Browsing by Subject "fluorescence"

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Characterizing mVenus adsorption to photodegraded polyethylene using circular dichroism and fluorescence spectroscopy
(2022-08) Amaris, Althea
Due to their versatility and relative cost-effectiveness, plastics as a material have gained increasing popularity and are heavily utilized by almost every major industry in the modern day. Their exponential rate of production coupled with a lack of proper disposal methods, however, have resulted in the global environmental issue of plastic pollution. Upon entering the ecosystem, plastic surfaces can act as a foundation for the formation of microbial communities known as biofilms. An initial key step to biofilm growth is the attachment of bacterial surface proteins onto the polymer. In this study, we examine structural changes of a “hard” model protein in the presence of environmentally relevant plastics. Using the intrinsic probes of the mVenus protein, a model yellow fluorescent protein (YFP), we study its structural response to variably photo-aged polyethylene (PE) through circular dichroism (CD) and tryptophan (W)/YFP-fluorescence spectroscopy. Upon binding to aged PE, mVenus undergoes mild secondary structure rearrangement. Interestingly, a forbidden transition in W-fluorescence is observed, evolving from the interaction between the sole tryptophan in mVenus and the increasingly hydrophilic surface of PE as the polymer is progressively photo-oxidized. The beta barrel and beta sheet structure of mVenus retains the overall stability of the protein, whereas the local structure and turn regions accommodate the protein-polymer interactions based on polymer surface chemistry. We can therefore start to predict that proteins bind variably during the initial docking of cells as the secondary structure behaves distinctly based on the age of the film to which it attaches. The dependence of protein docking on the extent of PE-irradiation reveals that film age, polymer type, and structural stability can either accelerate or inhibit biofilm growth.
Eco-Friendly Boron Complex Addition to Aldehydes
(2019-08) Merritt, Marcy
A series of aldehyde ligands were synthesized to increase yield and decrease resource use and waste production. Boron diphenyl complexes were then separately added to each of the ligands greatly increasing and shifting their intensity and fluorescence wavelength. The complexes were characterized via NMR and GCMS. The stability of these luminescent boron complexes and ability to be vacuum sublimated onto a glass surface makes them suitable for OLED application.
Exploration and Synthesis of Fluorescent Oxadiazole and Thiadiazole Boronyls
(2020-09) Stadem, Samuel
In the last decade, OLEDs have become increasingly ubiquitous in the market, already available in top-range monitors, televisions, and mobile phones. With the push for cheaper devices, much research has focused on finding new dopants to be used in these displays. Simple azole-based organoboron dyes have shown promise in this application, and our lab has investigated tetra-coordinated boron complexes of the oxadiazole and thiadiazole family. These ligands were chosen for their excellent electron transport capability and ability to make use of boron’s unique capability to form B(N,O)X type complexes. Our investigation focused on limiting the rotational sources of internal quenching, and some evidence suggests a notable bathochromic shift when chelated to with BPh2 while chelation to BF2 showed signs of a hypsochromic shift. Unfortunately, a lack of suitable instrumentation forced an end to the exploration of thiadiazole complexes. The onset of COVID-19 further stymied compound characterization, though NMR and limited fluorescence spectroscopic data on BPh2(ODP) was collected.
Metabolic-response assessment of metastatic murine breast cancer in 2D and 3D cultures using intrinsic NADH as a natural biomarker
(2019-08) Cong, Anh
The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture towards pathophysiological investigation, drug screenings, and cancer diagnostics. There is strong evidence, however, that suggests cellular behavior and metabolism in 2D cell culture is too simplistic of a model as compared with those in vivo tumor cells. In this project, we hypothesize that cancer cell metabolism and metabolic responses to external stimuli (e.g. drug treatments) are distinctly different in threedimensional (3D), tumor-like model as compared with that of the conventional 2D monolayer culture. To test this hypothesis, we employed two-photon (2P) fluorescence lifetime imaging microscopy (2P-FLIM) and time-resolved 2P-fluorescence anisotropy of the reduced nicotinamide adenine dinucleotide (NADH) in metastatic murine breast cancer cells 4T1. In addition, we investigated the cellular metabolic response of 4T1 cells in 2D monolayer and 3D collagen matrix cultures to drug treatment using two novel metabolic drugs, namely MD1 and TPPBr. Both 2P-FLIM and complementary time-resolved anisotropy approaches reveal significant differences between metabolic activities of 4T1 cells in 2D and 3D cultures. Our results suggest that these 4T1 cells in 3D culture adapt an oxidative shift but glycolysis dominances the metabolic state of 2D cells. In addition, 4T1 cells in 3D culture appear to adapt more quickly and exhibit enhanced metabolic activities in response to drug treatment. In contrast, 4T1 cells in 2D monolayer culture exhibit a mute response and are less sensitive to drug treatments. While the tumor-like 3D collagen matrix model may not be an exact replica of in vivo tumors, these studies represent a critical step towards the development of a fundamental understanding of cellular behaviors and metabolism in the more complex in vivo models. These studies would also help advance our understanding of how the cancer cell heterogeneity and microenvironmental conditions impact metabolism and metabolic plasticity in tumor growth and metastatic progression.
