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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
Atomic force microscopy (AFM) was used to study structural differences in the chromatin of cancerous (CP-D) and non-cancerous (EPC2) cell lines. Chromatin samples were extracted using a salt fractionation protocol and subject to Mnase digestion for 2, 4, 8, and 16 minutes. Samples were then immobilized on APTES-functionalized mica sheets. Images were produced using the tapping mode capabilities of the AFM and structural differences between cell lines were quantified using image processing software. Vast differences in chromatin structure were observed between cancerous and non-cancerous cell lines and it was discovered that CP-D chromatin is present as scattered nucleosomes and nucleosome aggregates while EPC2 chromatin is present in intricate arrays. It was also observed that in both the CP-D and EPC2 cell lines, nucleosomes were more isolated and less apparent at longer Mnase digestion times. These findings lead to the conclusion that as the DNA becomes sufficiently digested, chromatin and nucleosomal arrays begin to deteriorate and lose their complex and elaborate structure.
ContributorsPiper, Miles Jeffrey (Author) / Ros, Robert (Thesis director) / Lindsay, Stuart (Committee member) / School of Molecular Sciences (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Proteins continually and naturally incur evolutionary selection through mutagenesis that optimizes their fitness, which is primarily determined by their function. It is known that allosteric regulation alters a protein's conformational dynamics leading to functional changes. We have computationally introduced a mutation at a predicted regulatory site of a short, 46 residue-long, protein interaction module composed of a WW domain and corresponding polyproline ligand (PDB id: 1k9r). The dynamic flexibility index (DFI) was computed for the binding site of the wild type and mutant WW domains to quantify the mutations effect on the rigidity of the binding pocket. DFI is used as a metric to quantify the resilience of a given position to perturbation along the chain. Using steered molecular dynamics (SMD), we also measure the effect of the point mutation on allosteric regulation by approximating the binding free energy of the system calculated using Jarzynski's Equality. Calculation of the DFI shows that the overall flexibility of the protein complex increases as a result of the distal point mutation. Total change in DFI percentile of the binding site showed a 0.067 increase suggesting an allosteric, loss of function mutation. Furthermore, we see that the change in the binding free energy is greater for that of the mutated complex supporting the idea that an increase in flexibility is correlated to a decrease in proteinlig and binding affinity. We show that sequence mutation of an allosteric site affects the mechanical stability and functionality of the binding pocket.
ContributorsMarianchuk, Tegan (Author) / Ozkan, Sefika (Thesis director) / Ros, Robert (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor)
Created2018-05
Description
The goal of this study was to investigate the possibility of catch bond formation between nectin and actin during cellular adhesion by modeling the actin-filament binding protein, afadin, out of equilibrium. This was done through the in silico methodology of Molecular Dynamics (MD); more specifically using Steered Molecular Dynamics (SMD) and Replica Exchange Molecular Dynamics (REMD). The methodology of this experiment centered around generating physiologically probable structures through REMD, then using MD and SMD methods to generate structures in the absence and presence of force respectively. These structures were then analyzed through Solvent Accessible Surface Area (SASA) measurements to assess the overall compactness of the structure, which led to implicit observations on the overall resistance of force that this structure has. Overall, it was found that the structure displayed more compact conformations in the presence of force as the SASA values of the binding pocket and individual residues involved in the system tend to decrease as force was applied. This is indicative of more stable conformations and a force resistant quality that is indicative of catch bonding, thus leading to the natural conclusion that this structure displays catch bond character.
ContributorsChapman, Jonathan (Author) / Singharoy, Abhishek (Thesis director) / Beckstein, Oliver (Committee member) / Ros, Robert (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2024-05