Doctor of Philosophy (PhD), Ohio University, 2023, Chemistry and Biochemistry (Arts and Sciences)

In recent years, there took place a notable advancement observed in single-molecule fluorescence microscopy methods and its use in various biomolecular research. This technique allows for direct visualization of dynamics and its detailed complexities of various biological processes at the molecular level which is not possible in bulk measurement. Usually, in experiments related to single-molecule fluorescence measurements, the abundance of key molecules is intentionally minimized, which reduces the noise and improves the quality of imaging. However, such a strategy does not work when experiments involve weak interaction between biomolecules. In such a situation nonspecific interaction between molecules of interest and glass would lead to an unwanted fluorescence background signal, which compromises the imaging quality and reduces the measurement accuracy. In this work, glass surfaces have been functionalized in multiple steps. In the initial step, the glass coverslip surface is modified with (3-aminopropyl) triethoxysilane (APTES), and in the next step, the surface is functionalized using methoxy-terminated polyethylene glycol (mPEG) and biotin-terminated polyethylene glycol (bPEG) molecules. Each surface is characterized using dye-labeled protein molecules called neutravidin. for a variety of single-molecule fluorescence studies as PEG molecules are known to repel any nonspecific molecules binding on the functionalized surfaces. Then the surface is used for two-end immobilization of lambda DNA using biotin and neutravidin interaction. Once the surface is functionalized and characterized, lambda DNA is two ends immobilized on the surface using biotin and neutravidin interaction. Then we use that platform to study the intercalation and de-intercalation kinetics of various intercalating dyes such as single- intercalator (YO-PRO-1) and doubleintercalator (YOYO-1) at various experimental conditions of ionic strength and flow speed of the buffer (open full item for complete abstract)

Committee: Jixin Chen (Advisor) Subjects: Chemistry; Physical Chemistry

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Engineering

Quantitative colocalization analysis is a standard method in the life sciences used for evaluating the global spatial proximity of labeled biomolecules captured by fluorescence microscopy images. It is typically performed by characterizing the pixel-wise signal overlap or intensity correlation between spectral channels. However, this approach is critically flawed due to its focus on individual pixels which limits assessment to a single spatial scale constrained by the pixel's size, thus making the analysis dependent on the achieved optical resolution and ignorant of the spatial information presented by non-overlapping signals. In this dissertation, I present an improved method for quantifying biomolecule spatial proximity using a novel application of point process analysis adapted for discrete image data, and subsequently utilize it to address two novel cardiac conundrums. The tool, called Spatial Pattern Analysis using Closest Events (SPACE), leverages the distances between signal-positive pixels to statistically characterize the spatial relationship between labeled biomolecules from fluorescence microscopy images. In chapter two, SPACE's underlying theory and its adaption for discrete image-based data is described. Additionally, I characterize its sensitivity to segmentation parameters, image resolution, and signal sample size, and demonstrate its advantages over standard colocalization methods. With this tool, I hope to provide microscopists an improved method to robustly characterize spatial relationships independent of imaging modality or achieved resolution. In chapter three, SPACE is used to elucidate a novel, microtubule-based system for the distributed synthesis of membrane proteins in cardiomyocytes. Canonically, these cells are thought to produce membrane proteins in the peri-nuclear rough endoplasmic reticulum, then leverage the secretory-protein-trafficking pathway to transport nascent proteins to their sites of membrane insertion. By labeling car (open full item for complete abstract)

Committee: Rengasayee Veeraraghavan (Advisor); Przemysław Radwański (Committee Member); Peter Craigmile (Committee Member); Seth Weinberg (Committee Member) Subjects: Biology; Biomedical Engineering; Biomedical Research; Biophysics; Biostatistics; Cellular Biology; Engineering; Scientific Imaging; Statistics

