The detector operates at the Large Hadron Collider (LHC) at CERN. It was conceived to study proton-proton (and lead-lead) collisions at a centre-of-mass energy of 14 TeV (5.5 TeV nucleon-nucleon) and at luminosities up to 10(34)cm(-2)s(-1) (10(27)cm(-2)s(-1)). At the core of the CMS detector sits a high-magnetic-field and large-bore superconducting solenoid surrounding an all-silicon pixel and strip tracker, a lead-tungstate scintillating-crystals electromagnetic calorimeter, and a brass-scintillator sampling hadron calorimeter. The iron yoke of the flux-return is instrumented with four stations of muon detectors covering most of the 4 pi solid angle. Forward sampling calorimeters extend the pseudo-rapidity coverage to high values (vertical bar eta vertical bar <= 5) assuring very good hermeticity. The overall dimensions of the CMS detector are a length of 21.6 m, a diameter of 14.6 m and a total weight of 12500 t.

Performance of CMS muon reconstruction in pp collision events at √s = 7TeV

The performance of muon reconstruction, identification, and triggering in CMS has been studied using 40 pb-1 of data collected in pp collisions at s = 7 TeV at the LHC in 2010. A few benchmark sets of selection criteria covering a wide range of physics analysis needs have been examined. For all considered selections, the efficiency to reconstruct and identify a muon with a transverse momentum pT larger than a few GeV/c is above 95% over the whole region of pseudorapidity covered by the CMS muon system, < 2.4, while the probability to misidentify a hadron as a muon is well below 1%. The efficiency to trigger on single muons with pT above a few GeVc is higher than 90% over the full η range, and typically substantially better. The overall momentum scale is measured to a precision of 0.2% with muons from Z decays. The transverse momentum resolution varies from 1% to 6% depending on pseudorapidity for muons with pT below 100GeV/c and, using cosmic rays, it is shown to be better than 10% in the central region up to pT = 1 TeV/c. Observed distributions of all quantities are well reproduced by the Monte Carlo simulation. © 2012 IOP Publishing Ltd and Sissa Medialab srl.

Alignment of the CMS tracker with LHC and cosmic ray data

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hydrogeologic Setting and Ground-Water Flow Simulations of the Eastern High Plains Regional Study Area, Nebraska

The transport of anthropogenic and natural contaminants to public-supply wells was evaluated in a part of the High Plains aquifer near York, Nebraska, as part of the U.S. Geological Survey National Water-Quality Assessment Program. The aquifer in the Eastern High Plains regional study area is composed of Quaternary alluvial deposits typical of the High Plains aquifer in eastern Nebraska and Kansas, is an important water source for agricultural irrigation and public water supply, and is susceptible and vulnerable to contamination. A six-layer, steady-state ground-water flow model of the High Plains aquifer near York, Nebraska, was constructed and calibrated to average conditions for the time period from 1997 to 2001. The calibrated model and advective particle-tracking simulations were used to compute areas contributing recharge and travel times from recharge areas to selected public-supply wells. Model results indicate recharge from agricultural irrigation return flow and precipitation (about 89 percent of inflow) provides most of the ground-water inflow, whereas the majority of ground-water discharge is to pumping wells (about 78 percent of outflow). Particle-tracking results indicate areas contributing recharge to public-supply wells extend northwest because of the natural ground-water gradient from the northwest to the southeast across the study area. Particle-tracking simulations indicate most ground-water travel times from areas contributing recharge range from 20 to more than 100 years but that some ground water, especially that in the lower confined unit, originates at the upgradient model boundary instead of at the water table in the study area and has travel times of thousands of years.

Model for the Dynamics of the Cytoskeleton.

