Among the central constituents of the innate immune system are macrophages, which are known for phagocytosis or ‘eating’ foreign particles or pathogens. Macrophages express several cell-surface proteins including transmembrane and membrane-anchored receptors, which play a vital role in their response to pathogenic stimuli. The plasma membrane is a highly fluid and dynamic environment, which facilitates the diffusion of lipids and proteins within the plane of the membrane. This study aims to measure the lateral diffusion of two types of plasma membrane receptors on macrophages, toll-like receptor II (TLR2) and cluster of differentiation 14 (CD14), to answer three main research questions: 1) Which type of fluorescence-based microscopy techniques is best suited for measuring the lateral diffusion of TLR2 and CD14 on macrophage plasma membrane? 2) Does culturing macrophages on different surface topographies impact the diffusion of TLR2 in the plasma membrane and its pro-inflammatory response, along with morphological changes? 3) Does aging alter the lateral diffusion of TLR2 in the plasma membrane of macrophages? To date, a variety of fluorescence-based methods have been developed to study the dynamics of cell membrane constituents. These techniques are based on either ensemble or single particle measurements. We have used single particle tracking methods to track the mobility of fluorescently labeled membrane receptors on murine bone marrow-derived macrophages. Total internal reflection fluorescence microscopy (TIRF) was used to visualize and capture the dynamics in live cells. Using a custom routine algorithm we detected, localized, and tracked the particles to calculate their diffusion coefficient, extracted from the mean-squared displacement as the most common measure of diffusion. We also measured the diffusion coefficient using an ensemble-based technique known as Raster Image Correlation Spectroscopy (RICS) with a confocal laser-scanning microscope. The use of confocal eliminates the out-of-focus signal and enables measurements that are confined to a narrow plane in the cell. Also, the ability of RICS to separate the slow and immobile fractions of particles makes it possible to detect heterogeneities in diffusion. To our knowledge, this is the first study that has utilized both SPT and RICS to directly compare receptors’ diffusion in different membrane sections. Moreover, this is the first study that has examined the diffusion of receptors on macrophages adhered to different surface topographies, and the first that has investigated the receptors’ diffusion in young and old macrophages. / Thesis / Doctor of Philosophy (PhD) / The immune system is highly dependent on a specialized subset of white blood cells known as macrophages that are capable of clearing damaged and dead cells as well as a wide range of invading micro-organisms. Specific receptor proteins present on the membrane of macrophages are involved in the recognition of particles and subsequent signaling to recruit other immune cells or to promote healing and wound repair. To date, a variety of fluorescence-based microscopy methods have been used to study the dynamics of cell membrane components. The mobility of several membrane receptors in macrophages has been studied using microscopy techniques, which have provided valuable insights into their function. However, there is still insufficient information about the behavior of two key receptors (TLR2 and CD14) that participate in signaling in response to bacterial products. This thesis aims to answer three major questions with regard to receptor mobility (i.e., diffusion) within macrophage membrane: 1) Which type of fluorescence-based microscopy technique is more suitable for measuring the mobility of TLR2 and CD14 receptors on macrophage membranes? 2) What is the impact of different surface topographies on TLR2 diffusion in adhered macrophages, as well as cell shape, and the ability of macrophages to internalize particles? 3) Does aging alter TLR2 mobility in the membrane of macrophages? The following chapters provide detailed answers to these questions. In brief, we have demonstrated that TLR2 and CD14 diffusion measurements in adhered macrophages highly depend on the membrane section chosen. In addition, our results show that micro- and nanostructured surface topographies alter the shape of adhered macrophages and yield higher bacteria internalization, while the diffusion of TLR2 is not changed. When comparing macrophages derived from young and old mice, we find similar diffusion rate of TLR2 in macrophages of the two age groups.