Bachelor of Sciences, Ohio University, 2023, Biological Sciences

Globally, metastasis causes approximately 90% of mortality in cancer, making it a leading cause of death. In the United States, in both men and women, lung cancer is the second most prevalent cancer, with over 283,000 new cases estimated to be diagnosed in 2023. Both the tumor microenvironment (TME) and macropinocytosis have been shown to play a role in invasion, proliferation, and recurrence of cancers. Dr. Xiaozhuo Chen's lab at Ohio University studied the effects of the TME on cancer cells by performing RNA sequencing on A549. A549 are non-small cell human lung cancer (NSCLC) cells, which showed a consistent, significant upregulation of Stanniocalcin 1(STC1) gene expression when treated with extracellular ATP (eATP) and TGF-β. STC1 is a protein hormone involved in the regulation of the calcium phosphate balance, as well as ATP synthesis in mitochondria within the cell. Further studies showed that knock down of the STC1 gene led to reduced invasion and proliferation when compared to the untreated A549 cells. The aim of this project was to perform two main studies; one, to identify and assess macropinocytosis in a variety of cancer cell lines, and two, to investigate the effects of the STC1 gene on macropinocytosis. Using ATP concentration assays and IPA3 inhibition assays, macropinocytosis was examined in 11 cancer cell lines of varying cancer types. Macropinocytosis was confirmed with fluorescence microscopy by the colocalization of green fluorescent ATP and red fluorescent dextran. The impact of the knock-out of STC1 on macropinocytosis in A549 cells was investigated and quantified using ImageJ. The fluorescence microscopy study revealed that STC1 gene did play a role in macropinocytosis as predicted, which may be important for its effect on proliferation and invasion, the first step of metastasis.

Committee: Xiaozhuo Chen PhD (Advisor); Janet Duerr PhD (Advisor) Subjects: Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy, University of Akron, 2022, Chemistry

Methanotrophic bacteria absorb methane and oxidize it as their sole source of carbon and energy. Almost all methanotrophic bacteria contain an extensive network of Intracytoplasmic membrane (ICM). The ICMs contain particulate methane monooxygenase (pMMO), which is the initial enzyme in the metabolism of methane. Due to the accumulation of high lipid content in the form of ICM and the formation of polyhydroxybutyrate (PHB), there is a growing interest in utilizing these bacteria to convert the ICM and PHB to biofuel. Structural aspects of the ICM have been characterized by transmission electron microscopy. However, the dynamics and functional role of ICMs remain elusive. A rapid fluorescence microscopy method to visualize ICMs in situ using lipophilic dyes was developed in Chapter III. The extent to which ICM formation occurs in cells depends on the concentration of copper. The ICM formation was visualized and quantified in type I methanotroph Methylotuvimicrobium alcaliphilum (comb. Nov. 20Z) by tracking the bulk copper conversion spectroscopically and by live single-cell confocal imaging. Both methods showed a lag phase prior to the increase in ICM amounts over time. During the ICM formation, there was a significant amount of cell to cell heterogeneity. Further, rapid in-vivo quantification of the PHB method was developed to determine the conditions that enhance the PHB accumulation in methanotrophs. A rapid and cost-effective single cell PHB analysis through fluorescence microscopy by staining via Nile Blue A (NBA) in type II methanotroph Methylocystis sp. Rockwell was described in Chapter IV. NBA stained both the outer membrane of the cell and individual granules of PHB, distinctly but not the ICMs. The ICMs in Methylocystis. sp. Rockwell resides peripheral to the inner membrane whereas PHB is present in the cytoplasmic region. Methylocystis sp. Rockwell accumulated PHB when grown in ammonium mineral slats (AMS) medium, regardless of nitrogen or carbon stress. PH (open full item for complete abstract)

Committee: Adam Smith (Advisor); Sailaja Paruchuri (Committee Member); Nic Leipzig (Committee Member); Chrys Wesdemiotis (Committee Member); Yi Pang (Committee Member) Subjects: Biology; Chemistry

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2022, Photochemical Sciences

H. Peter Lu, Advisor Single-molecule fluorescence spectroscopy has been developed as a powerful technique that provides details of protein-protein and protein-DNA interactions, enzyme reactions, and conformational dynamics. It is highly informative to study protein's conformational dynamics during their activity or under the enzymatic condition to understand their function. The real-time monitoring of the enzyme's active site conformational dynamics and simultaneously activity is informative towards understanding the mechanism of action. Studying how conformational fluctuation dynamics play role in protein or enzyme activity can shed light on the field of enzymology. Particularly in this dissertation, we have employed Forster Resonance Energy Transfer (FRET) as a powerful tool to study the conformational dynamics of Calmodulin (CaM) during its interaction with an autoinhibitory domain (C28W peptide) of the Plasma Membrane Calcium ATPase (PMCA). FRET between donor dye-labeled N-domain of the calmodulin interacting with acceptor dye-labeled peptide reports the real-time conformational dynamics of this interaction which is essential for PMCA activation. Interestingly, by using a unique statistical method, the results provide a mechanistic understanding of CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics. In another study, the conformational dynamics of recognition proteins are studied to understand the mechanism of identification of DNA damage by two recognition proteins, Replication Protein A (RPA) and Xeroderma Pigmentosum Protein A (XPA). We use single-molecule fluorescence fluctuation measurements of a dye, labeled at a damaged position on DNA, to understand the interaction of the damage site with RPA14 and XPA. Our res (open full item for complete abstract)