Particle tracking of microbeads attached to the cytoskeleton (CSK) reveals an intermittent dynamic. The mean squared displacement (MSD) is subdiffusive for small Δt and superdiffusive for large Δt, which are associated with periods of traps and periods of jumps respectively. The analysis of the displacements has shown a non-Gaussian behavior, what is indicative of an active motion, classifying the cells as a far from equilibrium material. Using Langevin dynamics, we reconstruct the dynamic of the CSK. The model is based on the bundles of actin filaments that link themself with the bead RGD coating, trapping it in an harmonic potential. We consider a one- dimensional motion of a particle, neglecting inertial effects (over-damped Langevin dynamics). The resultant force is decomposed in friction force, elastic force and random force, which is used as white noise representing the effect due to molecular agitation. These description until now shows a static situation where the bead performed a random walk in an elastic potential. In order to modeling the active remodeling of the CSK, we vary the equilibrium position of the potential. Inserting a motion in the well center, we change the equilibrium position linearly with time with constant velocity. The result found exhibits a MSD versus time ’tau’ with three regimes. The first regime is when ‘tau’ < ‘tau IND 0’, where ‘tau IND 0’ is the relaxation time, representing the thermal motion. At this regime the particle can diffuse freely. The second regime is a plateau, ‘tau IND 0’ < ‘tau’ < ‘tau IND 1’, representing the particle caged in the potential. Here, ‘tau IND 1’ is a characteristic time that limit the confinement period. And the third regime, ‘tau’ > ‘tau IND 1’, is when the particles are in the superdiffusive behavior. This is where most of the experiments are performed, under 20 frames per second (FPS), thus there is no experimental evidence that support the first regime. We are currently performing experiments with high frequency, up to 100 FPS, attempting to visualize this diffusive behavior. Beside the first regime, our simple model can reproduce MSD curves similar to what has been found experimentally, which can be helpful to understanding CSK structure and properties.

The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrPC). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20–50, 100–200, and 300–400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrPC. STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrPC-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1–PrPC signaling

Photo-induced reversible assembly of colloidal polymer nanoparticles

Polymer nanoparticles functionalized on the surface with photo-responsive labels were synthesized. In a first synthetic step, polystyrene was copolymerized with the cross-linker divinylbenzene and poly(ethylene glycol) acrylate in a miniemulsion, to produce nano-sized spheres (~ 60 nm radius) with terminal hydroxyl groups, which were functionalized in a subsequent synthetic step with photo-responsive labels. For this purpose, two photo-active molecular structures were separately used: anthracene, which is well known to form covalently bonded dimers upon photo-excitation; and pyrene, which only forms short lived excited state dimers (excimers). Acid derivatives of these labels (9-anthracene carboxylic acid and 1-pyrene butyric acid) were bonded to the hydroxyl terminal groups of the nanoparticles through an esterification reaction, via the intermediate formation of the corresponding acid chloride.rnThe obtained labeled nanoparticles appeared to be highly hydrophobic structures. They formed lyophobic suspensions in water, which after analysis by dynamic light scattering (DLS) and ultramicroscopic particle tracking, appeared to equilibrate as a collection of singly dispersed nanoparticles, together with a few nanoparticle aggregates. The relative amount of aggregates decreased with increasing amounts of the surfactant sodium dodecyl sulfate (SDS), thus confirming that aggregation is an equilibrated state resulting from lyophobicity. The formation of such aggregates was corroborated using scanning electron microscopy (SEM). The photo-irradiation of the lyophobic aqueous suspensions of anthracene labeled nanoparticles (An-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. The obtained state of aggregation could be reverted by sonication. The possibility to re-aggregate the system in subsequent photo-excitation and sonication cycles was established. Likewise, the photo-irradiation of lyophobic aqueous suspensions of pyrene-labeled nanoparticles (Py-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. These appeared to remain aggregated due to hydrophobic interactions. This system could also be re-dispersed by sonication and re-aggregated in subsequent cycles of photo-excitation and sonication.

Non-invasive method to predict viscosity and size using total internal reflection fluorescence microscopy (TIRFM) system

This study develops an automated analysis tool by combining total internal reflection fluorescence microscopy (TIRFM), an evanescent wave microscopic imaging technique to capture time-sequential images and the corresponding image processing Matlab code to identify movements of single individual particles. The developed code will enable us to examine two dimensional hindered tangential Brownian motion of nanoparticles with a sub-pixel resolution (nanoscale). The measured mean square displacements of nanoparticles are compared with theoretical predictions to estimate particle diameters and fluid viscosity using a nonlinear regression technique. These estimated values will be confirmed by the diameters and viscosities given by manufacturers to validate this analysis tool. Nano-particles used in these experiments are yellow-green polystyrene fluorescent nanospheres (200 nm, 500 nm and 1000 nm in diameter (nominal); 505 nm excitation and 515 nm emission wavelengths). Solutions used in this experiment are de-ionized (DI) water, 10% d-glucose and 10% glycerol. Mean square displacements obtained near the surface shows significant deviation from theoretical predictions which are attributed to DLVO forces in the region but it conforms to theoretical predictions after ~125 nm onwards. The proposed automation analysis tool will be powerfully employed in the bio-application fields needed for examination of single protein (DNA and/or vesicle) tracking, drug delivery, and cyto-toxicity unlike the traditional measurement techniques that require fixing the cells. Furthermore, this tool can be also usefully applied for the microfluidic areas of non-invasive thermometry, particle tracking velocimetry (PTV), and non-invasive viscometry.

