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- Creators: Barrett, The Honors College
- Creators: Levitus, Marcia
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
Fluorescence spectroscopy is a popular technique that has been particularly useful in probing biological systems, especially with the invention of single molecule fluorescence. For example, Förster resonance energy transfer (FRET) is one tool that has been helpful in probing distances and conformational changes in biomolecules. In this work, important properties necessary in the quantification of FRET were investigated while FRET was also applied to gain insight into the dynamics of biological molecules. In particular, dynamics of damaged DNA was investigated. While damages in DNA are known to affect DNA structure, what remains unclear is how the presence of a lesion, or multiple lesions, affects the flexibility of DNA, especially in relation to damage recognition by repair enzymes. DNA conformational dynamics was probed by combining FRET and fluorescence anisotropy along with biochemical assays. The focus of this work was to investigate the relationship between dynamics and enzymatic repair. In addition, to properly quantify fluorescence and FRET data, photophysical phenomena of fluorophores, such as blinking, needs to be understood. The triplet formation of the single molecule dye TAMRA and the photoisomerization yield of two different modifications of the single molecule cyanine dye Cy3 were examined spectroscopically to aid in accurate data interpretation. The combination of the biophysical and physiochemical studies illustrates how fluorescence spectroscopy can be used to answer biological questions.
ContributorsShepherd Stennett, Elana Maria (Author) / Levitus, Marcia (Thesis advisor) / Ros, Robert (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2015
Description
In this project, we introduce a type of microscopy which produces correlated topography and fluorescence lifetime images with nanometer resolution. This technique combines atomic force microscopy (AFM) and time resolved confocal fluorescence microscopy to conduct biological and materials research. This method is used to investigate nanophotonic effects on single fluorophores, including quantum dots and fluorescent molecules. For single fluorescent molecules, we investigate the effects of quenching of fluorescence with the probe of an atomic force microscope which is combined and synchronized with a confocal fluorescence lifetime microscope. For quantum dots, we investigate the correlation between the topographic and fluorescence data. With this method of combining an atomic force microscope with a confocal microscope, it is anticipated that there will be applications in nanomaterial characterization and life sciences; such as the determination of the structure of small molecular systems on surfaces, molecular interactions, as well as the structure and properties of fluorescent nanomaterials.
ContributorsWard, Alex Mark (Author) / Ros, Robert (Thesis director) / Shumway, John (Committee member) / Schulz, Olaf (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Physics (Contributor)
Created2013-05
Description
The use of saliva sampling as a noninvasive way for drug analysis as well as the monitoring systems within the body has become increasingly important in recent research. Because of the growing interest in saliva, this project proposes a way to analyze sodium ion concentration in a saliva solution based on its fluorescence level when in the presence of a sodium indicator dye and recorded with a smartphone camera. The dyed sample was placed in a specially designed housing to exclude all ambient light from the images. A source light of known wavelength was used to excite the fluorescent dye and the smartphone camera images recorded the emission light wavelengths. After analysis of the images using ImageJ, it was possible to create a model to determine the level of fluorescence based on sodium ion concentration. The smartphone camera image model was compared to readings from a standard fluorescence plate recorder to test the accuracy of the model. The study found that the model was accurate within 5 % as compared to the fluorescence plate recorder. Based on the results, it was concluded that the method and resulting model proposed in this study is a valid was to analyze saliva or other solutions for their sodium ion concentration via images recorded by a smartphone camera.
ContributorsSmith, Catherine Julia (Author) / Antonio, Garcia (Thesis director) / Caplan, Michael (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
Photophysical Studies of the DNA Microenvironment and Small Molecule-DNA Interactions
The photophysical properties of ethidium in a variety of organic solvents, as well as several dsDNAs, were measured. We report that the fluorescence quantum yield of intercalated ethidium is .30(.03), which falls between previous stated measurements of .14 and .60. We believe this to be the most accurately measured fluorescence quantum yield to date, as verified by Strickler-Berg analyses, which exhibit excellent agreement with experimental fluorescence lifetimes. A marked hypochromism upon binding to DNA is noted due to interactions of the dye’s and nucleobases’ respective π-stacks. This more than counteracts the expected increase in transition dipole due to increased conjugation caused by twisting of the phenyl moiety upon intercalation.