Committee: Hong Peter Lu Ph.D. (Advisor); Vivian J Miller Ph.D. (Other); John R Cable Ph.D. (Committee Member); Mikhail A Zamkov Ph.D. (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2021, Physics

The ability to apply and measure high forces (≥10pN) on the nanometer scale is critical to the ongoing development of nanomedicine, molecular robotics, and the understanding of biological processes such as chromatin condensation, membrane deformation, and molecular motors [1] [2] [3]. Current force spectroscopy techniques rely on micron-sized handles to apply forces, which can limit applications within nanofluidic devices or cellular environments [4]. To overcome these limitations, I used deoxyribonucleic (DNA) origami to self-assemble a nanocaliper, building on previous designs[5] [6]. I characterize the nanocaliper via a short double-stranded (ds)DNA with each strand attached to opposite arms of the device, via device equilibrium state, output force, and dynamics, to understand the effects of sequence, vertex design, and strut length on the device properties. I also produce nucleosomes, hexasomes, and an alternate dsDNA, which were then measured in the device, yielding mechanistic insight into the free energy landscape of each. I measure forces greater than 20 pN applied by the device with a nanometer dynamic range and 1 to 10 pN/nm stiffness. These high performing characteristics which expand the capabilities of existing force spectroscopy techniques as well as those of DNA origami devices.

Committee: Michael Poirier (Advisor); Ralf Bundschuh (Committee Member); Carlos Castro (Committee Member); Ezekiel Johnston-Halperin (Committee Member) Subjects: Biochemistry; Biophysics; Nanoscience; Physics

Doctor of Philosophy, University of Akron, 2020, Chemistry

Single-cell fluorescence microscopy is a powerful tool which can be used to investigate the nature of cellular responses to external stimuli. The application of single-cell fluorescence microscopy to the growing problem of antimicrobial susceptibility detection stands to improve and refine our ability to address bacterial resistance in human infections. In cases of severe bacterial infection, determining the correct antibiotic to use to combat an infection is a race against the patient's dwindling lifespan. By combining the RedoxSensor™ Green fluorescent dye with live single-cell imaging, we have developed a method for antimicrobial susceptibility testing which can identify susceptibility to a given antibiotic within 100 minutes of treatment. We show that this method is reproducible and can identify susceptibility to several bacterial cell wall targets and bacterial cell membrane targeted antibiotics. Our methodology has considerable applicability within the sphere of rational antibiotic drug design as well in its ability to identify antibiotic efficacy as a function of time instead of antibiotic concentration. We use our method to compare the efficacy of 4 recently synthesized polyurethane antimicrobials. Our work lays the framework for expansion upon our method into microfluidics systems and use in screening candidate antimicrobial drugs. Mitochondrial morphological analysis within living eukaryotic cells represents another challenge which requires careful application of single-cell fluorescence microscopy. The cuprizone mouse treatment model for multiple sclerosis is known to generate enlarged mitochondria within oligodendrocytes, but much remains unknown about the dynamics of their formation. Cultured MO3.13 oligodendrocyte cells treated with cuprizone are shown to undergo mitochondrial enlargement within 8 hours of direct cuprizone exposure via single-cell mitochondrial size determination with MitoTracker Red fluorescent dye. Cuprizone treatment is also shown (open full item for complete abstract)

Committee: Michael Konopka (Advisor); Shriver Leah (Committee Member); Smith Adam (Committee Member); Paruchuri Sailaja (Committee Member); Joy Abraham (Committee Member) Subjects: Biochemistry; Chemistry; Microbiology

Doctor of Philosophy (PhD), Ohio University, 2019, Chemistry and Biochemistry (Arts and Sciences)

