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Autofluorescence-Based Diagnostic UV Imaging of Tissues and CellsRenkoski, Timothy Eli January 2013 (has links)
Cancer is the second leading cause of death in the United States, and its early diagnosis is critical to improving treatment options and patient outcomes. In autofluorescence (AF) imaging, light of controlled wavelengths is projected onto tissue, absorbed by specific molecules, and re-emitted at longer wavelengths. Images of re-emitted light are used together with spectral information to infer tissue functional information and diagnosis. This dissertation describes AF imaging studies of three different organs using data collected from fresh human surgical specimens. In the ovary study, illumination was at 365 nm, and images were captured at 8 emission wavelengths. Measurements from a multispectral imaging system and fiber optic probe were used to map tissue diagnosis at every image pixel. For the colon and pancreas studies, instrumentation was developed extending AF imaging capability to sub-300 nm excitation. Images excited in the deep UV revealed tryptophan and protein content which are believed to change with disease state. Several excitation wavelength bands from 280 nm to 440 nm were investigated. Microscopic AF images collected in the pancreas study included both cultured and primary cells. Several findings are reported. A method of transforming fiber optic probe spectra for direct comparison with imager spectra was devised. Normalization of AF data by green reflectance data was found useful in correcting hemoglobin absorption. Ratio images, both AF and reflectance, were formulated to highlight growths in the colon. Novel tryptophan AF images were found less useful for colon diagnostics than the new ratio techniques. Microscopic tryptophan AF images produce useful visualization of cellular protein content, but their diagnostic value requires further study.
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Targeting Melanocortin and Cholecystokinin Receptors via Multivalent Molecules Bearing Peptide LigandsNakath Gamlath Ralalage, Dayan Elshan January 2014 (has links)
Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.
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OPTICAL METHODS FOR MOLECULAR SENSING: SUPPLEMENTING IMAGING OF TISSUE MICROSTRUCTURE WITH MOLECULAR INFORMATIONWinkler, Amy January 2010 (has links)
More and more researchers and clinicians are looking to molecular sensing to predict how cells will behave, seeking the answers to questions like "will these tumor cells become malignant?" or "how will these cells respond to chemotherapy?" Optical methods are attractive for answering these questions because optical radiation is safer and less expensive than alternative methods, such as CT which uses X-ray radiation, PET/SPECT which use gamma radiation, or MRI which is expensive and only available in a hospital setting. In this dissertation, three distinct optical methods are explored to detect at the molecular level: optical coherence tomography (OCT), laser-induced fluorescence (LIF), and optical polarimetry. OCT has the capability to simultaneously capture anatomical information as well as molecular information using targeted contrast agents such as gold nanoshells. LIF is less useful for capturing anatomical information, but it can achieve significantly better molecular sensitivity with the use of targeted fluorescent dyes. Optical polarimetry has potential to detect the concentration of helical molecules, such as glucose. All of these methods are noninvasive or minimally invasive.The work is organized into four specific aims. The first is the design and implementation of a fast, high resolution, endoscopic OCT system to facilitate minimally invasive mouse colon imaging. The second aim is to demonstrate the utility of this system for automatically identifying tumor lesions based on tissue microstructure. The third is to demonstrate the use of contrast agents to detect molecular expression using OCT and LIF. The last aim is to demonstrate a new method based on optical polarimetry for noninvasive glucose sensing.
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Heterologous Expression of Alpha 6*- Nicotinic Acetylcholine Receptors and the Natural Distribution of Alpha 6 SubunitsBuhlman, Lori Marie January 2007 (has links)
Nicotinic acetylcholine receptors (nAChR) are neurotransmitter-gated ion channels that exist as a family of subtypes defined by unique subunit compositions. nAChR containing α6 subunits (α6*-nAChR) have attracted interest because α6 subunits are thought to be localized in brain regions implicated in reward, mood and drug dependence. To provide new information necessary toward a more complete understanding of roles of α6*-nAChR in neuropsychiatric health and disease, three lines of investigation were pursued. A set of stably transfected, human, immortalized cell lines were generated that heterologously express nAChR α6 subunits in combination with other nAChR subunits found in reward brain regions (nAChR subunit combinations α6β2, α6β4, α6β2β3, α6β4β3, α6β2β3α5, α6β4β3α5, α6α4β2β3 and α6α4β4β3). The α6α4β2β3 combination may have a functional response to epibatidine that differs from that of the α4β2 nAChR. A unique binding site was identified in cells transfected with the α6β4β3α5 nAChR subunit combination. Messenger RNA fluorescence in situ hybridization (mRNA FISH) studies established regional and celluar distribution of nAChR α6 subunit mRNA in the mouse brain. The third line of study extended this work to examine potential co-expression of nAChR α6 subunits and glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH) as labels of GABAergic and dopaminergic/catecholaminergic neurons respectively, using tandem mRNA FISH and fluorescence immunohistochemistry. nAChR α6 subunit signal in the substantia nigra (SN) and ventral tegmental area (VTA) was congruent with previous studies. Message was also detected in the amydala, dentate gyrus, striatum, zona incerta, and cingulate, entorhinal, perirhinal, piriform, and prelimbic cortices. nAChR α6 mRNA was coexpressed with GAD in the amygdala, dentate gyrus, striatum, SN, VTA and cingulate, entorhinal, prelimbic and prelimbic cortices. TH was exclusively co-localized with nAChR α6 mRNA in the SN and VTA. Findings suggest extended roles for α6*-nAChR in the brain, particularly in the control of GABAergic neuronal activity and/or GABA release. These studies provide new insights into the composition of α6*-nAChR, the localization and cellular origins of nAChR α6 subunit expression. Data collected suggest roles for α6*-nAChR in many brain regions, including those involved in higher order processes involved in drug dependence and reward, and in modulation of inhibitory neurotransmission.
