For cold chain tracking systems, precision and versatility across varying time intervals and temperature ranges remain integral to effective application in clinical, commercial, and academic settings. Therefore, while electronic and chemistry/physics based cold chain tracking mechanisms currently exist, both have…
For cold chain tracking systems, precision and versatility across varying time intervals and temperature ranges remain integral to effective application in clinical, commercial, and academic settings. Therefore, while electronic and chemistry/physics based cold chain tracking mechanisms currently exist, both have limitations that affect their application across various biospecimens and commercial products, providing the initiative to develop a time temperature visual indicator system that resolves challenges with current cold chain tracking approaches. As a result, a permanganate/oxalic acid time temperature visual indicator system for cold chain tracking has been proposed. At thawing temperatures, the designed permanganate/oxalic acid reaction system undergoes a pink to colorless transition as permanganate, Mn(VII), is reduced to auto-catalytic Mn(II), while oxalate is oxidized to CO2. Therefore, when properly stored and vitrified or frozen, the proposed visual indicator remains pink, whereas exposure to thawing conditions will result in an eventual, time temperature dependent, designed color transition that characterizes compromised biospecimen integrity. To design visual indicator systems for targeted times at specific temperatures, absorbance spectroscopy was utilized to monitor permanganate kinetic curves by absorbance at 525 nm. As a result, throughout the outlined research, the following aims were demonstrated: (i) Design and functionality of 1x (0.5 mM KMnO4) visual indicator systems across various time intervals at temperatures ranging from 25°C to -20°C, (ii) Design and functionality of high concentration, 5x, visual indicator systems across varying targeted time intervals at temperatures ranging from 25°C to 0°C, (iii) Pre-activation stability and long-term stability of the proposed visual indicator systems.
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Dielectrophoresis is an analytical technique which involves electroosmotic flow, electrophoresis, and dielectrophoretic force. These factors, when in correct proportions for a given analyte, allow for dielectrophoretic trapping, otherwise known as dielectrophoretic capture. Non-uniform electric fields are required for this phenomenon,…
Dielectrophoresis is an analytical technique which involves electroosmotic flow, electrophoresis, and dielectrophoretic force. These factors, when in correct proportions for a given analyte, allow for dielectrophoretic trapping, otherwise known as dielectrophoretic capture. Non-uniform electric fields are required for this phenomenon, and the device in this trial used to induce such an electric field was a gradient insulator-based design. Similar devices have been previously used to separate or identify a wide variety of analytes within solution. Much of the previous work has been focused on the differences in dielectrophoretic trapping between strains of bacteria, whereas this experiment focused on the differentiation of phenotypes within a single bacterial strain, Staphylococcus aureus isolate 35984. A control sample was tested, as well as a sample heated at 70oC for 15 minutes to induce phenotypic changes. The control sample was found to exhibit dielectrophoretic capture at a given gate at a potential of 800V and higher, whereas the heated sample was not observed to capture at any potential in this experiment, which reached a maximum of 1200V. Notably, neither of the samples were found to capture at or below 600V. The results of this experiment were encouraging, though it is worth noting that several experimental trials failed to produce any noteworthy results. As such, the procedure of this experiment should be refined to increase reproducibility of results.
