The hydration of paper studied with solid-state magnetisation-exchange H-1 NMR spectroscopy

The wide-line H-1 nuclear magnetic resonance (NMR) spectrum of paper in equilibrium with ambient humidity consists of super-imposed relatively broad and narrow lines. The narrower line is of the order of 2 kHz wide at half the maximum height, while the broader line is of the order of 40 kHz in width at half height. On the basis of these line widths, the narrow line is assigned to water sorbed to the paper, and the broad line to the polymeric constituents of the paper. It was not possible to distinguish between the various polymeric components of paper contributing to the H-1 NMR spectra. A modified Goldman-Shen pulse sequence was used to generate a spatial magnetisation gradient between the polymer and water phases. The exchange of magnetisation between protons associated with water and those associated with the macromolecules in paper was observed. The exchange of magnetisation is discussed within a heat transfer model for homonuclear dipolar coupling, with exchange being characterised by a spin-diffusion coefficient. Consideration of the magnitude of the initial rate of the exchange process and estimates of the spin-spin relaxation times based on H-1 line widths indicate that some water must exist in a sufficiently immobile state as to allow homonuclear dipolar interactions between adjacent polymer and water protons. Thus, water sorbed onto paper must exist in at least two states in mass exchange with each other. This observation allows certain conclusions to be drawn about the ratio of free/bound water as a function of moisture content and the dispersal of water within the polymer matrix.

Quantitative fluorescence spectra and quantum yield map of synthetic pheomelanin

Spectroscopic studies of pheomelanin and its constituents have been sparse. These data present what is by far the most complete description of the fluorescence characteristics of synthetic pheomelanin. Emission spectra between 260 and 600 nm were acquired,for excitation wavelengths between 250 and 500 nm at 1-nm intervals. A quantum yield map is also presented, correcting the fluorescence intensities for differences in species concentration and molar absorptivity. These fluorescence features exhibit interesting similarities and differences to eumelanin, and these data are interpreted with respect to possible chemical structures. Overall, these data suggest that pheomelanin oligomers may be more tightly coupled than those of eumelanin. Finally, the quantum yield is shown to be on the order of 10(-4) and exhibit a complex dependence on excitation energy, varying by a factor of 4 across the energies employed here. (c) 2006 Wiley Periodicals, Inc.

NMR microscopy: A tool for measuring monomer diffusion in supercritical CO2

The diffusion of styrene into linear low density polyethylene in a solution of supercritical CO2 was investigated using NMR microimaging. For both pure styrene and styrene dissolved in supercritical CO2, the diffusion was found to follow Fickian kinetics. Supercritical CO2 was found to enhance the rate and extent of diffusion of styrene into the substrate by up to three times under the conditions of this investigation, compared to pure styrene. NMR imaging was used to measure the concentration profiles of the styrene penetrants in real time, and the results were fitted to a Fickian model for diffusion. At a CO2 pressure of 150 bar and temperature of 40 degrees C, the diffusion coefficient of a 30 wt-% solution of styrene into LLDPE was calculated to be 1 X 10(-11) m(2). s(-1). This is significantly faster than the diffusion coefficient measured for pure styrene diffusion at 40 degrees C (3 x 10(-12) m(2). s(-1)). The diffusion coefficients determined by gravimetric analysis were slightly higher than those determined by the imaging method. This was probably due to residual styrene and/or polystyrene adhering to the surface of the films in the gravimetric technique.

Assembly of Human Papillomavirus Type-16 Virus-Like Particles: Multifactorial Study of Assembly and Competing Aggregation

Pentameric capsomeres of human papillomavirus capsid protein L1 expressed in Escherichia coli self-assemble into virus-like particles (VLPs) in vitro. A multifactorial experimental design was used to explore a wide range of solution conditions to optimize the assembly process. The degree of assembly was measured using an enzyme-linked immunosorbent assay, and a high-throughput turbidity assay was developed to monitor competing aggregation. The presence of zinc ions in the assembly buffer greatly increased the incidence of aggregation and had to be excluded from the experiment for meaningful analysis. Assembly of VLPs was optimal at a pH of about 6.5, calcium and sodium ions had no measurable effect, and dithiothreitol and glutathione inhibited assembly. Tryptophan fluorescence spectroscopy demonstrated that an increase in urea concentration reduced the rate of VLP formation but had no effect on the final concentration of assembled VLPs. This study demonstrates the use of the hanging-drop vapor-diffusion crystallization method to screen for conditions that promote aggregation and the use of tryptophan fluorescence spectroscopy for real-time monitoring of the assembly process.

