Time-resolved surface enhanced resonance Raman spectro-electrochemistry of heme proteins

The membrane protein Cytochrome c Oxidase (CcO) is one of the most important functional bio-molecules. It appears in almost every eukaryotic cell and many bacteria. Although the different species differ in the number of subunits, the functional differences are merely marginal. CcO is the terminal link in the electron transfer pathway of the mitochondrial respiratory chain. Electrons transferred to the catalytic center of the enzyme conduce to the reduction of molecular oxygen to water. Oxygen reduction is coupled to the pumping of protons into the inter-membrane space and hence generates a difference in electrochemical potential of protons across the inner mitochondrial membrane. This potential difference drives the synthesis of adenosine triphosphate (ATP), which is the universal energy carrier within all biological cells. rnrnThe goal of the present work is to contribute to a better understanding of the functional mechanism of CcO by using time-resolved surface enhanced resonance Raman spectroscopy (TR-SERRS). Despite intensive research effort within the last decades, the functional mechanism of CcO is still subject to controversial discussions. It was the primary goal of this dissertation to initiate electron transfer to the redox centers CuA, heme a, heme a3 and CuB electrochemically and to observe the corresponding redox transitions in-situ with a focus on the two heme structures by using SERRS. A measuring cell was developed, which allowed combination of electrochemical excitation with Raman spectroscopy for the purpose of performing the accordant measurements. Cytochrome c was used as a benchmark system to test the new measuring cell and to prove the feasibility of appropriate Raman measurements. In contrast to CcO the heme protein cc contains only a single heme structure. Nevertheless, characteristic Raman bands of the hemes can be observed for both proteins.rnrnIn order to investigate CcO it was immobilized on top of a silver substrate and embedded into an artificial membrane. The catalytic activity of CcO and therefore the complete functional capability of the enzyme within the biomimetic membrane architecture was verified using cyclic voltammetry. Raman spectroscopy was performed using a special nano-structured silver surface, which was developed within the scope of the present work. This new substrate combined two fundamental properties. It facilitated the formation of a protein tethered bilayer lipid membrane (ptBLM) and it allowed obtaining Raman spectra with sufficient high signal-to-noise ratios.rnSpectro-electrochemical investigations showed that at open circuit potential the enzyme exists in a mixed-valence state, with heme a and and heme a3 in the reduced and oxidized state, respectively. This was considered as an intermediate state between the non-activated and the fully activated state of CcO. Time-resolved SERRS measurements revealed that a hampered electron transfer to the redox center heme a3 characterizes this intermediate state.rn

Bathorhodopsin structure in the room-temperature rhodopsin photosequence: picosecond time-resolved coherent anti-Stokes Raman scattering.

Structural changes in the retinal chromophore during the formation of the bathorhodopsin intermediate (bathoRT) in the room-temperature rhodopsin (RhRT) photosequence (i.e., vision) are examined using picosecond time-resolved coherent anti-Stokes Raman scattering. Specifically, the retinal structure assignable to bathoRT following 8-ps excitation of RhRT is measured via vibrational Raman spectroscopy at a 200-ps time delay where the only intermediate present is bathoRT. Significant differences are observed between the C=C stretching frequencies of the retinal chromophore at low temperature where bathorhodopsin is stabilized and at room temperature where bathorhodopsin is a transient species in the RhRT photosequence. These vibrational data are discussed in terms of the formation of bathoRT, an important step in the energy storage/transduction mechanism of RhRT.

Time-resolved fluorescence observation of di-tyrosine formation in horseradish peroxidase upon ultrasound treatment leading to enzyme inactivation

The application of ultrasound to a solution can induce cavitional phenomena and generate high localised temperatures and pressures. These are dependent of the frequency used and have enabled ultrasound application in areas such as synthetic, green and food chemistry. High frequency (100 kHz to 1 MHz) in particular is promising in food chemistry as a means to inactivate enzymes, replacing the need to use periods of high temperature. A plant enzyme, horseradish peroxidase, was studied using time-resolved fluorescence techniques as a means to assess the effect of high frequency (378 kHz and 583 kHz) ultrasound treatment at equivalent acoustic powers. This uncovered the fluorescence emission from a newly formed species, attributed to the formation of di-tyrosine within the horseradish peroxidase structure caused by auto-oxidation, and linked to enzyme inactivation.