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In cold chain tracking systems, accuracy and flexibility across different temperatures ranges plays an integral role in monitoring biospecimen integrity. However, while two common cold chain tracking systems are currently available (electronic and physics/chemical), there is not an affordable cold…
In cold chain tracking systems, accuracy and flexibility across different temperatures ranges plays an integral role in monitoring biospecimen integrity. However, while two common cold chain tracking systems are currently available (electronic and physics/chemical), there is not an affordable cold chain tracking mechanism that can be applied to a variety of temperatures while maintaining accuracy for individual vials. Hence, our lab implemented our understanding of biochemical reaction kinetics to develop a new cold chain tracking mechanism using the permanganate/oxalic acid reaction. The permanganate/oxalic acid reaction is characterized by the reduction of permanganate (MnVII) to Mn(II) with Mn(II)-autocatalyzed oxidation of oxalate to CO2, resulting in a pink to colorless visual indicator change when the reaction system is not in the solid state (i.e., frozen or vitrified). Throughout our research, we demonstrate, (i) Improved reaction consistency and accuracy along with extended run times with the implementation of a nitric acid-based labware washing protocol, (ii) Simulated reaction kinetics for the maximum length reaction and 60-minute reaction based on previously developed MATLAB scripts (iii) Experimental reaction kinetics to verify the simulated MATLAB maximum and 60-minute reactions times (iv) Long-term stability of the permanganate/oxalic acid reaction with water or eutectic solutions of sodium perchlorate and magnesium perchlorate at -80°C (v) Reaction kinetics with eutectic solvents, sodium perchlorate and magnesium perchlorate, at 25°C, 4°C, and -8°C (vi) Accelerated reaction kinetics after the addition of varying concentrations of manganese perchlorate (vii) Reaction kinetics of higher concentration reaction systems (5x and 10x; for darker colors), at 25°C (viii) Long-term stability of the 10x higher concentration reaction at -80°C.
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Glycans are complex biological sugar polymers that are commonly found covalently attached to proteins, lipids, and lipoproteins. About 50% of all mammalian proteins are glycosylated. Aberrant glycosylation is a hallmark of most types of cancer, and glycosylation changes that occur…
Glycans are complex biological sugar polymers that are commonly found covalently attached to proteins, lipids, and lipoproteins. About 50% of all mammalian proteins are glycosylated. Aberrant glycosylation is a hallmark of most types of cancer, and glycosylation changes that occur in this disease are known to facilitate tumor development. In this dissertation, a bottom-up approach to glycomics, “glycan node analysis”, which is a method based on glycan linkage analysis that quantifies unique glycan features, such as “core fucosylation”, “α2-6 sialylation”, “β1-6 branching”, and “bisecting GlcNAc”, as single analytical signals by gas chromatography-mass spectrometry (GC-MS), was applied to cancer cell lines, antibodies, extracellular vesicles, and low density lipoproteins to understand the mechanisms leading to aberrant glycosylation in cancer, and to understand the role of blood plasma glycan sialylation in cancer immunity. Specific tumor antigens such as β1-6-branching, β1-4-branching, bisecting GlcNAc, antennary fucosylation, and Tn antigen (GalNAc-Ser/Thr), were found to be regulated by IL-6 in HepG2 cells; fewer glycan features were regulated by IL-1β. Additionally, neuraminidase enzyme treatment of alpha-1 antitrypsin IgG demonstrates how glycan node analysis can be used to detect relative changes in “α2-6-sialylation” along with corresponding increases in terminal galactose.
Extracellular vesicles (EVs) derived from metastatic and non-metastatic cancer cell lines displayed upregulated or downregulated expression of several specific glycan nodes, particularly 3-GlcNAc, which represents hyaluronic acid. EVs displayed several glycan features that distinguished them from the whole blood plasma glycome. These results were promising for developing new diagnostic strategies in cancer.
A “liquid phase permethylation” procedure for glycan node analysis that does not require spin columns was applied for the first time to whole biological specimens, and it demonstrated potential clinical utility in detecting specific tumor antigens. Significantly different glycan node profiles were found among three cancer cell lines and in peripheral blood mononuclear cells from healthy donors.
Changes in glycosylation and mechanisms regulating glycan changes were studied extensively in cancer cells. Subsequently, it is reported how glycosylation changes can have an impact in cancer immunity. A novel role for oxidized-desialylated low density lipoprotein in cancer immunity is reported, and its implications in cancer and atherosclerosis are discussed.