Myosin structural dynamics: mechanistic insights and therapeutic technology developments
(2019-03) Rohde, John
A major focus in molecular biophysics is to understand how protein structural isomerizations correspond to cellular and organismal physiology. The heart generates force to perfuse the body with oxygenated blood through contractile units in myocytes called sarcomeres. The primary force-generating protein in this contractile apparatus is myosin. Our lab has developed a strategic tool called transient time-resolved FRET, (TR)2FRET, to measure directly, with sub-nanometer and sub-millisecond resolution, the structural and biochemical kinetics of muscle myosin. This tool allows us to directly determine how myosin’s power stroke is coupled to the thermodynamic drive for force generation—the entropically-favored dissociation of inorganic phosphate. My research revealed that actin initiates the force-generating power stroke before phosphate dissociation, revealing how power output and efficiency are regulated by the distribution of myosin’s structural states. (TR)2FRET is also a powerful tool to examine small-molecule perturbations of structural transitions within myosin’s kinetic cycle. Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. My results demonstrate that OM stabilizes myosin’s pre-powerstroke structural state and significantly slows the actin-induced powerstroke. I also used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective therapeutic for hypertrophic cardiomyopathy. I found that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not present in the single-headed myosin motor fragment. From these results, we predicted that cardiac myosin is regulated by an interaction between its two heads and the thick filament, and proposed that mavacamten stabilizes this state. I also investigated two mutations in the converter domain of myosin V to examine how point mutations alter specific structural transitions in the myosin motor’s ATPase cycle. Transient kinetics analyses and FRET-based experiments demonstrated that one mutation slowed the recovery-stroke rate constants, while a second mutation enhanced these steps. These mutations correspond to human mutations that give rise to dilated or hypertrophic cardiomyopathies, respectively. Together these experiments reveal new and important mechanistic insights into myosin’s structural dynamics and provide proof-of-concept results for developing therapeutic technology.
Quantifying Polymer Surface Degradation Using Fluorescence Spectroscopy
(2023) Tigner, Jonathan
One solution to minimizing plastic pollution is to improve reuse and recycling strategies. Recycling, however, is limited by the overall degradation of plastics being used. Photochemical or thermal driving forces facilitate the incorporation of oxygen into the backbone and chain cleavage; yet, current techniques for monitoring this plastic degradation fail to observe early stages of degradation, which is key for optimizing reusability. This research seeks to develop a cheap, reproducible, and nondestructive technique for monitoring degradation of polyethylene and polypropylene materials using Nile red as a fluorescent probe. Changes in Nile red’s fluorescence spectra were observed upon exposure to stained, aged polyethylene and polypropylene samples. As the surface hydrophobicity of the plastic decreases, Nile red’s fluorescence signal undergoes corresponding signal shift to longer wavelengths (lower energy). The trends seen in the fluorescent profile were related to more commonly used measurements of plastic degradation, namely carbonyl index from infrared spectroscopy and bulk crystallinity from calorimetry. Results demonstrate clear trends in fluorescence spectra shifts as related to the chemical and physical changes to the plastics, with trends dependent on polymer type but independent of polymer film thickness. The strength of this technique is divided into two defined fits of the fluorescence signal; one fit characterizes the degradation throughout the whole range of degradative oxidation and the other is tailored to provide insight into the early stages of degradation. Overall, this work establishes a characterization tool that assesses the extent of plastics’ degradation, which may ultimately impact our ability to recover plastics and minimize plastic waste.
Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins
(2015-05) Smith, Elizabeth
The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.
Spectral Deconvolution and Quantification of Natural Organic Material and Fluorescent Tracer Dyes
(Proceedings of the Tenth Multidisciplinary Conference on Sinkholes and the Engineering and Environmental Impacts of Karst. © 2005 American Society of Civil Engineers. Published online: April 26, 2012, 2005-09-28) Alexander, Scott C
Fluorescent dyes have become an integral part of the study and management of ground water in karst environments. Researchers have striven to reduce detection limits and analyze multiple dyes in a single sample while minimizing dye concentrations for environmental, aesthetic and health reasons. The unambiguous separation from background, identification and quantification of fluorescent tracer dyes has increasingly taken on legal implications. Synchronous fluorescence spectroscopy and curve fitting software represent a major advances in the quantitative analysis of low levels of tracer dyes against naturally occurring background fluorescence. Determination of levels of detection (LOD) and levels of quantification (LOQ) are an important part of dye trace design and implementation. Factors that impact LOD and LOQ include levels of natural fluorescent compounds, absolute fluorescence of the specific dyes, the presence of multiple dyes with overlapping peaks and instrumental noise. Characterization of the spectral shapes and concentration dependences of the natural fluorescence background and applied tracer dyes are important to the determination of a positive dye trace result. Rather than representing noise, the natural fluorophores contain information about the flow environment. Spectral deconvolution with curve fitting software is an important tool in the karst researcher’s toolbox.
Synthesis, Characterization, and Investigation of Non-covalent Interactions Between Novel Pyrene-appended Porphyrins and C60
(2015-09) Wertish, Anthony
Two different sets of novel pyrene-containing porphyrins were synthesized. The first set, which consists of asymmetric A3B1 and A2B2 porphyrins, was synthesized by the condensation of t-butylphenyl dipyrromethane and newly reported 4-(1-pyrenylmethoxy)benzaldehyde. The second set consists of asymmetric A3B1 and A2B2 porphyrins, which were synthesized by condensation of the dipyrromethane of the newly reported 4-(1-pyrenylmethoxy)benzaldehyde and 1-ferrocenecarboxaldehyde. All four porphyrins were fully characterized by UV-Vis-NIR spectroscopy, NMR spectroscopy, MCD spectroscopy and high-resolution mass spectrometry. Fluorescence spectroscopy studies were performed to qualitatively observe the interactions of the porphyrins with C60 fullerene. It was observed that C60 significantly quenches the fluorescence of pyrene, thus blocking fluorescence, suggesting there is a large amount of interaction between pyrene and C60. DFT and TDDFT calculations were performed in order to further investigate the electronic structure and nature of the excited states of the target porphyrins by first optimizing the equilibrium geometries at the DFT level using the CAM-B3LYP exchange-correlation functional and furthermore using TDDFT. Transient absorption spectroscopy data is being analyzed to elucidate the electron transfer properties of the new porphyrins.

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