Single molecule fluorescence microscopy has been used in the past two decades to improve resolution and view information unattainable from bulk studies. As a relatively new field there is still a lot of areas to explore. This dissertation focuses on single molecule studies on two groups of dyes, including the YOYO-POPO-TOTO family of DNA intercalating dyes and fluorescent polymer dots. The motivation is to understand their special properties in single-molecule microscopy. In this dissertation I study YOYO-1's quenching mechanism, its binding and bleaching in DNA, and its production of reactive oxygen species (ROS) under light illumination. I studied YOYO-1's quenching mechanism by immersing it in air, water, and hexane. The difference in fluorescence in each medium suggest a different quenching mechanism than the currently accepted intramolecular rotation and the energy transfer mechanism. This finding has led to further mechanistic studies. I also study the photobleaching of YOYO-1 at different laser power densities. This is used to relate the bleaching lifetime to the binding of YOYO-1 to DNA to maintain a sparse single molecule density along the DNA strand during point accumulation for imaging in nanoscale topography (PAINT) imaging. A generalized set of equations is provided to calculate the minimum laser power density required to sufficiently bleach YOYO-1 fast enough to acquire a high-quality super resolved image of DNA. Both YOYO-1 and POPO-1 in DNA are used as a platform to study the generation of ROS at the single DNA level by the activation of CellRox, a dye that becomes fluorescent upon its reaction with superoxide or hydroxyl radicals. The generation of ROS was successfully monitored through the fluorescence increase of CellRox. A theoretical resolution limit for detecting the location of the generation of the ROS was suggested to be ~140 nm. Novel fluorescent polymer dots were characterized at the single molecule level. We found that the two polymer dot (open full item for complete abstract)

Committee: Jixin Chen (Advisor) Subjects: Biophysics; Chemistry; Physical Chemistry

Doctor of Philosophy, The Ohio State University, 2018, Physics

Individual cells decipher and react to both their chemical and mechanical environment. Clathrin-mediated endocytosis (CME) is a major process by which cells internalize macromolecules. The triskelion-shaped clathrin protein assembles on the membrane as a spherical lattice enveloping the membrane until scission begets internalization. The membrane curvature generated by the invaginations during endocytosis associate CME with the mechanical environment of the cell. Fluorescence microscopy is used to study the dynamics of CME, and in particular to discern the time it takes for CME events to complete (i.e. their lifetime). It is our hypothesis that the lifetime of CME events relates inversely to the cell membrane tension. We will support this hypothesis with live-cell imaging on glass substrates and in living organisms. We suggest a new methodology for studying CME dynamics that enables higher spatial and temporal resolution than lifetime analysis. We will also characterize the tension response of CME by using various cell manipulation techniques. In addition, we will demonstrate the ability of CME dynamics to predict cell movement and relate gradients in clathrin coat growth rates to previously established tension gradients in cultured cells and living organisms. Finally, we will demonstrate that this process is in a state of continual curvature generation.

Committee: Comert Kural (Advisor); Ralph Bundschuh (Committee Member); Michael Poirier (Committee Member); Sooryakumar Ratnasingham (Committee Member) Subjects: Biophysics; Physics

Doctor of Philosophy, University of Akron, 2018, Chemistry

Methanotrophs are a group of bacteria that are able to utilize methane as their sole carbon and energy source. The vast majority of these bacteria make extensive intracytoplasmic membrane (ICM) structures which house the particulate methane monooxygenase enzyme that converts methane to methanol. Methanotrophs are divided into two classes based on metabolic preferences and ICM architecture, with type I methanotrophs producing ICMs resembling stacked disks, and those of type II methanotrophs resembling concentric rings. Because these ICMs and other useful biomolecules, such as polyhydroxybutyrate (PHB), are synthesized from an inexpensive starting material they are of interest as a cost-effective source of biofuels. Thus far these membranes have been studied using primarily transmission electron microscopy which prevents in vivo analysis of these membranes and limits throughput. Here we demonstrate an alternative analysis for these ICMs using fluorescence microscopy and lipophilic dyes. Using confocal microscopy, ICMs can be quantified on a single cell level by measuring internal fluorescent area. We show that these measurements are sensitive to changes in ICM quantity and can be performed on live cells to monitor membrane dynamics. Furthermore, PHB can be imaged and quantified in a similar way. During imaging differences in ICM staining between type I and type II methanotrophs can be seen. These differences lead us to believe that ICMs in type I methanotrophs exist primarily as invaginations of the cytoplasmic membrane, while ICMs in type II methanotrophs exist as fully isolated structures. We also implement MARTINI coarse-grained molecular dynamics simulations to simulate interactions between model lipid bilayers and methane molecules in solution. These simulations show us that methane inserts itself into lipid bilayers with high efficiency and is able to passively diffuse into fully internalized membrane regions. The fluorescence methods detailed her (open full item for complete abstract)