Chromogenic in situ hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridisation and immunohistochemistry

Aims: The objective of this study was to evaluate the accuracy, ease of use and reproducibility of chromogenic in situ hybridisation (CISH) for HER2 testing by studying its inter-laboratory concordance in five Australian pathology laboratories. Methods: The HER2 status of 49 breast cancers was determined by CISH twice in two different laboratories. Each sample had previously been tested by immunohistochemistry (IHC; 2+ and 3+ cases selected) and fluorescence in situ hybridisation ( FISH). Participating laboratories were blinded to these test results. Oestrogen receptor ( ER) status was also evaluated for each cancer. Results: High correlation was observed between FISH and CISH results. No cases showing high gene amplification by FISH were scored as non-amplified by CISH ( kappa coefficient=1). High correlation was observed between IHC and CISH, all IHC 3+ samples showing amplification by CISH. Inter-laboratory CISH concordance was also good ( kappa coefficient=0.67). Fifty-six per cent of HER2-amplified samples tested ER positive, while 42% of ER-positive cases showed HER2 gene amplification, confirming that HER2 testing should not be confined to ER-negative breast cancers. Conclusions: These findings demonstrate that CISH is a robust test to assess HER2 status in breast cancer and therefore is an important addition to the HER2 testing algorithm.

Diffuse beta-amyloid (A beta) deposits and neurons: in situ secretion or diffusion of A beta?

The association between diffuse-type beta -amyloid (AP) deposits and neuronal cell bodies in Alzheimer's disease (AD) and Down's syndrome (DS) could result from the secretion of AP from clusters of neurons in situ or the diffusion of A beta from cell processes, glial cells or blood vessels. To decide between these hypotheses, spatial pattern analysis was used to study the relationship between the degree of clustering of neuronal cell bodies and the presence of diffuse deposits in the temporal lobe of patients with DS. Significant clustering of neuronal cell bodies was present in 17/24 (71%) of brain areas studied. in addition, in 23/24 (96%) of brain areas, there was a positive correlation between the presence of diffuse deposits and the density of neurons. Hence, the data support the hypothesis that diffuse deposits develop in situ mainly as a result of the secretion of A beta by local clusters of neurons rather than by significant diffusion. Furthermore, the size of a diffuse deposit is likely to be determined by the number of neurons within a cluster which secrete A beta. The number and density of neurons could also be a factor determining the evolution of a diffuse into a mature amyloid deposit.

NMR spectroscopy in the presence of ultrasound and other perturbations

The work described in this thesis has been concerned with exploring the potential uses of ultrasound in Nuclear Magnetic Resonance (NMR) spectroscopy, The NMR spectra of liquids provide detailed structural information that may be deduced from the chemical shifts and spin-spin coupling, that are evident in the narrow resonances, arising from some of the nuclear broadening interactions being reduced to zero. In the solid state, all of the nuclear broadening interactions are present and broad lines in the NMR spectrum are observed. Current techniques employed to reduce the line widths in solids are based on coherent averaging techniques such as MAS NMR1,2 which can remove first order interactions. Recently DOR3 and DAS4 have become available to remove higher order interactions. SINNMR (Sonically Induced Narrowing of the NMR spectra of solids) has been reported by Homer et al5 and developed by Homer and Howard6 to reduce the line widths of solids. The basis of their work is the proposal that a colloidal suspension of solid particles can be made to move like large molecules by using ultrasonic agitation. The advantage of the technique is that the particles move incoherently removing all of the nuclear interactions responsible for broad lines. This thesis describes work on the extension of SINNMR by showing that the line width of 27AI and 11B for the glass Na20/B203/AI203 can be reduced by placing solid particles in a colloidal suspension. Further line width reduction is possible by applying ultrasound, at 2 MHz, of sufficient intensity. It is proposed that a cavitation field is responsible for imparting sufficient rotational motion to the solid particles to partially average the nuclear interactions responsible for broad lines. Rapid stirring of the colloidal suspension generates turbulent flow, however, the motion is insufficient to narrow the line widths for 27AI in the glass. Investigations of sonochemical reactions for in situ rate measurements by NMR have been made. 8y using the Weissler reaction7, it has been shown that ultrasonic cavitation is possible up to 10MHz. Preliminary studies have been carried out into the rate of ultrasonic polymerisation of methylmethacrylate by NMR. Long range order in liquid crystals can imposed when they are aligned in the presence a magnetic field. The degree of alignment can be monitored by NMR using, for example a deuterated solute added to the liquid crystal8. Ultrasonic streaming can then be employed to deflect the directors of the liquid crystal from their equilibrium position, resulting in a change In the NMR spectrum. The angle of deflection has been found for the thermotropic liquid crystal (I35) to be ca, 35° and for the lyotropic (ZLI-1167) to be ca, 20°, Mechanical stirring can used to re- orientate the liquid crystal but was found to give a smaller deflection, In a separate study, that did not use ultrasound, it has been found that the signal to noise ratio of 13C NMR signals can be enhanced by rapidly stirring a Iiquid. Accelerating the diffusion of nuclei out of the coil region enables M0 to be re-established more rapidly than the normal relaxation process. This allows the pulse repetition rate to be reduced without saturating the spin system. The influence of varying the relaxation delay, acquisition time and inter-pulse delay have been studied and parameters optimised. By studying cholesterol the technique was found to be most effective for nuclei with long relaxation times, such as quaternary carbon sites. Key Worde: NMR, Ulf.rasciund, 1,.lqi.fi!:l cryllltalt!h SCll1C1otlemlstryl I!r1hano~d algnflllf