Fluorescence correlation spectroscopy (FCS) analyzes the fluctuations in the fluorescence intensity, which is emitted from a tiny excition volume, to obtain information about the concentration, the mobility, and the molecular interactions of labeled molecules. The more advanced fluorescence cross-correlation spectroscopy (FCCS) increases the precision in the determination of fl ow velocities and binding constants compared to standard FCS. The miniaturization in biomedical and chemical engineering has been developing rapidly, propelled by the vision of a fully functional laboratory on a single chip and its use in human therapeutics, for example, as implanted drug delivery system. A key requirement to fulfill this vision is the ability to handle small fl uid volumes. Handling liquids using the electrohydrodynamical principle circumvents many of the disadvantages of other systems. The complex flow pattern in the active region of such a pump could not be resolved by common tracking techniques. In this thesis, two-focus FCCS (2f-FCCS) was used to map the flow pro file inside a micropump. The high precision of 2f-FCCS in the determination of fl ow measurements even with small fluorescent particles allowed the measurement of the flow velocities induced by electrohydrodynamic forces acting on the solvent, while excluding the effects of dielectrophoretic forces acting on larger particles. Analysis of the fl ow data indicates a fl ow pattern that consists of two vortices of different size and opposite direction of rotation. The flow pattern derived by 2f-FCCS explains the observed complex particle trajectories in the force field and the accumulation of particles in well-de fined regions above the microelectrode array. In the second part of this thesis, the mechanism of RNA interference (RNAi) was studied by dual-color FCCS in vivo. RNAi is an evolutionary conserved gene silencing mechanism, which uses short double-stranded RNA molecules, called short interfering RNAs (siRNAs), as effector molecules. Due to its speci city and simplicity, RNAi yields a great potential for a widespread therapeutic use. To broaden the therapeutic applications, the in vivo stability of siRNAs has to be improved by chemical modi cations, but some of these modi fications inhibit the gene silencing mechanism. The presented FCCS assays are very well suited to investigate the individual assembly steps of RNAi machinery with very high specifi city and sensitivity in real time and to study the cleavage activity of the activated RNAi machinery. A direct correlation between activity of the RNAi machinery and the results from the FCCS measurements could be shown. The in fluence of several chemical modi cations on the assembly and activity of the RNAi machinery was investigated with these assays. / Fluoreszenz-Korrelations-Spektroskopie (FCS) analysiert die Fluktuationen im Fluoreszenzsignal eines kleinen angeregten Volumens, um Informationen über die Konzentration, die Bewegung und die Interaktionen der markierten Moleküle zu erhalten. Die Fluoreszenz-Kreuzkorrelations-Spektroskopie (FCCS) erhöht die Genauigkeit bei der Messung von Fließgeschwindigkeiten und Bindungskonstanten im Vergleich zur Standard-FCS. Die Miniaturisierung der Biomedizin und Chemie hat sich rapide entwickelt, angetrieben von der Vision eines kompletten Labors auf einem Chip und dem Einsatz dieses in der medizinischen Therapie, zum Beispiel als implantierter Medikamentenspender. Ein Schlüsselelement zur Erfüllung dieser Vision ist der Transport von kleinsten Flüssigkeitsmengen in diesen miniaturisierten Systemen. Der Transport von Flüssigkeiten mittels des elektrohydrodynamischen Prinzips umgeht viele Nachteile von anderen Systemen, allerdings zeigt eine solche Pumpe ein kompliziertes Strömungsbild in der aktiven Region, welches sich mit herkömmlichen Methoden wie Teilchenverfolgung nicht vermessen ließ. Hier wurde Zwei-Fokus-FCCS (2f-FCCS) genutzt, um das Strömungsbild in der Pumpe zu vermessen. Die hohe Genauigkeit der 2f-FCCS bei der Bestimmung von Fließgeschwindigkeiten auch mit kleinen fluoreszierenden Teilchen ermöglichte die Messung der Fließgeschwindigkeiten, aufgrund