Premovement activity of the pre-supplementary motor area and the readiness for action: Studies of time-resolved event-related functional MRI

The supplementary motor area (SMA) is thought to play in important role in the preparation and organisation of voluntary movement. It has long been known that cortical activity begins to increase up to 2 s prior to voluntary self-initiated movement. This increasing premovement activity measured in EEG is known as the Bereitschaftspotential or readiness potential. Modern functional brain imaging methods, using event-related and time-resolved functional MRI techniques, are beginning to reveal the role of the SMA, and in particular the more anterior pre-SMA, in premovement activity associated with the readiness for action. In this paper we review recent studies using event-related time-resolved fMRI methods to examine the time-course of activation changes within the SMA throughout the preparation, readiness and execution of action. These studies suggest that the preSMA plays a common role in encoding or representing actions prior to our own voluntary self-initiated movements, during motor imagery, and from the observation of others' actions. We suggest that the pre-SMA generates and encodes motor representations which are then maintained in readiness for action. (c) 2005 Elsevier B.V. All rights reserved.

The selection of intended actions and the observation of others' actions: A time-resolved fMRI study

Whenever we plan, imagine, or observe an action, the motor systems that would be involved in preparing and executing that action are similarly engaged. The way in which such common motor activation is formed, however, is likely to differ depending on whether it arises from our own intentional selection of action or from the observation of another's action. In this study, we use time-resolved event-related functional MRI to tease apart neural processes specifically related to the processing of observed actions, the selection of our own intended actions, the preparation for movement, and motor response execution. Participants observed a finger gesture movement or a cue indicating they should select their own finger gesture to perform, followed by a 5-s delay period; participants then performed the observed or self-selected action. During the preparation and readiness for action, prior to initiation, we found activation in a common network of higher motor areas, including dorsal and ventral premotor areas and the pre-supplementary motor area (pre-SMA); the more caudal SMA showed greater activation during movement execution. Importantly, the route to this common motor activation differed depending on whether participants freely selected the actions to perform or whether they observed the actions performed by another person. Observation of action specifically involved activation of inferior and superior parietal regions, reflecting involvement of the dorsal visual pathway in visuomotor processing required for planning the action. In contrast, the selection of action specifically involved the dorsal lateral prefrontal and anterior cingulate cortex, reflecting the role of these prefrontal areas in attentional selection and guiding the selection of responses. (c) 2005 Elsevier Inc. All rights reserved.

Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

Does Semantic Context Benefit Speech Understanding through "Top-Down" Processes? Evidence from Time-resolved Sparse fMRI.

When speech is degraded, word report is higher for semantically coherent sentences (e.g., her new skirt was made of denim) than for anomalous sentences (e.g., her good slope was done in carrot). Such increased intelligibility is often described as resulting from "top-down" processes, reflecting an assumption that higher-level (semantic) neural processes support lower-level (perceptual) mechanisms. We used time-resolved sparse fMRI to test for top-down neural mechanisms, measuring activity while participants heard coherent and anomalous sentences presented in speech envelope/spectrum noise at varying signal-to-noise ratios (SNR). The timing of BOLD responses to more intelligible speech provides evidence of hierarchical organization, with earlier responses in peri-auditory regions of the posterior superior temporal gyrus than in more distant temporal and frontal regions. Despite Sentence content × SNR interactions in the superior temporal gyrus, prefrontal regions respond after auditory/perceptual regions. Although we cannot rule out top-down effects, this pattern is more compatible with a purely feedforward or bottom-up account, in which the results of lower-level perceptual processing are passed to inferior frontal regions. Behavioral and neural evidence that sentence content influences perception of degraded speech does not necessarily imply "top-down" neural processes.