Committee: Michael Konopka (Advisor); Leah Shriver (Committee Member); David Modarelli (Committee Member); Sailaja Paruchuri (Committee Member); Hazel Barton (Committee Member) Subjects: Biochemistry; Biophysics

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2016, Photochemical Sciences

This dissertation work investigated the parameters affecting the interfacial electron transfer (ET) dynamics in dye–semiconductor nanoparticles (NPs) system by using single-molecule fluorescence spectroscopy and imaging combined with electrochemistry. The influence of the molecule–substrate electronic coupling, the molecular structure, binding geometry on the surface and the molecule-attachment surface chemistry on interfacial charge transfer processes was studied on zinc porphyrin–TiO2 NP systems. The fluorescence blinking measurement on TiO2 NP demonstrated that electronic coupling regulates dynamics of charge transfer processes at the interface depending on the conformation of molecule on the surface. Moreover, semiconductor surface charge induced electronic coupling of molecule which is electrostatically adsorbed on the semiconductor surface also predominantly alters the ET dynamics. Furthermore, interfacial electric field and electron accepting state density dependent ET dynamics has been dissected in zinc porphyrin–TiO2 NP system by observing the single-molecule fluorescence blinking dynamics and fluorescence lifetime with and without applied bias. The significant difference in fluorescence fluctuation and lifetime suggested the modulation of charge transfer dynamics at the interface with external electric field perturbation. Quasi-continuous distribution of fluorescence intensity with applied negative potential was attributed to the faster charge recombination due to reduced density of electron accepting states. The driving force and electron accepting state density ET dependent dynamics has also been probed in zinc porphyrin–TiO2 NP and zinc porphyrin–indium tin oxide (ITO) systems. Study of a molecule adsorbed on two different semiconductors (ITO and TiO2), with large difference in electron densities and distinct driving forces, allows us to observe the changes in rates of back electron transfer process reflected by the suppressed fluorescence blin (open full item for complete abstract)

Committee: H. Peter Lu Dr. (Advisor); Yu Zhou Dr. (Other); Ksenija D. Glusac Dr. (Committee Member); Alexey T. Zayak Dr. (Committee Member) Subjects: Chemistry; Physical Chemistry; Physics

Master of Science, University of Akron, 2015, Physics

Cellular membranes are complex assemblies and a clear understanding of the physical interactions during their function is of paramount importance. Here, we perform two separate studies for a better understanding of the interactions between membrane compartments and other biomolecules. In the first study, we developed a coupler to integrate a high sensitivity spectrometer with an epi-fluorescence microscope to measure fluorescence spectra of small area samples (400 micrometer squared). We applied our measurements on standard samples, performed three corrections on them and after a linear demixing process, the percentage of FRET efficiency was obtained. The development of this method will be advantageous in future single cell studies for detecting population heterogeneity. In the second study, we investigated the dynamics of membrane lipids in a supported lipid bilayer. Single particle tracking total internal reflection fluorescence microscopy (TIRF) was used to study the lateral mobility of phosphatidylinositol phosphate (PIP) lipids with and without an adsorbed polycationic polymer, quaternized polyvinylpyridine (QPVP). Diffusion coefficients were determined with Brownian and anomalous models. Our results indicate a decrease in diffusion coefficient of the lipids in the presence of QPVP in comparison to its absence, revealing their interaction.

