Master of Science (M.S.), University of Dayton, 2022, Chemistry

The spectroscopic properties of a fluoranthene pyrrole based, meso-mesityl substituted dipyrromethene (FlMst) were investigated.This novel compound produced visible absorption and substantial fluorescent properties. This compound is the first non-bridged dipyrromethene to produce substantial flourescence that has been recorded in literature. FlMst produced an absorption peak at 583 nm, and an emission peak at 640 nm, with a fluorescence quantum yield of 0.07. The effects of pH on optical behavior was studied through both a titration using TFA, as well as electrochemical experiments using cyclic voltammetry. The titration with TFA resulted in the loss of fluorescent, and a shift of the absorbance peak to 630 nm. Cyclic voltammetry provided supporting evidence for the effect of TFA on FlMst, offering data on molecular orbital shifts and stabilizations congruent with the titration data. This demonstrated the “On/Off switch” like behavior of FlMst in environments of varying pH. This work has a primary focus on the attributes on the FlMst dipyrromethene, but this thesis also presents a preliminary investigation of the synthesis, spectroscopy, and electrochemistry of the FlMst BODIPY.

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)

Doctor of Philosophy, University of Akron, 2019, Chemistry

Fluorescence molecular probes have become one of the most popular tools in many aspects of chemistry and biology. Many fluorescence probes have been developed for the detection of different cations, anions, and some essential small molecules in the live cells. Some of the probes are useful for visualization of the cell organelles, which could be powerful tools to aid the study in cellular activities. Near-infrared (NIR) emitting probes are desirable for biological applications as they offer deep tissue penetration and reduce the autofluorescence and background noise. Toward this direction, several new NIR emitting fluorescence probes have been developed via combining excited-state intramolecular mechanism (ESIPT) with cyanine, and other different architectures. In chapter II, a cyanine dye 2.1 (λem~700 nm) with a large Stokes' shift (Δλ ≈ 234 nm) is synthesized by coupling 2-(2'-hydroxyphenyl)benzothiazole (HBT) with a benzothiazolium-derived unit. The phenolic proton, which controls ESIPT, also acts as a switch to respond to an acidic environment (pH=4-6). Staining the normal human lung fibroblast cells (NHLF) reveals that 2.1 is selectively staining lysosome without “alkalinizing effect.” Chapter III demonstrates the synthesis of NIR-emitting dyes 3.1 and 3.2 with large Stokes' shifts (Δλ ≈ 231 - 246 nm), whose structures consist of a hemicyanine unit (benzothiazolium and indolium respectively) and an ESIPT unit [2-(benzo[d]oxazol-2-yl)-4-methylphenol (HBO)]. Compound 3.1 (Ф ≈ 0.28 – 0.35 in CH2Cl2) exhibited excellent selectivity for staining intracellular lysosomes. However, probe 3.2 (Ф ≈ 0.27, in CH2Cl2) has excellent selectivity towards mitochondria. Chapter IV includes a near infrared-emitting pyridinium cyanine with a large Stokes shift (Δλ ≈ 230 nm) which has been synthesized by coupling pyridinium cyanine with ESIPT unit HBT. The developed new probe 4.1a exhibits a significantly higher fluorescence quantum yield (Фfl≈0.34 in Dichloromethane) than c (open full item for complete abstract)