Time resolved construction of a bimodal laccolith (Torres del Paine, Patagonia)

Granitic and mafic magma pulses were sequentially accreted in the spectacularly exposed shallow crustal Torres del Paine laccolith, in southern Patagonia. This 12.5 Ma pluton forms a composite intrusion with a subvertical feeding system in the west and a laccolith in the east. A key unknown in the formation of sill complexes is how individual magma pulses are assembled over time and the geometry and localization of their feeding system. High resolution zircon CA-ID-TIMS U-Pb dating shows that the laccolith grew first by under-accretion of granitic sills over 90 +/- 30 ka, linked to a `sheet-like' feeding system, followed by underplating of mafic sills after similar to 20 ka of quiescence. In the mafic sills complex, individual sills were injected by over-accretion during 41 +/- 11 ka. Our data show that successive granitic and mafic magmas emplacement generated a volume of similar to 88 km(3) in 162 +/- 11 ka. (C) 2012 Elsevier B.V. All rights reserved.

Morphodynamic analysis of cerebral aneurysm pulsation from time-resolved rotational angiography

This paper presents a technique to estimate and model patient-specific pulsatility of cerebral aneurysms over onecardiac cycle, using 3D rotational X-ray angiography (3DRA) acquisitions. Aneurysm pulsation is modeled as a time varying-spline tensor field representing the deformation applied to a reference volume image, thus producing the instantaneousmorphology at each time point in the cardiac cycle. The estimated deformation is obtained by matching multiple simulated projections of the deforming volume to their corresponding original projections. A weighting scheme is introduced to account for the relevance of each original projection for the selected time point. The wide coverage of the projections, together with the weighting scheme, ensures motion consistency in all directions. The technique has been tested on digital and physical phantoms that are realistic and clinically relevant in terms of geometry, pulsation and imaging conditions. Results from digital phantomexperiments demonstrate that the proposed technique is able to recover subvoxel pulsation with an error lower than 10% of the maximum pulsation in most cases. The experiments with the physical phantom allowed demonstrating the feasibility of pulsation estimation as well as identifying different pulsation regions under clinical conditions.

Time-resolved lanthanide luminescence for lab-on-a-chip detection of biomarkers on cancerous tissues.

PDMS-based microfluidic devices combined with lanthanide-based immunocomplexes have been successfully tested for the multiplex detection of biomarkers on cancerous tissues, revealing an enhanced sensitivity compared to classical organic dyes.

Time-resolved imaging of the laser forward transfer of liquids

Time-resolved imaging is carried out to study the dynamics of the laser-induced forward transfer of an aqueous solution at different laser fluences. The transfer mechanisms are elucidated, and directly correlated with the material deposited at the analyzed irradiation conditions. It is found that there exists a fluence range in which regular and well-defined droplets are deposited. In this case, laser pulse energy absorption results in the formation of a plasma, which expansion originates a cavitation bubble in the liquid. After the further expansion and collapse of the bubble, a long and uniform jet is developed, which advances at a constant velocity until it reaches the receptor substrate. On the other hand, for lower fluences no material is deposited. In this case, although a jet can be also generated, it recoils before reaching the substrate. For higher fluences, splashing is observed on the receptor substrate due to the bursting of the cavitation bubble. Finally, a discussion of the possible mechanisms which lead to such singular dynamics is also provided.

Control and femtosecond time-resolved imaging of torsion in a chiral molecule

We study how the combination of long and short laser pulses can be used to induce torsion in an axially chiral biphenyl derivative (3,5-difluoro-3 ,5 -dibromo-4 -cyanobiphenyl). A long, with respect to the molecular rotational periods, elliptically polarized laser pulse produces 3D alignment of the molecules, and a linearly polarized short pulse initiates torsion about the stereogenic axis. The torsional motion is monitored in real-time by measuring the dihedral angle using femtosecond time-resolved Coulomb explosion imaging. Within the first 4 picoseconds (ps), torsion occurs with a period of 1.25 ps and an amplitude of 3◦ in excellent agreement with theoretical calculations. At larger times, the quantum states of the molecules describing the torsional motion dephase and an almost isotropic distribution of the dihedral angle is measured.We demonstrate an original application of covariance analysis of two-dimensional ion images to reveal strong correlations between specific ejected ionic fragments from Coulomb explosion. This technique strengthens our interpretation of the experimental data