Committee: Adam Smith (Advisor); Jutta Luettmer-Strathmann (Committee Chair); Sergei Lyuksyutov (Committee Member) Subjects: Biophysics; Chemistry; Physical Chemistry; Physics

PHD, Kent State University, 2013, College of Arts and Sciences / Department of Physics

A Langmuir film, which is a molecularly thin insoluble film on a liquid substrate, is one practical realization of a quasi-two dimensional matter. The major advantages of this system for the study of phase separation and phase co-existence are (a) it allows accurate control of the components and molecular area of the film and (b) it can be studied by various methods that require very flat films. Phase separation in molecularly thin films plays an important role in a range of systems from biomembranes to biosensors. For example, phase-separated lipid nano-domains in biomembranes are thought to play crucial roles in membrane function. I use Brewster Angel Microscopy (BAM) coupled with Fluorescence Microscopy (FM) and static Light Scattering Microscopy (LSM) to image phases and patterns within Langmuir films. The three microscopic techniques – BAM, FM and LSM - are complimentary to each other, providing distinct sets of information. They allow direct comparison with literature results in lipid systems. I have quantitatively validated the use of detailed hydrodynamic simulations to determine line tension in monolayers. Line tension decreases as temperature rises. This decrease gives us information on the entropy associated with the line, and thus about line structure. I carefully consider the thermodynamics of line energy and entropy to make this connection. In the longer run, LSM will be exploited to give us further information about line structure. I have also extended the technique by testing it on domains within the curved surface of a bilayer vesicle. I also note that in the same way that the presence of surface-active agents, known as surfactants, affects surface energy, the addiction of line active agents alters the inter-phase line energy. Thus my results set to stage to systematically study the influence of line active agents -`linactants’ - on the inter-phase line energy. Hierarchal self-assembled chiral patterns were observed as a fun (open full item for complete abstract)

Committee: Elizabeth Mann Dr. (Committee Chair); David Allender Dr. (Committee Member); Hamza Balci Dr. (Committee Member); J. Mann Dr. (Committee Member); Hiroshi Yokoyama Dr. (Committee Member); Qi-Huo Wei Dr. (Committee Member) Subjects: Biophysics; Physics

Master of Science (MS), Bowling Green State University, 2013, Chemistry

Conformational dynamics plays an important role in determining protein reactivity including affinity to substrate and product, catalytic rate constant and product release process. Whereas the influence of conformational dynamics on kinetic parameters of enzymatic reaction have been widely studied by ensemble averaging techniques, in these experiments conformational variations tend to average out and therefore ensemble averaging methods lack the level of resolution necessary to characterize time dependent behavior of individual enzyme molecules, which is essential to understanding enzymatic catalysis. This thesis consists of two parts. The first part focuses on the new development of integrated spectroscopy system, which combined the advantages of wide plane view field of objective-type TIRFM imaging with high time-resolution confocal single-molecule spectroscopy to address the challenge of probing both temporally and spatially stochastic events of the single-molecule reactions, such as fluorogenic enzymatic reactions with enzyme tethered to a solution/glass interface. Tthe calibration of this integrated apparatus is demonstrated with fluorescence microspheres and the feasibility by spectroscopy measurement of single molecule Rhodamine 6G on glass surface. Finally, the application of the apparatus is demonstrated by studying spatially and temporally randomly distributed single-molecule enzymatic reaction of fluorogenic substrate, horseradish peroxidase-catalyzed oxidation of amplex red. The second part focuses on exploratory studies of enzyme Alkaline Phosphatase from E. coli and its potential to be studied on single molecule level using fluorogenic substrate 3-o-methylfluorescein phosphate (MFP) and two polarization total internal reflection fluorescence microscopy apparatus. Ensemble averaged kinetics data and several single molecule trajectories are presented here.

Committee: Peter Lu (Advisor); Neocles Leontis (Committee Member); George Bullerjahn (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2002, Biophysics

Reactive oxygen species (ROS) play an important role in many biological systems. Skeletal muscles have been shown to generate considerable ROS in resting and in contracting conditions. In this study, I tested the hypothesis that increased ROS production in skeletal muscles is also associated with exposure to two other conditions of stress which are common in normal skeletal muscle during exercise, namely heat stress and hypoxia. There is no previous direct evidence that ROS are produced during these stresses, particularly in skeletal muscle, but they may play important roles in normal contractile and cell signaling responses. Two assays for superoxide (O2•-) formation were used in rodent diaphragm, the cytochrome c assay for extracellular O2•- release and the hydroethidine oxidation for intracellular O2•- formation. The results demonstrate the following: 1) Markedly increased intra- and extracellular ROS formation was observed, particularly O2•-, at temperatures known to be physiologically relevant to exercise (i.e. 42°C). 2) The process of O2•-, release (extracellular formation) is not directly related to mitochondria, NADPH oxidase, or anion channels, though these are normally believed to be involved in either O2•- generation or the exit pathway of O2•- through membranes. 3) Upstream pathways of arachidonic acid (AA) metabolism, both phospholipase A2 and nitric oxide synthase are associated with O2•- release. 4) Downstream pathways of AA metabolism are also involved. Though blockage of either cyclooxygenase or cytochrome P450-dependent enzymes does not cause any inhibition of O2•- release, blockage of lipoxygenase (LOX) results in near elimination of the signal. This suggests that O2•- release is dependent on AA metabolism through the LOX pathway. However, confocal measurements of intracellular O2•- formation suggest that intracellular ROS are produced by a separate mechanism. 5) Tissue fluorometry techniques, using a fluorescence probe sensitive to hydrogen perox (open full item for complete abstract)