Doctor of Philosophy, University of Akron, 2018, Chemistry

G protein-coupled receptors (GPCRs) make up the largest family of cell surface protein receptors and are involved in a number of diverse biological processes. The association of GPCRs, whether they be monomeric, dimeric, or oligomeric, is hypothesized to alter their signaling. Attaining crystallographic evidence of the dimeric or oligomeric associations of Class A GPCRs, specifically (non)visual opsins, remains a difficulty, as does establishing the stability of these associations. The purpose of this research was to quantify the association of (non)visual opsins, in situ, in the plasma membrane of live cells. We used a time-resolved fluorescence approach to accomplish this purpose. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) offered a way in which the dynamic interactions of (non)visual opsins could be quantified. Throughout this dissertation, three projects will be presented. The first project focused on the dimeric association of rhodopsin, the light sensitive protein involved in scotopic vision. By transfecting low concentrations of rhodopsin into mammalian cells, we found a modest affinity for dimerization. The second project focused on the proteins involved in trichromatic photopic vision, cone opsins. Two of the three human cone opsins, OPN1LW (red) and OPN1MW (green) share a 95% sequence homology. Despite having such a homology, red and green cone opsin showed different affinities for dimerization. Red cone opsin was observed to have the highest affinity for dimeric association among the GPCRs studied. Green cone opsin was shown to primarily exist as a monomer. Mutagenesis was performed on both red and green cone opsin in an attempt to decrease red cone opsin dimerization affinity and increase green cone opsin dimerization affinity. The third project focused on melanopsin, a non-visual human opsin. Melanopsin is expressed in the ganglion cell layer (GCL) of the retina and plays a role in both circadian rhythm and t (open full item for complete abstract)

Doctor of Philosophy, University of Akron, 2017, Chemistry

The cell membrane is a complex environment made up of thousands of molecular components. The dynamic assembly of these components regulates a myriad of cellular functions, but it is difficult to measure in a biologically relevant context. Pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) is a time resolved fluorescence technique that was used to obtain concentration, mobility and co-diffusion (fc) of membrane proteins in live cells. Several lines of evidence support the hypothesis that homo-dimerization (or even oligomerization) facilitates the function of membrane proteins. The goal of this research was to elucidate the dynamic organization and relative affinity of membrane protein-protein interactions. In order to accomplish this goal, a mathematical model was developed to interpret the cross-correlation value obtained from PIE-FCCS and to quantify the dynamic interactions of membrane receptors in a more rigorous way. This thesis describes three main projects. The first project focused on determining the homo-dimerization of the neuronal membrane protein PlexinA4 before and after ligand stimulation. PIE-FCCS measurements of protein controls with varying degrees of homo-multimerization were used to determine that PlexinA4 receptors assemble into preformed dimers. In the next project, a mathematical model was developed to interpret the PIE-FCCS figure of merit, fc. Several controls systems with varying dimerization affinity and degrees of oligomerization were measured and analyzed to verify the accuracy of the model. Lastly, the organization of two class A G protein-coupled receptors (GPCRs) was investigated using PIE-FCCS and a new labeling strategy. The organization of these membrane proteins showed a high degree of cell to cell variability. A simple monomer-dimer equilibrium model failed to describe the range of single cell data, so the mathematical model was altered to describe a cluster model that agreed with the experiment (open full item for complete abstract)

Master of Science, University of Akron, 2017, Physics

Fluorescence correlation spectroscopy (FCS) is a useful experimental technique, that uses statistical analysis of fluorescence intensity fluctuations and a correlation function. The method permits studying the kinetics and interactions of particles not only on the surface of the solution, but also in the bulk. Another benefit of FCS is that it does not require the selection of specific molecules or time intervals for its measurements. By analyzing biological dynamics, we can learn about concentration, viscosity, individual movement and diffusion of particles as well as interactions and the possibility of binding between them. In this work, we present a brief explanation on the analytic formalism of the governing concepts of FCS, as well as detail the experimental setups. We also discuss protein-vesicle interactions and the kinematics of double-stranded DNA and labeled nucleotides. The brightness and size of molecules, before and after adding vesicles to the protein solution, are calculated. Finally the possibility of vesicle-protein binding is validated.

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.