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Probing the denatured state ensemble with fluorescenceAlston, Roy Willis 30 September 2004 (has links)
To understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, λmax for the proteins and model compounds differed very little, 349.3 ± 1.2 nm. However, significant differences were observed in the fluorescence intensity at λmax (IF), suggesting that IF is more sensitive to the immediate environment than λmax. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.
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Visualisering av mikroorganismer i hårfolliklar från patienter med follikulit / Visualizationof Microorganisms in Hair Follicles from Patients with FolliculitisBerg, Johanna January 2012 (has links)
No description available.
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Direct observation of correlated motions in colloidal gels and glassesGao, Yongxiang. January 2008 (has links)
Dynamical heterogeneity (DH) has been observed in many systems approaching the glass or jamming transition. Whether DH has a structural origin is under heated debate. To provide a deeper understanding, in this thesis I investigate the microscopic dynamics in weakly attractive colloidal systems by confocal fluorescence microscopy. The van Hove density-density correlation function is applied to our systems. Separable fast and slow populations emerge in the self part (svH), while the distinct part shows a strong signature of DH close to the gel transition. At intermediate time, svH shows a purely exponential tail, mainly arising from the fast population. I show that this broad tail is a direct consequence of the occurrence of rare large jumps that are statistically distributed. The slow population tends to form a space-spanning backbone, and its mean squared displacement close to the gel transition exhibits a plateau, whose height is consistent with the range of attraction, suggesting a bonding mechanism for the dynamical arrest. I further examine various quantities characterizing local structure and local dynamics and a strong correlation is identified between them. Subsequently, I develop order parameters for quantifying amorphous structure and apply them to our systems. I find that attractive colloidal systems exhibit higher order under higher attraction tension, while hard spheres become more ordered under higher compression. Finally, I investigate the effect of the range of attraction on the structure and dynamics of attractive colloidal systems. I observe that the system with shorter range of attraction forms a denser and more heterogeneous structure. Meanwhile, I observe an even stronger dynamical heterogeneity. These observations provide further evidence of a connection between structural heterogeneity and dynamical heterogeneity in these systems, providing guidance for a theoretical description of the dynamical arrest as well as the relaxation mechanisms upon gelation and its relation to solidification in glasses. / In order to do all of this, I first implemented full 3D subpixel resolution localization of particles and improved particle tracking algorithms tailored for the sorts of heterogenous dynamics these systems exhibit, that otherwise confounds existing methods such that the very relaxation mechanisms would be missed. This allows us to obtain unprecedented precision in positions of all of the particles and complete tracking, both of which are essential for correctly determining system properties that depend on measured particle dynamics.
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Characterization of the Self-Assembly of Pyrene-Labelled Macromolecules in WaterSiu, Howard Chun-Kui January 2010 (has links)
The self-assembly of several pyrene-labelled amphiphilic macromolecules in water was characterized by fluorescence. Information on their self-assembly was obtained by monitoring the level of pyrene aggregation in solution. A measure of the level of association was obtained by determining the fraction of aggregated pyrene of the labelled macromolecules from the global analysis of their monomer and excimer fluorescence decays. Global analysis limits the degrees of freedom of the analysis thus reducing the error on the parameters retrieved from the analysis. Extensive developments in the global analysis of the pyrene monomer and excimer decays enabled the first characterization of the molar absorbance coefficient of the pyrene aggregates formed by aqueous solutions of pyrene-labelled poly(N,N-dimethylacrylamide) (PyPDMA) and poly(ethylene oxide) (PyPEO). The molar absorbance coefficients of the pyrene aggregates determined for PyPDMA and PyPEO were both found to be broader and red-shifted compared to that of unaggregated pyrene. These results agree with observations found in the scientific literature made by using absorption and excitation fluorescence measurements. Attempts to determine the molar absorbance coefficient of pyrene-labelled hydrophobically-modified alkali-swellable emulsion (PyHASE) polymers were unsuccessful. The inability to characterize the pyrene aggregates of PyHASE was attributed to the greater complexity of the PyHASE polymer compared to PyPDMA and PyPEO. For these simpler pyrene-labelled polymers, a protocol has been established which uses the global analysis of the pyrene monomer and excimer decays to determine quantitatively the level of association of pyrene-labelled polymers as well as the molar absorbance coefficient of their aggregates.