der auf das Lösungsmittel wirkenden elektrohydrodynamischen Kräfte, unter Ausschluss der auf größere Teilchen wirkenden dielektrophoretischen Kräfte. Die Analyse der Daten ergab, dass das Strömungsbild aus zwei entgegengesetzt rotierenden unterschiedlich großen Wirbeln besteht. Dieses Strömungsbild erklärt die komplizierten Teilchenbewegungsbahnen und die Anreicherung der Teilchen in klar abgegrenzten Bereichen über den Mikroelektroden. Im zweiten Teil dieser Arbeit wurde der RNAi-Mechanismus in lebenden Zellen mittels Zwei-Farben-FCCS untersucht. RNA Interferenz (RNAi) ist ein evolutionär erhaltener Geninaktivierungsmechanismus, der kurze doppelsträngige RNA Moleküle, so genannte kurze interferierende RNAs (siRNAs), als Effektormoleküle nutzt. Die Spezifi tät und Einfachheit der RNAi hat ihr ein weites Feld in der medikamentösen Therapie geöffnet. Zur Erweiterung dieses Feldes ist es nötig die Stabilität der siRNAs im Körper mittels chemischer Modi fikationen zu erhöhen. Einige dieser Modifikationen hemmen aber den RNAi-Mechanismus. Die hier vorgestellten FCCS Experimente sind sehr gut geeignet, um die einzelnen Schritte des Zusammenbaus der RNAi Maschinerie mit hoher Empfi ndlichkeit und Spezi fität in Echtzeit zu untersuchen und die Aktivität der RNAi Maschinerie zu studieren. Es konnte ein Zusammenhang zwischen der Aktivität der RNAi Maschinerie und den Ergebnissen der FCCS Messungen hergestellt werden. Der Einfluss von verschiedenen chemischen Modikationen auf den Zusammenbau und die Aktivität der RNAi Maschinerie wurde mit diesen neuartigen Methoden untersucht.

No description available.

This thesis describes the development and applications of an extension of DWS which enables the recovery of ‘localized’ mechanical properties, in a specified region of a complex jelly-like object which is inhomogeneous, marked out by the focal volume of an ultrasound transducer, also called the region-of-interest (ROI). Introduction of the sinusoidal forcing creates a sinusoidal phase variation in the detected light in a DWS experiment which modulates the measured intensity autocorrelation, g2 (τ ). Decay in the modulation depth with τ is used to recover the visco-elastic spectrum of the material in the ROI. En route to this, growth of the mean-squared dis- placement (MSD) with time is extracted from the modulation depth decay, which was verified first by the usual DWS experimental data from an homogeneous object with properties matching those in the ROI of the inhomogeneous object and then those obtained by solving the generalized Langevin equation (GLE) modelling the dynamics of a typical scattering centre in the ROI. A region-specific visco-elastic spectral map was obtained by scanning the inhomogeneous object by the ultrasound focal volume. Further, the resonant modes of the vibrating ROI were measured by locating the peaks of the modulation depth variation in g2(τ ) with respect to the ultrasound frequency. These resonant modes were made use of to recover elasticity of the material of the object in the ROI. Using a similar strategy, it was also shown that flow in pipe can be detected and flow rate computed by ‘tagging’ the photons passing through the pipe with a focussed ultrasound beam. It is demonstrated, both through experiments and simulations that the ultrasound-assisted technique devel- oped is better suited to both detect and quantitatively assess flow in a background of Brownian dynamics than the usual DWS. In particular, the MSD of particles in the flow, which shows forth a super-diffusive dynamics with MSD growing following τ α with α < 2, is captured over larger intervals of τ than was possible using existing methods. On the theoretical front, the main contribution is the derivation of the GLE, with multiplicative noise modulating the interaction ‘spring constant’. The noise is derived as an average effect of the micropolar rotations suffered by the ‘bath’ particles on the ‘system’ particle modelled. It has been shown that the ‘local’ dynamics of the system particle is nontrivially influenced by the dynamics, both translation and rotation, of ‘nonlocal’ bath particles.

