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- All Subjects: Biophysics
- All Subjects: Science
- Creators: School of Molecular Sciences
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Description
Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.
ContributorsRamirez, Christopher Nicholas (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in which photons of light are captured, converted into chemically useful forms, and stored for biological use, an investigation into the reaction center protein, specifically into its cascade of cofactors, was undertaken. The purpose of this experimentation was to advance our knowledge and understanding of how differing protein environments and variant cofactors affect the spectroscopic aspects of and electron transfer kinetics within the reaction of Rh. sphaeroides. The native quinone, ubiquinone, was extracted from its pocket within the reaction center protein and replaced by non-native quinones having different reduction/oxidation potentials. It was determined that, of the two non-native quinones tested—1,2-naphthaquinone and 9,10- anthraquinone—the substitution of the anthraquinone (lower redox potential) resulted in an increased rate of recombination from the P+QA- charge-separated state, while the substitution of the napthaquinone (higher redox potential) resulted in a decreased rate of recombination.
ContributorsSussman, Hallie Rebecca (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Lin, Su (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
In vitro fertilization, or IVF, is currently a worldwide medical procedure designed to give infertile men and women the ability to have children of their own. An IVF procedure takes place outside of a woman’s body, often in a laboratory setting. However, before scientists used the procedure on humans, they initially performed IVF on animals for selective breeding and agricultural purposes. After scientists realized that the procedure had potential to become a treatment option for infertility, they expanded their research subjects to include using the technique on humans. During the procedure’s initial development, scientists began to conduct numerous IVF trials on humans that often ended in early miscarriages. This thesis shows us the history of how some of the first attempts at IVF in humans using various options such as donated egg cells and cryopreserved embryos, often ended in early miscarriages. At that time, most members of the scientific community and general public responded to those trials by regarding them as insignificant. In 1998, the success rate of women under the age of 38 having children with the use of IVF was 22.1%. Over time, scientists began to acknowledge those published findings that detailed various “failed” human IVF experiments. Scientists learned to use them as a guide for what to do differently in future IVF experiments. Because of that, scientists have since developed more effective IVF methods which have ultimately improved the procedure’s success rate. In 2016, the success rate of IVF had increased to 39.6% for women. Therefore, what we might initially think of as a “failure” is in reality not a failure at all, but rather is a “purported failure” because we can use it as a stepping-stone towards an end goal. By looking at the history of IVF research, my thesis illustrates how some of the most important science comes from acknowledging the purported failures along with the triumphant successes.
ContributorsTuoti, Whitney (Author) / Maienschein, Jane (Thesis director) / Abboud, Carolina (Committee member) / Fitzpatrick, Susan (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Nanosphere lithography is a high throughput procedure that has important implications
for facile, low cost scaling of nanostructures. However, current benchtop experiments have
limitations based on the placement of molecular species that exhibit greater than singlemolecular binding. In addition, reliance upon bottom-up self-assembly of close-packed
nanospheres makes it problematic to resolve images using low-cost light microscopes due to the
spacing limitations smaller in magnitude than light wavelength. One method that is created to
resolve this issue is iterative size reduction (ISR), where repetitive ‘iterative’ processes are
employed in order to increase the precision at which single molecules bind to a given substrate.
ISR enables inherent separation of nanospheres and therefore any subsequent single molecule
binding platforms. In addition, ISR targets and encourages single-molecule binding by
systematically reducing binding site size. Results obtained pursuing iteratively reduced
nanostructures showed that many factors are needed to be taken into consideration, including
functionalization of nanosphere particles, zeta potential, and protonation-buffer reactions.
Modalities used for observation of nanoscale patterning and single-molecule binding included
atomic force microscopy (AFM) and ONI super-resolution and fluorescence microscopy. ISR
was also used in conjunction with zero mode waveguides, which are nanoapertures enabling realtime single molecule observation at zeptoliter volumes. Although current limitations and
obstacles still exist with reproducibility and scalability of ISR, it nonetheless exhibits limitless
potential and flexibility in nanotechnology applications.
for facile, low cost scaling of nanostructures. However, current benchtop experiments have
limitations based on the placement of molecular species that exhibit greater than singlemolecular binding. In addition, reliance upon bottom-up self-assembly of close-packed
nanospheres makes it problematic to resolve images using low-cost light microscopes due to the
spacing limitations smaller in magnitude than light wavelength. One method that is created to
resolve this issue is iterative size reduction (ISR), where repetitive ‘iterative’ processes are
employed in order to increase the precision at which single molecules bind to a given substrate.
ISR enables inherent separation of nanospheres and therefore any subsequent single molecule
binding platforms. In addition, ISR targets and encourages single-molecule binding by
systematically reducing binding site size. Results obtained pursuing iteratively reduced
nanostructures showed that many factors are needed to be taken into consideration, including
functionalization of nanosphere particles, zeta potential, and protonation-buffer reactions.
Modalities used for observation of nanoscale patterning and single-molecule binding included
atomic force microscopy (AFM) and ONI super-resolution and fluorescence microscopy. ISR
was also used in conjunction with zero mode waveguides, which are nanoapertures enabling realtime single molecule observation at zeptoliter volumes. Although current limitations and
obstacles still exist with reproducibility and scalability of ISR, it nonetheless exhibits limitless
potential and flexibility in nanotechnology applications.
ContributorsLe, Eric K (Author) / Hariadi, Rizal (Thesis director) / Kishnan, Devika (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
OP50 Esherichia coli is a Gram-negative bacterium with a fast replication rate and can be easily manipulated, making it a model species for many science disciplines. To probe this bacterium’s search strategy, cultures were starved and the cell velocity was probed at various points later in time after perturbing the buffer in which the bacteria were located. To start, we added E.coli OP50 filtrate. In yet another experiment filtrate from a Bdellovibrio bacteriovorus (Gram-negative predator) culture was added to monitor the OP50’s differential response to cues from its environment. Using MATLAB code, thousands of E.coli tracks were measured.