Doctor of Philosophy, University of Akron, 2014, Chemistry

As the limitations of fossil fuels and the effects they have on the environment become more apparent, the need for new alternative energy sources is becoming increasingly obvious. One potential source of renewable energy converts solar energy using organic and inorganic based photovoltaic (PV) cells into electrical energy. In this dissertation, I have used fluorescence spectroscopy to study two photoactive organic compounds that have been shown to undergo self-assembly, and two organic-inorganic hybrids that were also designed for use in PV cells. I also developed a new technique to study sub-picosecond fluorescence anisotropy. The organic compounds studied in this work were amphiphilic N,N-disubstituted naphthalene diimides that were shown to self-assemble into highly ordered aggregates that eventually formed nanostructures such as twisted nanotapes, twisted nanoribbons, and nanotubes. In this work, picosecond time correlated single photon counting (TCSPC) was used to investigate the fluorescence lifetimes and time-dependent fluorescence anisotropies nanostructures. For two of these compounds (Dipeptide B and Bola 1), the fluorescence lifetimes (tfl ~153 ps for Dipeptide B and tfl ~313 ps for Bola 1) were over an order of magnitude longer than that of a simple naphthalene diimide (N,N-dibutyl naphthalene diimide, tfl ~16.4 ps). The lifetime results were consistent with energy migration within highly ordered nanostructures. Further, the time-resolved fluorescence anisotropy results indicated an ultrafast depolarization of the fluorescence signal that could only be attributed to energy migration along a twisted or circular structure. The second portion of this work involved organic-inorganic hybrids consisting of a capped cadmium sulfide nanoparticle forming nanocomposites with a homo-poly(phenylene-ethynylene) polymer and an alternating co-poly(phenylene-ethynylene) polymer. In this work, picosecond TCSPC was used to investigate the average lumin (open full item for complete abstract)

Master of Science, The Ohio State University, 2009, Chemical Engineering

Cell assays for high-throughput screening (HTS) of potential drug candidates are important tools in the process of drug discovery. Most cellular assays are currently based on 2-D monolayer cell cultures, but 3-D cell cultures could better mimic the in vivo characteristics of actual organism tissues. Unfortunately, assays using 3-D culture models usually require significant manual manipulation and are therefore not suitable for HTS. Research under Dr. Shang-Tian Yang has resulted in a functioning system for high-throughput 3-D cellular assays using engineered cells to express enhanced green fluorescence protein (EGFP) quantifiable through fluorometry. System improvement to allow rapid assessment of cellular events, such as specific gene expression or cell cycle progress is limited by the long persistence of the current reporter protein in the cells. In this study a new fluorescence reporting cell line was established using a destabilized EGFP (d4EGFP) expressed in Chinese hamster ovary (CHO) cells. Correlating fluorescence with cell number for the d4EGFP cell line in 2-D assays indicated that d4EGFP expression may be too low for use in high-throughput cell number reporting. The fluorescence and cell number correlation in 3-D assays indicated better performance could be achieved in 3-D but the fluorescence was sensitive to duration between sampling, possibly due to oxygen transfer limitation, hindering reliable use for cell number reporting. Response to such factors could still serve a purpose for culture condition monitoring, and could be applied in culture development and optimization.

Doctor of Philosophy, The Ohio State University, 2004, Mechanical Engineering

A new trans-rotational temperature diagnostic with ±50K accuracy has been developed for use in nonequilibrium, low temperature, monatomic gases seeded with carbon monoxide (CO). The scheme utilizes single-photon laser induced fluorescence (LIF) of CO under vibrationally-excited conditions in which single-photon transitions from the CO X1Sig+ ground electronic state to upper electronic A1Pi or D'1Sig+ states become accessible to a tunable, narrowband ArF excimer laser at 193 nm. Two vibrationally excited environments in which the chemistry is well understood were used as a testbed; an optically-pumped 3% CO/Ar plasma at 100 torr and a 4% CO/He d.c. glow discharge at 8 torr. For the optically-pumped CO/Ar plasma, a spatially-averaged temperature of 536±103 K (2sig) was obtained from rotationally resolved X1Sig+(v"=20)-D'1Sig+(v'=2) LIF excitation spectra. Temperature measurements pumping the X1Sig+(v"=7)-A1Pi(v'=1) 4th Positive (528±51 K) were also found to compare well with line-of-sight Fourier Transform-InfraRed (FT-IR) emission measurements (536±10 K). Spatially averaged FT-IR spectroscopy of the CO 1st overtone was used to verify a vibrational population of ~0.1% within the positive column of the CO/He d.c. glow discharge. The A-X (7,1) transition was pumped and subsequent (8,1) emission at 200.8 nm collected. Spectral peaks were assigned and used to determine a spatially averaged rotational temperature of 432±44 K on the discharge centerline. This was found to be in good agreement with FT-IR spectroscopy measurements (395±10 K). As a prelude to Planar-LIF (PLIF) temperature measurements, vibrationally-resolved emission from laser excitation of various rotational lines within the A-X and D'-X bands were used to investigate spectral interferences. This information was used to determine that a simple aqueous organic filter (urea) in the A-X case, or commercial glass filter (UG-11) in the D'-X case, are adequate for rejecting elastically-scattered radiation and extr (open full item for complete abstract)