New optical technologies were developed to continuously measure cerebral blood flow (CBF) and oxygen metabolism (CMRO2) non-invasively through the skull. Methods and devices were created to improve the performance of near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS) for use in experimental animals and humans. These were employed to investigate cerebral metabolism and cerebrovascular reactivity under different states of anesthesia and during models of pathological states. Burst suppression is a brain state arising naturally in pathological conditions or under deep general anesthesia, but its mechanism and consequences are not well understood. Electroencephalography (EEG) and cortical hemodynamics were simultaneously measured in rats to evaluate the coupling between cerebral oxygen metabolism and neuronal activity in the burst suppressed state. EEG bursts were used to deconvolve NIRS and DCS signals into the hemodynamic and metabolic response function for an individual burst. This response was found to be similar to the stereotypical functional hyperemia evoked by normal brain activation. Thus, spontaneous burst activity does not cause metabolic or hemodynamic dysfunction in the cortex. Furthermore, cortical metabolic activity was not associated with the initiation or termination of a burst. A novel technique, time-domain DCS (TD-DCS), was introduced to significantly increase the sensitivity of transcranial CBF measurements to the brain. A new time-correlated single photon counting (TCSPC) instrument with a custom high coherence pulsed laser source was engineered for the first-ever simultaneous measurement of photon time of flight and DCS autocorrelation decays. In this new approach, photon time tags are exploited to determine path-length-dependent autocorrelation functions. By correlating photons according to time of flight, CBF is distinguished from superficial blood flow. Experiments in phantoms and animals demonstrate TD-DCS has significantly greater sensitivity to the brain than existing transcranial techniques. Intracranial pressure (ICP) modulates both steady-state and pulsatile CBF, making CBF a potential marker for ICP. In particular, the critical closing pressure (CrCP) has been proposed as a surrogate measure of ICP. A new DCS device was developed to measure pulsatile CBF non-invasively. A novel method for estimating CrCP and ICP from DCS measurement of pulsatile microvascular blood flow in the cerebral cortex was demonstrated in rats. / 2018-03-08T00:00:00Z

Neste trabalho investigou-se o comportamento sistemático do campo hiperfino magnético nos compostos intermetálicos Gd(Ni,Pd,Cu)In , Tb(Ni,Pd)In, Dy(Ni,Pd)In e Ho(Ni,Pd)In, com a técnica de Correlação Angular &gamma;-&gamma; Perturbada, nos sítios dos elementos terras raras com o núcleo de prova 140Ce e no sítio do In com o núcleo de prova 111Cd. Os resultados obtidos da dependência com a temperatura do Campo Hiperfino Magnético (Bhf) de cada composto foram primeiramente comparados com a teoria do campo molecular por meio da função de Brillouin, da qual foram extrapolados os valores de saturação de Bhf para cada composto estudado. Os valores extrapolados a zero Kelvin de Bhf (Bhf(T=0)) para os compostos estudados neste trabalho foram usados na comparação com valores de Bhf da literatura para outros compostos contendo o mesmo elemento R (R=Terra Rara). O resultado mostrou uma dependência linear com a temperatura de ordenamento magnético, o mesmo previsto pelo modelo RKKY o que indica que a principal contribuição para Bhf vem da polarização dos elétrons de condução (CEP) via interação de contato de Fermi. O comportamento de Bhf saturado para cada família de compostos de terras raras pesados RNiIn e RPdIn como função da projeção do spin 4f do elemento R mostrou também uma relação linear, com exceção dos resultados para os compostos RNiIn obtidos com 111Cd onde foi observado um leve desvio da linearidade. Foi feita uma investigação dos parâmetros hiperfinos elétricos em função da temperatura para os compostos RPdIn e GdNiIn com o núcleo de prova 111Cd, e os resultados mostram que para o composto GdPdIn pode ocorrer desordem Gd-In a altas temperaturas. / Systematic behavior of magnetic hyperfine field (Bhf) in the intermetallic compounds Gd(Ni,Pd,Cu)In , Tb(Ni,Pd)In, Dy(Ni,Pd)In and Ho(Ni,Pd)In was studied by Perturbed Gamma-Gamma Angular Correlation spectroscopy. The measurements of Bhf were carried out at the rare earth atom and In sites using the nuclear probes 140Ce and 111In respectively. The variation of hyperfine field with temperature, in most cases, follows the Brillouin function predicted from the molecular field theory. The hyperfine field values at rare earth atom sites obtained from 140Ce probe as well as at In sites obtained from 111In probe for each series of compounds were extrapolated to zero Kelvin Bhf(T=0) from these curves. These values were compared with the values of the literature for other compounds containing the same rare earth element and all of them show a linear relationship with the ordering temperature. This indicates that the main contribution to Bhf comes from the conduction electron polarization (CEP) through Fermi contact interaction and the principal mechanism of magnetic interaction in these compounds can be described by the RKKY type interaction. The values of Bhf(T=0) for each family of intermetallic compounds RNiIn and RPdIn when plotted as a function of 4f spin projection of rare earth element also shows a linear relationship. Exceptions are the results for the compounds RNiIn obtained with 111Cd probe where a small deviation from linearity is observed. The results of the measurements carried out with the 111Cd probe were also analyzed to obtain the hyperfine parameters of the quadrupole interaction as a function of temperature for RPdIn and GdNiIn compounds. The results show that for the compound GdPdIn there might be some Gd-In disorder at high temperature.