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Serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL) has enabled the determination of protein structures and protein reaction intermediates in millisecond to microsecond time resolutions. Mix-and-Inject crystallography (MISC) at XFELs enables fast mixing in the magnitude of milliseconds…
Serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL) has enabled the determination of protein structures and protein reaction intermediates in millisecond to microsecond time resolutions. Mix-and-Inject crystallography (MISC) at XFELs enables fast mixing in the magnitude of milliseconds in order to achieve desired reaction time points. For these experiments, numerical simulations of a hydrodynamic flow mixer capable of fast mixing by diffusion has been developed using both COMSOL Multiphysics 5.6 and QuickerSims Computational Fluid Dynamics (CFD) Toolbox for MATLAB. These simulation programs were compared by calculations of mixing times and concentration flow profiles. Mixing times in the range of 1-10 ms were calculated in COMSOL under certain flow rate conditions whereas mixing times in the range of 6-15 ms were calculated with QuickerSims. From these mixing times, reaction intermediates can be varied from sub-millisecond to several hundred millisecond time points for a MISC experiment. Explanations for the discrepancies between the two models were attributed to variations in parameter definitions and meshing. Further analysis on the mixing characteristics were investigated by calculating an analytical solution to the convection-diffusion equation for fluid flow in a two-dimensional rectangular channel. The concentration profile along the width of the channel for the analytical solution was compared with the numerical solution obtained with COMSOL and QuickerSims. Upon comparison, it was determined that the diffusion coefficient may not be a significant factor for the disagreement between the two hydrodynamic flow models.
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Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which are then fired out as a jet in the beam…
Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which are then fired out as a jet in the beam of an X-ray free electron laser (XFEL). A crucial part of the device is its method of droplet detection. This project presents a design for a capacitive sensor that uses a unique electrode configuration to detect the difference in capacitance between the aqueous and oil phases. This design was developed using MATLAB and COMSOL Multiphysics simulations and printed using high-resolution 3D printing. Results show that this design can successfully distinguish between the two immiscible liquids, confirming it as a possible detection method in future SFX experiments.
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The chemical, structural, and electrical properties of niobium-silicon, niobium-germanium, and YBCO-dielectric interfaces are characterized. Reduction in the concentration of interfacial defects in these structures can improve the performance of (i) many devices including low-loss coplanar, microstrip, and stripline microwave resonators…
The chemical, structural, and electrical properties of niobium-silicon, niobium-germanium, and YBCO-dielectric interfaces are characterized. Reduction in the concentration of interfacial defects in these structures can improve the performance of (i) many devices including low-loss coplanar, microstrip, and stripline microwave resonators used in next-generation cryogenic communication, sensor, and quantum information technologies and (ii) layers used in device isolation, inter-wiring dielectrics, and passivation in microwave and Josephson junction circuit fabrication.
Methods were developed to synthesize amorphous-Ge (a-Ge) and homoepitaxial-Si dielectric thin-films with loss tangents of 1–2×10 -6 and 0.6–2×10 -5 at near single-photon powers and sub-Kelvin temperatures (≈40 mK), making them potentially a better choice over undoped silicon and sapphire substrates used in quantum devices. The Nb/Ge interface has 20 nm of chemical intermixing, which is reduced by a factor of four using 10 nm Ta diffusion layers. Niobium coplanar resonators using this structure exhibit reduced microwave losses.
The nature and concentration of defects near Nb-Si interfaces prepared with commonly-used Si surface treatments were characterized. All samples have H, C, O, F, and Cl in the Si within 50 nm of the interface, and electrically active defects with activation energies of 0.147, 0.194, 0.247, 0.339, and 0.556 eV above the valence band maximum (E vbm ), with concentrations dominated by a hole trap at E vbm +0.556 eV (presumably Nb Si ). The optimum surface treatment is an HF etch followed by an in-situ 100 eV Ar ion mill. RCA etches, and higher energy ion milling processes increase the concentration of electrically active defects.