Doctor of Philosophy (PhD), Ohio University, 2007, Chemistry (Arts and Sciences)

The T box transcription antitermination system is present in many amino acid biosynthesis, metabolism, and transport, and tRNA synthetase genes in Gram-positive bacteria. The regulatory system involves a novel RNA/RNA interaction in which a complex set of conserved structural and sequence elements within the 5' untranslated leader region of the regulated gene transcript interacts with the uncharged form of the gene's cognate tRNA. As a part of the control mechanism for transcription of the mRNA, two mutually exclusive structures are formed within the 5' leader region – the terminator or antiterminator structure. The cognate tRNA binds to the specifier sequence in stem I of the leader region, but the antiterminator is only stabilized by uncharged cognate tRNA. If a charged tRNA is bound to the specifier sequence, the antiterminator is not stabilized and the terminator forms. Transcription read-through is allowed upon the formation of the antiterminator structure, which consists of a highly conserved seven-nucleotide bulge in which the first four bases of the bulge base pair with the 3' acceptor end of the tRNA. The current studies use models of the Bacillus subtilistyrosyl-tRNA synthetase gene ( tyrS) as a model system for fluorescence and NMR studies of the tRNA acceptor end interaction with the antiterminator. The fluorescence studies have shown that the core fold of the tRNA is necessary for tight binding of the tRNA with the antiterminator. Additional fluorescence studies have shown that the binding of the tRNA with the antiterminator is a two-step process, in which a small conformational change is induced by the first binding step and significant structural reorganization accompanies the second binding step. For this binding interaction to occur, a threshold level of Mg 2+is necessary for "cationic mediation" of the negatively-charged electrostatic repulsion that arises as the 5'-monophosphate at the end of the tRNA comes into close proximity with the phosphate (open full item for complete abstract)

Master of Science (MS), Bowling Green State University, 2012, Chemistry

One mechanism to achieve photon upconversion, the frequency conversion of low energy photons to those of higher energy, is sensitized triplet-triplet annihilation, a non-coherent (lasers not required) process. In this scheme, a triplet sensitizer is selectively excited at long wavelengths, eventually transferring its triplet energy to an appropriate acceptor molecule in a bimolecular energy transfer reaction. Finally, a second bimolecular energy transfer reaction occurring between two excited triplet acceptors pools the combined energy onto one molecule, producing the fluorescent excited singlet state of the acceptor molecule. This energized molecule radiatively decays back to its ground state releasing photon energies in excess of that of the excitation source, i.e. upconverted with respect to the incident light. This phenomenon has become realized in various combinations of chromophores resulting in wavelength shifting properties that range from the UV to the near-IR. Recently, the upconversion process has become a viable solution to drive fuel-forming chemistry in photoelectrochemical cells and for display applications in polymer host films. The concepts and experiments related to photon upconversion are facile and readily present an opportunity to educate young chemists in this field. Related to established green-to-blue upconversion systems, [Ru(bpy)3](PF6)2 and 9,10-diphenylanthracene (DPA) in deoxygenated dichloromethane is demonstrated here to be a suitable composition for an undergraduate laboratory experiment in physical and/or inorganic chemistry using a conventional fluorimeter. Quadratic incident light power dependence is displayed from the singlet fluorescence of DPA (λem max = 430 nm) resulting from selective excitation of [Ru(bpy)3]2+ at 500 nm using a conventional single photon counting fluorimeter equipped with a 75 W Xe arc lamp. This is easily justified by the fact that two sensitized triplets must be formed in order to ultimately generate the si (open full item for complete abstract)