Neste trabalho investigou-se o comportamento sistemático do campo hiperfino magnético nos compostos intermetálicos Gd(Ni,Pd,Cu)In , Tb(Ni,Pd)In, Dy(Ni,Pd)In e Ho(Ni,Pd)In, com a técnica de Correlação Angular &gamma;-&gamma; Perturbada, nos sítios dos elementos terras raras com o núcleo de prova 140Ce e no sítio do In com o núcleo de prova 111Cd. Os resultados obtidos da dependência com a temperatura do Campo Hiperfino Magnético (Bhf) de cada composto foram primeiramente comparados com a teoria do campo molecular por meio da função de Brillouin, da qual foram extrapolados os valores de saturação de Bhf para cada composto estudado. Os valores extrapolados a zero Kelvin de Bhf (Bhf(T=0)) para os compostos estudados neste trabalho foram usados na comparação com valores de Bhf da literatura para outros compostos contendo o mesmo elemento R (R=Terra Rara). O resultado mostrou uma dependência linear com a temperatura de ordenamento magnético, o mesmo previsto pelo modelo RKKY o que indica que a principal contribuição para Bhf vem da polarização dos elétrons de condução (CEP) via interação de contato de Fermi. O comportamento de Bhf saturado para cada família de compostos de terras raras pesados RNiIn e RPdIn como função da projeção do spin 4f do elemento R mostrou também uma relação linear, com exceção dos resultados para os compostos RNiIn obtidos com 111Cd onde foi observado um leve desvio da linearidade. Foi feita uma investigação dos parâmetros hiperfinos elétricos em função da temperatura para os compostos RPdIn e GdNiIn com o núcleo de prova 111Cd, e os resultados mostram que para o composto GdPdIn pode ocorrer desordem Gd-In a altas temperaturas. / Systematic behavior of magnetic hyperfine field (Bhf) in the intermetallic compounds Gd(Ni,Pd,Cu)In , Tb(Ni,Pd)In, Dy(Ni,Pd)In and Ho(Ni,Pd)In was studied by Perturbed Gamma-Gamma Angular Correlation spectroscopy. The measurements of Bhf were carried out at the rare earth atom and In sites using the nuclear probes 140Ce and 111In respectively. The variation of hyperfine field with temperature, in most cases, follows the Brillouin function predicted from the molecular field theory. The hyperfine field values at rare earth atom sites obtained from 140Ce probe as well as at In sites obtained from 111In probe for each series of compounds were extrapolated to zero Kelvin Bhf(T=0) from these curves. These values were compared with the values of the literature for other compounds containing the same rare earth element and all of them show a linear relationship with the ordering temperature. This indicates that the main contribution to Bhf comes from the conduction electron polarization (CEP) through Fermi contact interaction and the principal mechanism of magnetic interaction in these compounds can be described by the RKKY type interaction. The values of Bhf(T=0) for each family of intermetallic compounds RNiIn and RPdIn when plotted as a function of 4f spin projection of rare earth element also shows a linear relationship. Exceptions are the results for the compounds RNiIn obtained with 111Cd probe where a small deviation from linearity is observed. The results of the measurements carried out with the 111Cd probe were also analyzed to obtain the hyperfine parameters of the quadrupole interaction as a function of temperature for RPdIn and GdNiIn compounds. The results show that for the compound GdPdIn there might be some Gd-In disorder at high temperature.

Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the presence of denaturants or small molecule dissociators. These highly active small molecule dissociators, selected from the Apex Screen 5040 library, were previously identified by ELISA studies by the laboratory of Dr. Harry LeVine. We have used fluorescence correlation spectroscopy (FCS) to characterize the size distribution and mole fraction of synthetically prepared fluorescein labeled Aβ (1-42) oligomers. Our FCS results show that in the presence of denaturants or small molecule dissociators, oligomer dissociation may proceed by at least two different mechanisms; high order cooperative dissociation and linear dissociation. A cooperative mechanism is more desirable for therapeutics as oligomer directly dissociates into monomer rather than through various oligomer intermediates. Our FCS studies show the most efficient dissociators proceed through the cooperative dissociation mechanism. We also observed a large retardation of the oligomer dissociation in the presence of gallic acid. We also started preliminary work to develop a total internal reflection fluorescence (TIRF) spectroscopy method to image Aβ (1-42) oligomers. This technique if successful will help to verify the two distinct mechanisms seen by FCS or determine if there is one mechanism that occurs at different rates as TIRF allows for faster analysis.

Fluorescent molecules commonly shift to transient dark states, induced bylight or triggered by chemical reactions. The transient dark states can beused as probes of the local environment surrounding the fluorescent molecules,and are therefore attractive for use in biomolecular applications. Thisthesis explores the use and development of novel fluorescence spectroscopictechniques for monitoring transient dark states.This work demonstrates that kinetic information regarding photoinduced transient dark states of fluorescent molecules can be obtained from the time-averaged fluorescence intensity of fluorescent molecules subject totemporally modulated illumination. Methods based on this approach havethe advantage that the light detectors can have a low time resolution, which allows for parallelization and screening of biomolecular interactions withhigh throughput. Transient state images are presented displaying local environmental differences such as those in oxygen concentration and quencher accessibility.Analysis of the fluorescence intensity fluctuations resulting from thetransitions to and from transient dark states can be used to obtain information regarding the transition rates and occupancy of the transient darkstates. Fluorescence fluctuation analysis was used to reveal rates of protonbinding and debinding to single fluorescent molecules located close to biological membranes and protein surfaces. The results from these studies show that the proton exchange rate increases dramatically when the fluorescent molecule is close to the membrane. / QC 20100809

Single-molecule spectroscopy has provided a means to uncover pathways and heterogeneities that were previously hidden beneath the ensemble average. Such heterogeneity, however, is often obscured by the artifacts of experimental noise and the occurrence of undesired processes within the experimental medium. This has subsequently caused in the need for new analytical methodologies. It is particularly important that objectivity be maintained in the development of new analytical methodology so that bias is not introduced and the results improperly characterized. The research presented herein identifies two such sources of experimental uncertainty, and constructs objective approaches to reduce their effects in the experimental results. The first, photoblinking, arises from the occupation of dark electronic states within the probe molecule, resulting in experimental data that is distorted by its contribution. A method based in Bayesian inference is developed, and is found to nearly eliminate photoblinks from the experimental data while minimally affecting the remaining data and maintaining objectivity. The second source of uncertainty is electronic shot-noise, which arises as a result of Poissonian photon collection. A method based in wavelet decomposition is constructed and applied to simulated and experimental data. It is iii found that, while making only one assumption, that photon collection is indeed a Poisson process, up to 75% of the shot-noise contribution may be removed from the experimental signal by the wavelet-based procedure. Lastly, in an effort to connect model-based approaches such as molecular dynamics simulation to model-free approaches that rely solely on the experimental data, a coarse-grained molecular model of a molecular ionic fluorophore diffusing within an electrostatically charged polymer brush is constructed and characterized. It is found that, while the characteristics of the coarse-grained simulation compare well with atomistic simulations, the model is lacking in its representation of the electrostatically-driven behavior of the experimental system.