Committee: Thomas Clanton (Advisor) Subjects:

Master of Science (MS), Ohio University, 2006, Physics (Arts and Sciences)

Single molecule fluorescence microscopy is a powerful tool to investigate local environments at the nanometer scale. Oxidation of DNA is an important problem that leads to DNA mutations. In this project, single molecule fluorescence measurements were used to study the interaction between covalently bound nucleotides (dNTP) and fluorophores (Cyanine dye), dNTP-Cy3 that have been incorporated into DNA. By tracing changes in the fluorescence signal, evidence for single events of DNA oxidation were found that could otherwise not be observed at the ensemble level. The differences between the nucleotides and their quenching properties are shown here especially guanine's ability to temporarily quench the fluorescence of Cy3 completely. After some illumination time, the quenching of the dye by the guanine changes dramatically. We interpret this change to possible oxidation of the guanine base. This work could lead to a method for monitoring and investigating important DNA oxidation processes which are essential to Genomic and Cancer research.

Committee: Ido Braslavsky (Advisor) Subjects:

Master of Science (MS), Ohio University, 2005, Biological Sciences (Arts and Sciences)

NO is an important mediator of neuronal physiology (signaling) and pathology (neuronal death via apoptosis and necrosis). Previous studies using lung endothelial cell cultures, mouse fibroblast cell cultures and neuronal cell cultures have shown that there is an increase in intracellular Zn 2+ in the presence of relatively high concentrations of NO > 100μM. The current study done on primary cortical neuronal cultures uses fluorescence microscopy and the fluorescent Zn 2+ probe ZnAF-2F DA to prove that there is a significant increase in intracellular Zn 2+ in the presence of relatively low concentrations of NO (0.5μM-5μM) in the perfusion medium. The fluorescence microscopy experiments measure free intracellular Zn 2+ levels. Radioactive 65 Zn 2+ was used to determine the release of 65 Zn 2+ to the extracellular space upon treatment with 2μM NO. There was a small increase in extracellular 65 Zn 2+ after NO treatment was started but it did not reach statistical significance when compared with the control. There was no significant difference between the control and 2μM NO in the total 65 Zn 2+ release during treatment with TPEN. The residual amount of Zn 2+ present in the cells after the TPEN treatment was greater when 2μM NO was present, but did not reach statistical significance. Since 65 Zn 2+ labels all intracellular Zn 2+ and fluorescent Zn 2+ probes measure only free intracellular Zn 2+ , these results suggest that the Zn 2+ released intracellularly is sequestered by TPEN insensitive intraneuronal stores. Finally this study also revealed that there is no significant cell death at 0.5μM-5μM NO when intracellular Zn 2+ is elevated. This is most probably due to lack of peroxynitrite formation due to the antioxidants present in the neurobasal medium.

Committee: Robert Colvin (Advisor) Subjects:

Doctor of Philosophy in Medicinal Chemistry (Ph.D.), University of Toledo, 2011, College of Pharmacy