The reduced volume cylinder model was tested by the quenching of the fluorescence of an intercalator (ethidium bromide) by a groove binder (methyl viologen). We report that the model is not accurate over a relevant range of DNA concentrations.
The photophysical properties of ethidium in a variety of organic solvents, as well as several dsDNAs, were measured. We report that the fluorescence quantum yield of intercalated ethidium is .30(.03), which falls between previous stated measurements of .14 and .60. We believe this to be the most accurately measured fluorescence quantum yield to date, as verified by Strickler-Berg analyses, which exhibit excellent agreement with experimental fluorescence lifetimes. A marked hypochromism upon binding to DNA is noted due to interactions of the dye’s and nucleobases’ respective π-stacks. This more than counteracts the expected increase in transition dipole due to increased conjugation caused by twisting of the phenyl moiety upon intercalation.
The reduced volume cylinder model was tested by the quenching of the fluorescence of an intercalator (ethidium bromide) by a groove binder (methyl viologen). We report that the model is not accurate over a relevant range of DNA concentrations.
ContributorsEngelhart, Aaron (Author) / Gould, Ian (Thesis director) / Francisco, Wilson (Committee member) / Bednar, Valerie (Committee member) / Barrett, The Honors College (Contributor)
Created2005-05
Description
Contrast agents in medical imaging can help visualize structural details, distributions of particular cell types, or local environment characteristics. Multi-modal imaging techniques have become increasingly popular for their improved sensitivity, resolution, and ability to correlate structural and functional information. This study addresses the development of dual-modality (magnetic resonance/fluorescence) and dual-functional (thermometry/detection) nanoprobes for enhanced tissue imaging.
ContributorsHemzacek, Katherine Leigh (Author) / Kodibagkar, Vikram (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Fluorescence spectroscopy is a powerful tool for biophysical studies due to its high sensitivity and broad availability. It is possible to detect fluorescence from single molecules allowing researchers to see the behavior of subpopulations whose presence is obscured by “bulk” collection methods. The fluorescent probes used in these experiments are affected by the solution and macromolecular environments they are in. A misunderstanding of a probe’s photophysics can lead researchers to assign observed behavior to biomolecules, when in fact the probe is responsible. On the other hand, a probe’s photophysical behavior is a signature of the environment surrounding it; it can be exploited to learn about the biomolecule(s) under study. A thorough examination of a probe’s photophysics is critical to data interpretation in both cases and is the focus of this work. This dissertation investigates the photophysical behavior of symmetric and asymmetric cyanines in a variety of solution and biomolecular environments. Using fluorescent techniques—such as time-correlated single photon counting (TCSPC) and fluorescence correlation spectroscopy (FCS)—it was found that cyanines are influenced by the local environment. In the first project, the symmetric cyanines are found to be susceptible to paramagnetic species, such as manganese(II), that enhance the intersystem crossing (ISC) rate increasing triplet blinking and accelerating photobleaching. Another project found the increase in fluorescence of Cy3 in the protein induced fluorescence enhancement (PIFE) technique is due to reduced photoisomerization caused by the proximity of protein to Cy3. The third project focused on asymmetric cyanines; their photophysical behavior has not been previously characterized. Dy630 as a free dye behaves like Cy3; it has a short lifetime and can deactivate via photoisomerization. Preliminary experiments on Dy dyes conjugated to DNA show these dyes do not photoisomerize, and do not show PIFE potential. Further research will explore other conjugation strategies, with the goal of optimizing conditions in which Dy630 can be used as the red-absorbing analogue of Cy3 for PIFE applications. In summary, this dissertation focused on photophysical investigations, the understanding of which forms the backbone of rigorous fluorescent studies and is vital to the development of the fluorescence field.