BA, Oberlin College, 2013, Physics and Astronomy

The lineshapes of the D1 (22S1/2 → 22P1/2 ) and D2 (22S3/2 → 22P1/2) transitions in lithium were measured using a diode laser that was frequency-stabilized to a Ti:Sapphire 1 GHz optical frequency comb. The excitation was achieved by retroreflecting the diode laser, in effect producing the Doppler-free profiles for the center frequencies of transitions. The observed spectra were compared to density matrix calculations to gain insight into systematic effects including the dependence of Doppler-free profiles on power and polarization angle of the diode. For certain transitions, the method of saturated fluorescence spectroscopy inevitably leads to the presence of extra resonances known as crossover signals. Our preliminary data suggest that the presence of this complicating effect may render saturated fluorescence spectroscopy an ineffective technique for resolution of transitions whose relative separation is on the order of the natural linewidth of Li.

PhD, University of Cincinnati, 2024, Arts and Sciences: Geology

Classically, we describe the Eocene-Oligocene Boundary (EOB), which occurred ca. 34 million years ago (Ma), as a time of rapid cooling and aridification. However, while this trend was widespread, not all regions were impacted in the same way. In particular, plant and animal communities in low latitudes, regions that had already experienced aridification earlier in the Eocene, and coastal environments were sometimes less impacted than the rest of the planet. Due to a limited fossil record, Africa represents a continent-sized gap in our understanding of the EOB. Because Africa remained isolated from other continents during the early Cenozoic, it had unique mammal communities that were not comparable to other regions of the world.. In this dissertation I present three related projects that center around using carbon and oxygen isotope values in tooth enamel to improve our understanding of the early Cenozoic environmental conditions in northern Africa (specifically, the Fayum Depression in Egypt), while balancing the ethical necessity of minimizing destructive sampling of unique, scarce, or irreplaceable specimens. First, I examine the relationship between the relative abundance of calcium and phosphorous and the isotopic composition of fossil enamel. The goal of this project is to develop a non-destructive method that uses a handheld X-ray fluorescence spectrometer (hXRF) to evaluate specimen preservation. Such a tool could prevent unnecessary destructive sampling of fossils that have been too diagenetically altered to be useful in isotopic analyses, and could save time and expense. I next evaluate environmental change across the EOB at the Fayum Depression in Egypt using enamel carbon and oxygen isotope values for four orders of herbivorous mammals: proboscideans, embrithopods, hyraxes, and artiodactyls. The Fayum Depression is a uniquely fossiliferous region of northern Africa that was located near the southern coast of the Tethys Sea during the late Eocene (open full item for complete abstract)

Master of Science (M.S.), University of Dayton, 2024, Chemistry

Linear polyenes are a class of compounds containing two or more alternating carbon-carbon double and single bonds that have a range of applications from antioxidants in nature to non-linear optical switches. More recently, other related π conjugated systems (like cinnamaldehyde) with heteroatoms within the π-conjugation are being used as chemical additives in fracturing fluids (or “fracking”) in our continued exploration of crude oil. Though this class of chemicals is considered non-hazardous, they are susceptible to oxidative additions initiated with a free radical oxidant. Because of their relative insolubility in water and high solubility in non-polar solvents, the protonation of conjugated polyenes represents a potential pathway to create novel cyclic conjugated structures in one step or hazardous products in the environment. I will present a systematic approach to analyzing the numerous products from the reaction of linear polyenes with trifluoroacetic acid under Airfree conditions to compare to the relative product distributions. The data generated in this project also provides insight into the relative stability of carbocation intermediates that are potentially created. Based on the structure of the products, we will propose mechanisms for the resulting reactions based on multivariate spectroscopic data sets. Some of the spectroscopic techniques include 1D and 2D NMR, UV-vis spectroscopy, fluorescence spectroscopy, and Mass Spectroscopy. The products that were isolated thus far indicate that inter- and intramolecular cyclization are occurring after the protonation of the polyene occurs.