The primary cilium is an important sensory organelle present in most mammalian cells. The cilium is involved in regulating various essential cellular processes and by virtue of its structure and location; the most important function of the primary cilium is to act as a sensor. The cilium senses the conditions in the extra cellular matrix and transduces the message to the cell interior resulting in changes in gene expression and protein synthesis. Dysfunctional cilia result in a variety of diseases, collectively called as “ciliopathies”. Our current studies have a two-fold aim. First, we aim to show that pharmacological agents modulate cilia length. To prove this, we examined intracellular molecules that regulate cilia length and/or cilia function in vitro and ex vivo. For the first time, we show that intracellular cAMP and cAMP-dependent protein kinase (PKA) regulate both cilia length and function in vascular endothelial cells. Although calcium-dependent protein kinase (PKC) modulates cilia length, it does not play a significant role in cilia function. Cilia length regulation also involves mitogen-activated protein kinase (MAPK), protein phosphatase-1 (PP-1) and cofilin. Furthermore, cofilin regulates cilia length through actin rearrangement. Overall, our study suggests that the molecular interactions between cilia function and length can be independent of one another. We propose that cilia length and function are regulated by distinct, yet complex intertwined signaling pathways. Our second aim is to show that dysfunctional dopamine/dopamine receptors are related to hypertension observed in polycystic kidney disease (PKD). PKD is characterized by cardiovascular irregularities, including hypertension. Dopamine, a circulating hormone, is implicated in essential hypertension in humans and animal models. Vascular endothelial primary cilia are known to function as mechano-sensory organelles. Though both primary cilia and dopamine receptors play important roles in vascula (open full item for complete abstract)

Committee: Surya Nauli (Committee Chair); Paul Erhardt (Committee Member); William Messer (Committee Member); Zi-Jian Xie (Committee Member); Anthony Quinn (Committee Member); Patricia Komuniecki (Other) Subjects: Biomedical Research

BS, Kent State University, 2012, College of Arts and Sciences / Department of Physics

A single molecule FRET study was performed to gather structural and dynamic information about a G-Quadruplex (GQ) in the presence of potassium ions (K+) and Replication Protein A (RPA), a predominant single strand DNA binding protein. A GQ is a non-canonical DNA structure that forms in guanosine (G) rich sequences. The structure is composed of stacked tetrads, each formed by four G residues, and loops of unstructured ssDNA. This study concentrates on characterization of a three-layer GQ with loops that are three nucleotides long each. The results confirm the formation of the GQ structure, while revealing nearly 100% folding at the physiological concentration of K+ (150 mM). It was also found that RPA-mediated unfolding dominates folding by K+ under physiological conditions, giving some insight into this structure's physiological viability. The timescales of transitioning from an unfolded single strand structure to a folded GQ, along with the transition from a folded GQ to an RPA-bound unfolded structure were also measured in this single molecule assay. The transition from unfolded to folded GQ structure was measured to be 0.35±0.10 seconds, while the transition from a folded GQ to an RPA-bound unfolded structure was 0.28±0.10 seconds. These results suggest that the folding and unfolding dynamics of this structure take place on a similar timescale. The rest of this study compares the results from this specific GQ structure to results gathered from the same assays on other GQ structures with varying number of layers and loop lengths to draw conclusions about these structures' physiological relevance.

Committee: Hamza Balci PhD (Advisor); Elizabeth Mann PhD (Committee Member); Soumitra Basu PhD (Committee Member); Natasha Levinson PhD (Committee Member) Subjects: Biology; Biophysics; Optics; Physics

PHD, Kent State University, 2011, College of Arts and Sciences / Department of Physics

This dissertation looks at the phase-separation of two binary lipid systems: (a) an experimental study of binary lipid Langmuir monolayers and (b) the theoretical modeling of a binary system of lipids in a symmetric bilayer. Experimentally, monolayers of dihydrocholesterol (Dchol) and dimyristoylphosphatidylcholine (DMPC) at the air/water interface were studied with Brewster angle microscopy (BAM) and fluorescence microscopy (FM) in order to test the effect of fluorescent dyes, which may be strongly line active, on the phase diagram. The theoretical model of phase separation in mixed lipid bilayers emphasizes the asymmetry of the two layers of a bilayer. A Landau free energy in terms of order parameters the cholesterol mole fraction, and the hydrocarbon chain length is extended to look at both cubic and quartic terms. We found four states: (1) a single homogeneous phase, (2) two-phase coexistence, (3) three-phase coexistence, and (4) four-phase coexistence, depending on temperature and the composition of the layers. Three phase and four phase coexistence are of special interest because they have been little studied either theoretically or experimentally.

Committee: David Allender Dr. (Committee Chair); Elizabeth Mann Dr. (Committee Co-Chair); John Portman Dr. (Committee Member); Edgar Kooijman Dr. (Committee Member); Anatoly Khitrin Dr. (Committee Member) Subjects: Biophysics; Physics; Solid State Physics