ContributorsCiuba, Monika A (Author) / Levitus, Marcia (Thesis advisor) / Liu, Yan (Committee member) / Vaiana, Sara (Committee member) / Arizona State University (Publisher)
Created2017
Description
Type 1 diabetes is a metabolic disorder in which the pancreas produces little to no insulin due to the cells being destroyed by a person’s own body. A potential treatment for this disorder is the allogeneic transplantation of pancreatic beta cells. Unfortunately, this potential solution requires the use of immunosuppressants. For my project with the Weaver Lab, I will be assessing pseudoislet survival in macroencapsulation via injection molding. I will be analyzing survival and metabolic assays of the pseudoislets in the mold process. Pseudoislets in hydrogels usually undergo hypoxia-included cell death due to the diffusion distances oxygen has to travel. We will test the impact of macroencapsulation device geometry on hypoxia within encapsulated cells. I will be culturing pancreatic cells and encapsulating them in hydrogels. Macroencapsulation devices will be utilized to shield islets from the immune system and eliminate the need for immunosuppressants. In order to analyze the cells’ structure and to ensure their viability, confocal microscopy will be used. Staining for live cells will be done using calcein AM which produces green fluorescence and indicates live cells. Staining for dead cells on the other hand will be done using an ethidium homodimer which produces red fluorescence and indicates dead cells. To determine if the cells are metabolically active the Alamar Blue assay will be used.
ContributorsSaenz, Fidel Junior (Author) / Weaver, Jessica (Thesis director) / Emerson, Amy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05
Description
Upon cooling a semicrystalline polymer from its amorphous melt state, it undergoes melt crystallization where organized microstructures develop through a process of nucleation and crystal growth. Understanding the crystallization kinetics of a semicrystalline thermoplastic is key to tuning crystallinity and microstructure, which play integral roles in the material’s final properties such as toughness, gas permeability, and degradation rate. Nonisothermal crystallization, in particular, has great technological relevance to polymer engineering processes such as injection molding, film blowing, and fiber spinning, all of which rely on fast cooling rates. Spectroscopic, scattering, calorimetric, and rheological techniques have been conventionally used for studying nonisothermal crystallization, but can be limited in their sensitivity, tunability, and availability. Our group has recently developed a fluorescence technique for sensing the melting transitions of semicrystalline thermoplastics by incorporating fluorescent probes into polymer matrices. Herein, this methodology has been extended to an in-situ study of nonisothermal melt crystallization by monitoring the T-dependent fluorescence intensity of the fluorophores incorporated into a polymer matrix. As crystals form upon cooling from the amorphous melt state, the intramolecular motions of fluorophores are restricted and thus their T-dependent fluorescence intensity data exhibit a stepwise increase during nonisothermal crystallization. The first derivative of the T-dependent fluorescence intensity data can provide insight into the onset, peak, and endset crystallization temperatures, all of which align reasonably well with conventional differential scanning calorimetry measurements. This facile, sensitive, and contact-free fluorescence technique can access faster cooling rates (up to ~100 oC min-1) than many other existing methods for nonisothermal crystallization studies, which is more relevant to industrial polymer processing conditions. Additionally, the fluorescence detection mechanism shows great sensitivity not only to the degree of crystallinity but also to the crystalline microstructure formed during nonisothermal crystallization. Furthermore, unique fluorescent labeling is expected to foster novel studies on the local crystallization within heterogeneous polymeric systems including blends, composites, and multilayer films. Such local crystallization studies are out of reach for most conventional techniques that measure spatially averaged properties. Overall, this nonisothermal crystallization study expands the capabilities of this novel fluorescence technique for advancing the field of semicrystalline thermoplastic design and processing.
ContributorsCabello, Maya (Author) / Jin, Kailong (Thesis director) / Nile, Gabriel (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2022-05