Master of Science, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

High throughput screening (HTS) has emerged as an efficient method for drug discovery. Unlike traditional screening methods, HTS is a highly sensitive and inexpensive tool that rapidly tests drug libraries to identify lead compounds for the treatment of human disease. Due to the high demand for therapeutic drugs, researchers must optimize new approaches in HTS. A limitation in performing HTS when studying protein-protein interactions is the large amount of protein required. To overcome this limitation, we have designed an approach based on the sequential addition of molecules to a single reaction well. We validated this approach by developing an assay to identify inhibitors of the interaction between the clathrin-coated vesicle adaptor protein CALM and the SNARE protein VAMP8. First, we developed a protocol to produce and isolate high yields of CALM-ANTH domain. Then we designed a fluorescence polarization assay to measure the binding constant of CALM-ANTH domain with VAMP8. Finally, we used the assay to screen over 10,000 compounds from an FDA-approved and commercially purchased library. We have identified a compound that can interfere with CALM-VAMP8 interaction named proflavine, a known DNA intercalating agent that commonly serves as an antiseptic for wound repair. Moreover, we validated a novel strategy for HTS that requires lower amounts of protein when compared with traditional HTS approaches. Our method yields similar results and allows the investigator to obtain direct information on a single compound basis. These studies may lead to increased efficiency of HTS and improved drug identification.

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)

Doctor of Philosophy (Ph.D.), University of Dayton, 2023, Electrical Engineering

The recent pandemic of Corona-virus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. Further, foodborne and waterborne diseases are not only spreading faster, but also appear to be emerging more rapidly than ever before and are able to circumvent conventional control measures. The Polymerase Chain Reaction (PCR) system is a well-known diagnostic tool for many applications in medical diagnostics, environmental monitoring, and food and water quality assessment. Here, we describe the design, development, and testing of a portable, low-cost, and real-time PCR system that can be used in emergency health crises and resource-poor situations. The described PCR system incorporates real-time reaction monitoring using fluorescence as an alternative to gel electrophoresis for reaction analysis, further decreasing the need of multiple reagents, reducing sample testing cost, and reducing sample analysis time. The bill of materials cost of the described system is approximately $340. The described PCR system utilizes a novel progressive selective proportional–integral–derivative controller that helps in reducing sample analysis time. In addition, the system employs a novel primer-based approach to quantify the initial target amplicon concentration, making it well-suited for food and water quality assessment. The developed PCR system performed DNA amplification at a level and speed comparable to larger and more expensive commercial table-top systems. The fluorescence detection sensitivity was also tested to be at the same level as commercially available multi-mode optical readers, thus making the PCR system an attractive solution for medical point-of-care and food and water quality assessment. In general, sensitive testing methods require genetic material (open full item for complete abstract)

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)

Master of Science, Miami University, 2023, Physics

This study presents a series of experiments to extract the temperature-dependent radiative lifetime of the sodium molecules in the 6sΣ𝑔(𝑣=9,𝐽=31) electronic state. Using pulsed lasers, the time-resolved double-resonance spectroscopy method was employed for the excitation scheme. The molecular fluorescence was observed from the 6sΣ𝑔(𝑣=9,𝐽=31) to the 𝐴sΣ𝑢(𝑣′,𝐽′) electronic state and the lifetime measurement was done using a time-correlated photon counting technique. After obtaining the buffer-gas-free lifetime using a Stern-Volmer plot, the temperature dependence of the radiative lifetime was measured over a temperature range from 593 K to 653 K. The result agrees well within the error limits with the theoretical cal