ContributorsSanchez, Alec Jesus (Author) / Presse, Steve (Thesis director) / Gile, Gillian (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Integrin is a protein in cells that manage cell adhesion. They are crucial to the biochemical functions of cells. L 2 is one type of integrin. Its I domain is responsible for ligand binding. Scientists understand how Alpha L I domain binds Mg2+ at a pH of 7 but not in acidic environments. Knowing the specificity of integrin at a lower pH is important because when tissues become inflamed, they release acidic compounds. We have cloned, expressed, and purified L I-domain and using NMR analysis, we determined that wild type Alpha L I domain does not bind to Mg2+ at a pH of 5.
ContributorsALAM, RAHAT (Author) / Wang, Xu (Thesis director) / Podolnikova, Nataly (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex contains an (αβ)3 (hexamer) ring in which catalysis occurs, as well as a rotor comprised by subunit-ε in addition to the coiled-coil and globular foot domains of subunit-γ. The F1 complex can hydrolyze ATP in vitro in a manner that drives counterclockwise (CCW) rotation, in 120° power strokes, as viewed from the positive side of the membrane. The power strokes that occur in ≈ 300 μsec are separated by catalytic dwells that occur on a msec time scale. A single-molecule rotation assay that uses the intensity of polarized light, scattered from a 75 × 35 nm gold nanorod, determined the average rotational velocity of the power stroke (ω, in degrees/ms) as a function of the rotational position of the rotor (θ, in degrees, measured in reference to the catalytic dwell). The velocity is not constant but rather accelerates and decelerates in two Phases. Phase-1 (0° - 60°) is believed to derive power from elastic energy in the protein. At concentrations of ATP that limit the rate of ATP hydrolysis, the rotor can stop for an ATP-binding dwell during Phase-1. Although the most probable position that the ATP-binding dwell occurs is 40° after the catalytic dwell, the ATP-binding dwell can occur at any rotational position during Phase-1 of the power stroke. Phase-2 of the power stroke (60° - 120°) is believed to be powered by the ATP-binding induced closure of the lever domain of a β-subunit (as it acts as a cam shaft against the γ-subunit). Algorithms were written, to sort and analyze F1-ATPase power strokes, to determine the average rotational velocity profile of power strokes as a function of the rotational position at which the ATP-binding dwell occurs (θATP-bd), and when the ATP-binding dwell is absent. Sorting individual ω(θ) curves, as a function of θATP-bd, revealed that a dependence of ω on
θATP-bd exists. The ATP-binding dwell can occur even at saturating ATP concentrations. We report that ω follows a distinct pattern in the vicinity of the ATP-binding dwell, and that the ω(θ) curve contains the same oscillations within it regardless of θATP-bd. We observed that an acceleration/deceleration dependence before and after the ATP-binding dwell, respectively, remained for increasing time intervals as the dwell occurred later in Phase-1, to a maximum of ≈ 40°. The results were interpreted in terms of a model in which the ATP-binding dwell results from internal drag at a variable position on the γε rotor.
θATP-bd exists. The ATP-binding dwell can occur even at saturating ATP concentrations. We report that ω follows a distinct pattern in the vicinity of the ATP-binding dwell, and that the ω(θ) curve contains the same oscillations within it regardless of θATP-bd. We observed that an acceleration/deceleration dependence before and after the ATP-binding dwell, respectively, remained for increasing time intervals as the dwell occurred later in Phase-1, to a maximum of ≈ 40°. The results were interpreted in terms of a model in which the ATP-binding dwell results from internal drag at a variable position on the γε rotor.
ContributorsBukhari, Zain Aziz (Author) / Frasch, Wayne D. (Thesis director) / Allen, James P. (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
DescriptionThis project is designed to generate enthusiasm for science among refugee students in hopes of inspiring them to continue learning science as well as to help them with their current understanding of their school science subject matter.
ContributorsSipes, Shannon Paige (Author) / O'Flaherty, Katherine (Thesis director) / Gregg, George (Committee member) / School of Molecular Sciences (Contributor) / Division of Teacher Preparation (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Both technological and scientific fields continue to revolutionize in a similar fashion; however, a major difference is that high-tech corporations have found models to continue progressions while still keeping product costs low. The main objective was to identify which, if any, components of certain technological models could be used with the vaccine and pharmaceutical markets to significantly lower their costs. Smartphones and computers were the two main items investigated while the two main items from the scientific standpoint were vaccines and pharmaceuticals. One concept had the ability to conceivably decrease the costs of vaccines and drugs and that was "market competition". If the United States were able to allow competition within the vaccine and drug companies, it would allow for the product prices to be best affected. It would only take a few small companies to generate generic versions of the drugs and decrease the prices. It would force the larger competition to most likely decrease their prices. Furthermore, the PC companies use a cumulative density function (CDF) to effectively divide their price setting in each product cycle. It was predicted that if this CDF model were applied to the vaccine and drug markets, the prices would no longer have to be extreme. The corporations would be able to set the highest price for the wealthiest consumers and then slowly begin to decrease the costs for the middle and lower class. Unfortunately, the problem within the vaccine and pharmaceutical markets was not the lack of innovation or business models. The problem lied with their liberty to choose product costs due to poor U.S. government regulations.
ContributorsCalderon, Gerardo (Author) / Johnston, Stephen (Thesis director) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05