Development of a non-destructive fluorescence technique for analysis of contact lens deposition levels

The primary objective of this research has been to determine the potential of fluorescence spectroscopy as a method for analysis of surface deposition on contact lenses. In order to achieve this it was first necessary to ascertain whether fluorescence analysis would be able to detect and distinguish between protein and lipid deposited on a lens surface. In conjunction with this it was important to determine the specific excitation wavelengths at which these deposited species were detected with the greatest sensitivity. Experimental observations showed that an excitation wavelength of 360nm would detect lipid deposited on a lens surface, and an excitation wavelength of 280nm would detect and distinguish between protein and lipid deposited on a contact lens. It was also very important to determine whether clean unspoilt lenses showed significant levels of fluorescence themselves. Fluorescence spectra recorded from a variety of unworn contact lenses at excitation wavelengths of 360nm and 280nm indicated that most contact lens materials do not fluoresce themselves to any great extent. Following these initial experiments various clinically and laboratory based studies were performed using fluorescence spectroscopy as a method of analysing contact lens deposition levels. The clinically based studies enabled analysis of contact lenses with known wear backgrounds to be rapidly and individually analysed following discontinuation of wear. Deposition levels in the early stages of lens wear were determined for various lens materials. The effect of surfactant cleaning on deposition levels was also investigated. The laboratory based studies involved comparing some of the in vivo results with those of identical lenses that had been spoilt using an in vitro method. Finally, an examination of lysosyme migration into and out of stored ionic high water contact lenses was made.

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al.

Laser-induced breakdown spectroscopy quantitative analysis method via adaptive analytical line selection and relevance vector machine regression model

Abstract A new LIBS quantitative analysis method based on analytical line adaptive selection and Relevance Vector Machine (RVM) regression model is proposed. First, a scheme of adaptively selecting analytical line is put forward in order to overcome the drawback of high dependency on a priori knowledge. The candidate analytical lines are automatically selected based on the built-in characteristics of spectral lines, such as spectral intensity, wavelength and width at half height. The analytical lines which will be used as input variables of regression model are determined adaptively according to the samples for both training and testing. Second, an LIBS quantitative analysis method based on RVM is presented. The intensities of analytical lines and the elemental concentrations of certified standard samples are used to train the RVM regression model. The predicted elemental concentration analysis results will be given with a form of confidence interval of probabilistic distribution, which is helpful for evaluating the uncertainness contained in the measured spectra. Chromium concentration analysis experiments of 23 certified standard high-alloy steel samples have been carried out. The multiple correlation coefficient of the prediction was up to 98.85%, and the average relative error of the prediction was 4.01%. The experiment results showed that the proposed LIBS quantitative analysis method achieved better prediction accuracy and better modeling robustness compared with the methods based on partial least squares regression, artificial neural network and standard support vector machine.