Changes in the level of aggregation of pyrene-labelled lipids (PLLs) having head groups bearing an alcohol (PSOH) or imido diacetic acid (PSIDA) embedded in 1-palmitoyl-2-oleyl-3-sn-phosphatidylcholines (POPC) or distearylphosphatidylcholine (DSPC) liposomes were probed by fluorescence. Distribution of the PLLs in the fluid POPC membrane was found to be homogeneous while the PLLs phase-separated into amorphous channels created in the DSPC membranes. Multivalent cations Cu2+ and La3+ were found to bind to PSIDA, hindering diffusional encounters between unaggregated PSIDA but leaving the PLL aggregates intact. Using the fluorescence quenching ability of Cu2+, the viscosity of the amorphous channels of the DSPC membrane was determined to be about six times greater than that of the more fluid POPC membrane.
Simultaneous rheological and fluorescence measurements were achieved by interfacing a rheometer with time-resolved and steady-state fluorometers using fiber-optic cables. This joint set up enabled the simultaneous rheological and fluorescence measurements of PyHASE solutions having concentrations ranging from 0.5 w/w% to 5 w/w%. The level of association of the PyHASE solutions was tracked using fluorescence at shear rates of 0, 0.1 and 100 s–1. Despite the presence of shear thinning leading to viscosity drops of up to four orders of magnitude, no change in the fluorescence and hence the level of association was observed. The lack of change in level of association implied that the mechanism of shear thinning is due to a switching from inter- to intramolecular association rather than a drop in the level of association. This information will prove useful for future models attempting to predict the rheological behaviour of sheared associative polymers.
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Projected Barzilai-Borwein Method with Infeasible Iterates for Nonnegative Image DeconvolutionFraser, Kathleen 22 July 2011 (has links)
The Barzilai-Borwein (BB) method for unconstrained optimization has attracted attention for its "chaotic" behaviour and fast convergence on image deconvolution problems. However, images with large areas of darkness, such as those often found in astronomy or microscopy, have been shown to benefit from approaches which impose a nonnegativity constraint on the pixel values. We present a new adaptation of the BB method which enforces a nonnegativity constraint by projecting the solution onto the feasible set, but allows for infeasible iterates between projections. We show that this approach results in faster convergence than the basic Projected Barzilai-Borwein (PBB) method, while achieving better quality images than the unconstrained BB method. We find that the new method also performs comparably to the Gradient Projection-Conjugate Gradient (GPCG) method, and in most test cases achieves a lower restoration error, despite being a much simpler algorithm.
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Using Small Molecules to Inhibit an E2A-PBX1:CBP Interaction Involved in Acute Lymphoblastic LeukemiaPurvis, Amelia 03 September 2009 (has links)
E2A-PBX1 is expressed as a consequence of a recurring chromosomal translocation seen in 5% of acute lymphoblastic leukemia cases. We recently reported that substitution of a leucine residue (L20A) within the N-terminal transcriptional activation domain (AD1) of E2A-PBX1 markedly impairs binding to the KIX domain of CBP/p300 and, importantly, leukemia induction in a mouse bone marrow transplantation model. Since both the protein-protein interaction and consequent leukemogenesis rely on a focal contact point and might therefore be susceptible to antagonism by small molecules, we devised a cell-free assay based on fluorescence anisotropy (FA) to detect binding of a fluorescently labeled peptide derived from AD1 of E2A-PBX1 (FITC-E2A) with recombinantly expressed KIX domain. The optimized FA assay reveals a dissociation constant of 2 µM for the wild-type interaction and correctly detects disruption of the complex by naphthol AS-E phosphate, a compound previously shown to antagonize KIX binding. The optimized FA assay was used to screen the Prestwick, Spectrum and Chembridge libraries containing 12400 compounds in total. Of the initial 43 positive hits from the libraries, 10 caused a reproducible decrease in FA. Since intrinsic small molecule fluorescence can produce false positive results in the FA-based screen, intrinsically fluorescent compounds were excluded from further analysis unless they could be shown to bind to KIX. Two hits, L1 and C2, were intrinsically fluorescent but demonstrated KIX interactions and one hit, P9, was not intrinsically fluorescent. These three compounds were tested for their ability to inhibit binding of a larger portion of E2A (residues 1 to 483) to full length CBP in a pull down assay with only compound P9 demonstrating efficacy. Further characterization of P9 by NMR showed no binding to KIX, however evaluation by FA showed binding to FITC-E2A with a 20 µM affinity. A cell-based cytotoxicity assay demonstrated that compound P9 was slightly more toxic on leukemic cells that express E2A-PBX1, compared to leukemic cells lacking E2A-PBX1 expression. Mammalian two-hybrid analysis did not provide details of the effects of P9 on the E2A:KIX interaction. We expect the identification of a novel compound, P9, capable of disrupting the oncogenic E2A-PBX1:CBP interaction, to guide the development of effective, less toxic leukemia drugs and provide new tools for elucidating the molecular mechanisms of leukemia induction by E2A-PBX1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-08-31 11:13:19.517
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