A thin SrTiO 3 buffer layer used in YBa 2 Cu 3 O 7-δ superconductor/high-performance Ba(Zn 1/3 Ta 2/3 )O 3 and Ba(Cd 1/3 Ta 2/3 )O 3 microwave dielectric trilayers improves the structural quality of the layers and results in 90 K superconductor critical temperatures. This advance enables the production of more compact high-temperature superconductor capacitors, inductors, and microwave microstrip and stripline devices.
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Eosinophils are innate immune cells that are most commonly associated with parasite infection and allergic responses. Recent studies, though, have identified eosinophils as cells with diverse effector functions at baseline and in disease. Eosinophils in specific tissue immune environments are…
Eosinophils are innate immune cells that are most commonly associated with parasite infection and allergic responses. Recent studies, though, have identified eosinophils as cells with diverse effector functions at baseline and in disease. Eosinophils in specific tissue immune environments are proposed to promote unique and specific effector functions, suggesting these cells have the capacity to differentiate into unique subtypes. The studies here focus on defining these subtypes using functional, molecular, and genetic analysis as well as using novel techniques to image these subtypes in situ.
To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity.
To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies.
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The successful reduction of CO2 and protons by a light-induced cobalt porphyrin/cytb562 hybrid metalloenzyme in water is reported. Incorporation of the porphyrin into a protein scaffold results in increases in CO and H2 production over naked porphyrin. Rational point…
The successful reduction of CO2 and protons by a light-induced cobalt porphyrin/cytb562 hybrid metalloenzyme in water is reported. Incorporation of the porphyrin into a protein scaffold results in increases in CO and H2 production over naked porphyrin. Rational point mutations to the CoPPIX binding site of cytb562 modulate production, indicating possible further improvements in catalytic activity.
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Oxygen fugacity (ƒO2) is a thermodynamic variable used to represent the redox state of a material or a system. It is equivalent to the partial pressure of oxygen in a particular environment corrected for the non-ideal behavior of the gas.…
Oxygen fugacity (ƒO2) is a thermodynamic variable used to represent the redox state of a material or a system. It is equivalent to the partial pressure of oxygen in a particular environment corrected for the non-ideal behavior of the gas. ƒO2 is often used to indicate the potential for iron to occur in a more oxidized or reduced state at a particular temperature and pressure in a natural system. Secondary ion mass spectrometry (SIMS) is a powerful analytical instrument that can be used to analyze elemental and isotopic compositional information about microscopic features within solid materials. SIMS analyses of the secondary ion energy distribution of semi-pure metals demonstrate that the energy spectrum of individual mass lines can provide information about alterations in its surface environment.
The application of high-resolution (see Appendix C) energy spectrum calibrations to natural ilmenite led to the investigation of zirconium (90Zr+) and niobium (93Nb+) as potential indicators of sample ƒO2. Energy spectrum measurements were performed on an array of ilmenite crystals from the earth’s upper mantle retrieved from kimberlites and from a reduced meteorite. In all studied materials, variability in the peak shape and width of the energy spectra has been correlated with inferred sample ƒO2. The best descriptor of this relationship is the full-width at half-maximum (FWHM; see Appendix C) of the energy spectra for each sample. It has been estimated that a 1eV change in the FWHM of 93Nb+ energy spectra is roughly equivalent to 1 log unit ƒO2. Simple estimates of precision suggest the FWHM values can be trusted to 1eV and sample ƒO2 can be predicted to ±1 log unit, assuming the temperature of formation is known.
The work of this thesis also explores the applicability of this technique beyond analysis of semi-pure metals and ilmenite crystals from kimberlites. This technique was applied to titanium oxides experimentally formed at known ƒO2 as well as an ilmenite crystal that showed compositional variations across the grain (i.e., core to rim chemical variations). Analyses of titanium oxides formed at known ƒO2 agree with the estimation that 1 eV change in the FWHM of 93Nb+ is equivalent to ~1 log unit ƒO2 (in all cases but one); this is also true for analyses of a natural ilmenite crystal with compositional variations across the grain.
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