The correlation of structure and electronic properties near the surface of transition metal oxides

The strong couplings between different degrees of freedom are believed to be responsible for novel and complex phenomena discovered in transition metal oxides (TMOs). The physical complexity is directly responsible for their tunability. Creating surfaces/interfaces add an additional ' man-made' twist, approaching the quantum phenomena of correlated materials. ^ The dissertation focused on the structural and electronic properties in proximity of surface of three prototype TMO compounds by using three complementary techniques: scanning tunneling microscopy, angle-resolved photoelectron spectroscopy and low energy electron diffraction, particularly emphasized the effects of broken symmetry and imperfections like defects on the coupling between charge and lattice degrees of freedom. ^ Ca1.5Sr0.5RuO4 is a layered ruthenate with square lattice and at the boundary of magnetic/orbital instability in Ca2-xSrxRuO4. That the substitution of Sr 2+ with Ca2+ causing RuO6 rotation narrows the dxy band width and changes the Fermi surface topology. Particularly, the γ(dxy) Fermi surface sheet exhibited hole-like in Ca1.5Sr0.5RuO4 in contrast to electron-like in Sr2RuO4, showing a strong charge-lattice coupling. ^ Na0.75CoO2 is a layered cobaltite with triangular lattice exhibiting extraordinary thermoelectric properties. The well-ordered CoO2-terminated surface with random Na distribution was observed. However, lattice constants of the surface are smaller than that in bulk. The surface density of states (DOS) showed strong temperature dependence. Especially, an unusual shift of the minimum DOS occurs below 230 K, clearly indicating a local charging effect on the surface. ^ Cd2Re2O7 is the first known pyrochlore oxide superconductor (Tc ∼ 1K). It exhibited an unusual second-order phase transition occurring at TS1 = 200 K and a controversial first-order transition at TS2 = 120 K. While bulk properties display large anomalies at TS1 but rather subtle and sample-dependent changes at TS2, the surface DOS near the EF show no change at T s1 but a substantial increase below TS2---a complete reversal as the signature for the transitions. We argued that crystal imperfections, mainly defects, which were considerably enhanced at the surface, resulted in the transition at TS2. ^

Three-dimensional brain fiber tracking modeling in diffusion tensor imaging

This dissertation establishes the foundation for a new 3-D visual interface integrating Magnetic Resonance Imaging (MRI) to Diffusion Tensor Imaging (DTI). The need for such an interface is critical for understanding brain dynamics, and for providing more accurate diagnosis of key brain dysfunctions in terms of neuronal connectivity. ^ This work involved two research fronts: (1) the development of new image processing and visualization techniques in order to accurately establish relational positioning of neuronal fiber tracts and key landmarks in 3-D brain atlases, and (2) the obligation to address the computational requirements such that the processing time is within the practical bounds of clinical settings. The system was evaluated using data from thirty patients and volunteers with the Brain Institute at Miami Children's Hospital. ^ Innovative visualization mechanisms allow for the first time white matter fiber tracts to be displayed alongside key anatomical structures within accurately registered 3-D semi-transparent images of the brain. ^ The segmentation algorithm is based on the calculation of mathematically-tuned thresholds and region-detection modules. The uniqueness of the algorithm is in its ability to perform fast and accurate segmentation of the ventricles. In contrast to the manual selection of the ventricles, which averaged over 12 minutes, the segmentation algorithm averaged less than 10 seconds in its execution. ^ The registration algorithm established searches and compares MR with DT images of the same subject, where derived correlation measures quantify the resulting accuracy. Overall, the images were 27% more correlated after registration, while an average of 1.5 seconds is all it took to execute the processes of registration, interpolation, and re-slicing of the images all at the same time and in all the given dimensions. ^ This interface was fully embedded into a fiber-tracking software system in order to establish an optimal research environment. This highly integrated 3-D visualization system reached a practical level that makes it ready for clinical deployment. ^

Soot measurements in steady and pulsed ethylene/air diffusion flames using Laser-Induced Incandescence

Combustion-generated carbon black nano particles, or soot, have both positive and negative effects depending on the application. From a positive point of view, it is used as a reinforcing agent in tires, black pigment in inks, and surface coatings. From a negative point of view, it affects performance and durability of many combustion systems, it is a major contributor of global warming, and it is linked to respiratory illness and cancer. Laser-Induced Incandescence (LII) was used in this study to measure soot volume fractions in four steady and twenty-eight pulsed ethylene diffusion flames burning at atmospheric pressure. A laminar coflow diffusion burner combined with a very-high-speed solenoid valve and control circuit provided unsteady flows by forcing the fuel flow with frequencies between 10 Hz and 200 Hz. Periodic flame oscillations were captured by two-dimensional phase-locked LII images and broadband luminosity images for eight phases (0° – 360°) covering each period. A comparison between the steady and pulsed flames and the effect of the pulsation frequency on soot volume fraction in the flame region and the post flame region are presented. The most significant effect of pulsing frequency was observed at 10 Hz. At this frequency, the flame with the lowest mean flow rate had 1.77 times enhancement in peak soot volume fraction and 1.2 times enhancement in total soot volume fraction; whereas the flame with the highest mean flow rate had no significant change in the peak soot volume fraction and 1.4 times reduction in the total soot volume fraction. A correlation (fvRe-1 = a + b·Str) for the total soot volume fraction in the flame region for the unsteady laminar ethylene flames was obtained for the pulsation frequency between 10 Hz and 200 Hz, and the Reynolds number between 37 and 55. The soot primary particle size in steady and unsteady flames was measured using the Time-Resolved Laser-Induced Incandescence (TIRE-LII) and the double-exponential fit method. At maximum frequency (200 Hz), the soot particles were smaller in size by 15% compared to the steady case in the flame with the highest mean flow rate.