DEVELOPMENT OF CONFOCAL IMAGING TECHNIQUES FOR PROBING INTERFACIAL DYNAMICS IN MICROSCALE, GAS-LIQUID, TWO-PHASE FLOW

Micro-scale, two-phase flow is found in a variety of devices such as Lab-on-a-chip, bio-chips, micro-heat exchangers, and fuel cells. Knowledge of the fluid behavior near the dynamic gas-liquid interface is required for developing accurate predictive models. Light is distorted near a curved gas-liquid interface preventing accurate measurement of interfacial shape and internal liquid velocities. This research focused on the development of experimental methods designed to isolate and probe dynamic liquid films and measure velocity fields near a moving gas-liquid interface. A high-speed, reflectance, swept-field confocal (RSFC) imaging system was developed for imaging near curved surfaces. Experimental studies of dynamic gas-liquid interface of micro-scale, two-phase flow were conducted in three phases. Dynamic liquid film thicknesses of segmented, two-phase flow were measured using the RSFC and compared to a classic film thickness deposition model. Flow fields near a steadily moving meniscus were measured using RSFC and particle tracking velocimetry. The RSFC provided high speed imaging near the menisci without distortion caused the gas-liquid interface. Finally, interfacial morphology for internal two-phase flow and droplet evaporation were measured using interferograms produced by the RSFC imaging technique. Each technique can be used independently or simultaneously when.

Dispersal Modeling of Fish Early Life Stages: Sensitivity with Application to Atlantic Cod in the Western Gulf of Maine

As an initial step in establishing mechanistic relationships between environmental variability and recruitment in Atlantic cod Gadhus morhua along the coast of the western Gulf of Maine, we assessed transport success of larvae from major spawning grounds to nursery areas with particle tracking using the unstructured grid model FVCOM (finite volume coastal ocean model). In coastal areas, dispersal of early planktonic life stages of fish and invertebrate species is highly dependent on the regional dynamics and its variability, which has to be captured by our models. With state-of-the-art forcing for the year 1995, we evaluate the sensitivity of particle dispersal to the timing and location of spawning, the spatial and temporal resolution of the model, and the vertical mixing scheme. A 3 d frequency for the release of particles is necessary to capture the effect of the circulation variability into an averaged dispersal pattern of the spawning season. The analysis of sensitivity to model setup showed that a higher resolution mesh, tidal forcing, and current variability do not change the general pattern of connectivity, but do tend to increase within-site retention. Our results indicate strong downstream connectivity among spawning grounds and higher chances for successful transport from spawning areas closer to the coast. The model run for January egg release indicates 1 to 19 % within-spawning ground retention of initial particles, which may be sufficient to sustain local populations. A systematic sensitivity analysis still needs to be conducted to determine the minimum mesh and forcing resolution that adequately resolves the complex dynamics of the western Gulf of Maine. Other sources of variability, i.e. large-scale upstream forcing and the biological environment, also need to be considered in future studies of the interannual variability in transport and survival of the early life stages of cod.

Collimated Field Emission Beams from Metal Double-Gate Nanotip Arrays Optically Excited via Surface Plasmon Resonance

The generation of collimated electron beams from metal double-gate nanotip arrays excited by near infrared laser pulses is studied. Using electromagnetic and particle tracking simulations, we showed that electron pulses with small rms transverse velocities are efficiently produced from nanotip arrays by laser-induced field emission with the laser wavelength tuned to surface plasmon polariton resonance of the stacked double-gate structure. The result indicates the possibility of realizing a metal nanotip array cathode that outperforms state-of-the-art photocathodes.

Optimal fits of diffusion constants from single-time data points of Brownian trajectories

Experimental methods based on single particle tracking (SPT) are being increasingly employed in the physical and biological sciences, where nanoscale objects are visualized with high temporal and spatial resolution. SPT can probe interactions between a particle and its environment but the price to be paid is the absence of ensemble averaging and a consequent lack of statistics. Here we address the benchmark question of how to accurately extract the diffusion constant of one single Brownian trajectory. We analyze a class of estimators based on weighted functionals of the square displacement. For a certain choice of the weight function these functionals provide the true ensemble averaged diffusion coefficient, with a precision that increases with the trajectory resolution.