In vivo tissue diagnosis for myocardial infarction using optical spectroscopy with novel spectral interpretation algorithms

In recent decades, the rapid development of optical spectroscopy for tissue diagnosis has been indicative of its high clinical value. The goal of this research is to prove the feasibility of using diffuse reflectance spectroscopy and fluorescence spectroscopy to assess myocardial infarction (MI) in vivo. The proposed optical technique was designed to be an intra-operative guidance tool that can provide useful information about the condition of an infarct for surgeons and researchers. ^ In order to gain insight into the pathophysiological characteristics of an infarct, two novel spectral analysis algorithms were developed to interpret diffuse reflectance spectra. The algorithms were developed based on the unique absorption properties of hemoglobin for the purpose of retrieving regional hemoglobin oxygenation saturation and concentration data in tissue from diffuse reflectance spectra. The algorithms were evaluated and validated using simulated data and actual experimental data. ^ Finally, the hypothesis of the study was validated using a rabbit model of MI. The mechanism by which the MI was induced was the ligation of a major coronary artery of the left ventricle. Three to four weeks after the MI was induced, the extent of myocardial tissue injury and the evolution of the wound healing process were investigated using the proposed spectroscopic methodology as well as histology. The correlations between spectral alterations and histopathological features of the MI were analyzed statistically. ^ The results of this PhD study demonstrate the applicability of the proposed optical methodology for assessing myocardial tissue damage induced by MI in vivo. The results of the spectral analysis suggest that connective tissue proliferation induced by MI significantly alter the characteristics of diffuse reflectance and fluorescence spectra. The magnitudes of the alterations could be quantitatively related to the severity and extensiveness of connective tissue proliferation.^

Functional near infrared spectroscopy study of language, joint attention and motor skills

Near infrared spectroscopy (NIRS) is an emerging non-invasive optical neuro imaging technique that monitors the hemodynamic response to brain activation with ms-scale temporal resolution and sub-cm spatial resolution. The overall goal of my dissertation was to develop and apply NIRS towards investigation of neurological response to language, joint attention and planning and execution of motor skills in healthy adults. Language studies were performed to investigate the hemodynamic response, synchrony and dominance feature of the frontal and fronto-temporal cortex of healthy adults in response to language reception and expression. The mathematical model developed based on granger causality explicated the directional flow of information during the processing of language stimuli by the fronto-temporal cortex. Joint attention and planning/ execution of motor skill studies were performed to investigate the hemodynamic response, synchrony and dominance feature of the frontal cortex of healthy adults and in children (5-8 years old) with autism (for joint attention studies) and individuals with cerebral palsy (for planning/execution of motor skills studies). The joint attention studies on healthy adults showed differences in activation as well as intensity and phase dependent connectivity in the frontal cortex during joint attention in comparison to rest. The joint attention studies on typically developing children showed differences in frontal cortical activation in comparison to that in children with autism. The planning and execution of motor skills studies on healthy adults and individuals with cerebral palsy (CP) showed difference in the frontal cortical dominance, that is, bilateral and ipsilateral dominance, respectively. The planning and execution of motor skills studies also demonstrated the plastic and learning behavior of brain wherein correlation was found between the relative change in total hemoglobin in the frontal cortex and the kinematics of the activity performed by the participants. Thus, during my dissertation the NIRS neuroimaging technique was successfully implemented to investigate the neurological response of language, joint attention and planning and execution of motor skills in healthy adults as well as preliminarily on children with autism and individuals with cerebral palsy. These NIRS studies have long-term potential for the design of early stage interventions in children with autism and customized rehabilitation in individuals with cerebral palsy.