Biophysics Commons^™

Advancing Single-Molecule Nanopore Sensing: From Protein Conformation And Metal Binding To Dna Transport Via Hybrid Nanostructures, Matthew J. Odonohue

Multidisciplinary Studies Theses and Dissertations

This dissertation presents the development and application of solid-state and hybrid nanopore platforms for single-molecule analysis of biomolecular interactions. First, a silicon nitride nanopore was employed to characterize the conformational states of the transferrin receptor (TfR), enabling discrimination between its monomeric and dimeric forms, as well as from its ligand, human serum transferrin (hSTf), despite their similar molecular weights. In a separate study, the same nanopore configuration was used to track the real-time binding of titanium ions to transferrin, demonstrating single-molecule sensitivity to metal-induced conformational changes. A hybrid microtubule–solid-state nanopore (MT–SSN) was then engineered, repurposing microtubules as electrostatically anchored conduits …

Compliance And Roughness In Relation To Gecko Locomotion, Brooklynn Campbell

Williams Honors College, Honors Research Projects

Geckos move efficiently through a variety of substrates, from swaying branches to wooden fences to the sides of structures. Previous studies have studied locomotion and adhesion on manmade and rough substrates; however, the work is limited when discussing locomotion and adhesion as a relationship of compliance and roughness. Ease of movement was quantified by speed, under the assumption that the faster a gecko moves, the easier it is for the gecko to cross the substrate. Gehyra oceanica geckos from Moorea, French Polynesia were tested on substrates varying in compliance and roughness with four different treatments in total. Compliance, roughness, and …

Mechanistic Analysis Of Four-Way Dna Junctions And Cytokine-Binding Aptamers For Therapeutic Interventions, Roaa S. Mahmoud

Theses and Dissertations

DNA is inherently dynamic and topologically diverse and can fold into many different structures. Besides the canonical Watson-Crick structure, other higher-order structures such as G-quadruplexes (G4), i-motifs (iM), and four-way DNA junctions are possible. Although these high-order DNA structures are known to form transiently, they are important due to the crucial roles they play in many cellular processes including DNA replication, recombination, and repair. Among these DNA structures, 4-way junctions (also known as Holliday junctions, HJ) which are formed during the repair of double-strand DNA breaks (DSBs) and interact with proteins have garnered significant attention due to their central role …

Quantitative Raman Analysis Of Human Blood Serum Of Glaucoma Patients Supplemented With Macular Pigment Carotenoids., Joy Udensi, Ekaterina Loskutova, James Loughman, Hugh J. Byrne

Datasets

Carotenoids have been widely studied for their high dietary, antioxidant, and vision protection roles in the human body. Blood contains all major dietary carotenoids and hence presents as a suitable and efficient substrate for the estimation of dietary carotenoids in vivo. Following the 18-month supplementation of open angle glaucoma patients with macula pigment carotenoids (Lutein, Zeaxanthin and Meso-Zeaxanthin) in the European Nutrition in Glaucoma Management (ENIGMA) trial, Raman spectroscopic analysis of the pre-supplementation baseline and 18-month supplemented blood serum carotenoids from participants was carried out, to investigate the systemic impact of supplementation in the blood and explore a more direct …

Reducing Food Scarcity: The Benefits Of Urban Farming, S.A. Claudell, Emilio Mejia

Journal of Nonprofit Innovation

Urban farming can enhance the lives of communities and help reduce food scarcity. This paper presents a conceptual prototype of an efficient urban farming community that can be scaled for a single apartment building or an entire community across all global geoeconomics regions, including densely populated cities and rural, developing towns and communities. When deployed in coordination with smart crop choices, local farm support, and efficient transportation then the result isn’t just sustainability, but also increasing fresh produce accessibility, optimizing nutritional value, eliminating the use of ‘forever chemicals’, reducing transportation costs, and fostering global environmental benefits.

Imagine Doris, who is …

Shedding Light On Cellular Glycolysis Pathway Kinetics: Combining Kinetic, Mechanistic Modelling Approaches With Label Free Microspectroscopic Imaging. (Cytomechspec), Nitin Patil

Articles

Glycolysis pathway kinetics in cells are known to be modulated for several disorders (cancers, neurodegenerative disorders, diabetes, etc.) and are currently been targeted as a biomarker for disease diagnostics and therapy. Although there are several approaches for monitoring the pathway kinetics, ranging from simple kinetic biochemical assays to sophisticated omics techniques, there remains a significant gap in the field, as there is a noticeable compromise between the extent of insights achievable from approaches which are destructive to the sample, and the real-time, kinetic analysis of a system.

Vibrational spectroscopy is a powerful, label-free, non-invasive tool capable of sub-cellular resolution, the …

Analysis Of Biologically Effective Dose For Retroactive Yttrium-90 Trans-Arterial Radioembolization Treatment Optimization, Mj Lindsey

CMC Senior Theses

Trans-arterial radioembolization (TARE) is a protracted modality of radiation therapy where radionuclides labeled with Yttrium-90 (⁹⁰Y) are inserted inside a patient's hepatic artery to treat hepatocellular carcinoma (HCC). While TARE has been shown to be a clinically effective and safe treatment, there is little understanding of the radiobiological relationship between absorbed dose and tissue response, and thus there is no dosimetric standard for treatment planning. The Biologically Effective Dose (BED) formalism, derived from the Linear-Quadratic model of radiobiology, is used to weigh the absorbed dose by the time pattern of delivery. BED is a virtual dose that can …

Cellular Mechanisms Underlying State-Dependent Neural Inhibition With Magnetic Stimulation, Hui Ye, Vincent Chiun-Fan Chen, Jenna Hendee

Engineering Science Faculty Publications

Novel stimulation protocols for neuromodulation with magnetic fields are explored in clinical and laboratory settings. Recent evidence suggests that the activation state of the nervous system plays a significant role in the outcome of magnetic stimulation, but the underlying cellular and molecular mechanisms of state-dependency have not been completely investigated. We recently reported that high frequency magnetic stimulation could inhibit neural activity when the neuron was in a low active state. In this paper, we investigate state-dependent neural modulation by applying a magnetic field to single neurons, using the novel micro-coil technology. High frequency magnetic stimulation suppressed single neuron activity …

Somatic Inhibition By Microscopic Magnetic Stimulation, Hui Ye

Biology: Faculty Publications and Other Works

Electric currents can produce quick, reversible control of neural activity. Externally applied electric currents have been used in inhibiting certain ganglion cells in clinical practices. Via electromagnetic induction, a miniature-sized magnetic coil could provide focal stimulation to the ganglion neurons. Here we report that high-frequency stimulation with the miniature coil could reversibly block ganglion cell activity in marine mollusk Aplysia californica, regardless the firing frequency of the neurons, or concentration of potassium ions around the ganglion neurons. Presence of the ganglion sheath has minimal impact on the inhibitory effects of the coil. The inhibitory effect was local to the …

Ionic Mechanism Of Lysosomal Function And Cell Metabolism, Jian Xiong

Dissertations and Theses (Open Access)

Two Pore Channels (TPCs) are endolysosomal ion channels that are permeable to sodium and calcium. Defects in TPCs have been implicated to impair vesicle trafficking, autophagy and cell metabolism control; however, the detailed mechanism remains largely unknown. In this study, I show that TPCs are critical for appropriate cargo delivery to the lysosomes and deletion of either TPC1 or TPC2 leads to delayed clearance of autophagosomes, resulting in enlarged lysosomes and accumulated contents inside the lysosomes. Cells with both TPC deleted also exhibit 50% reduction in lysosomal amino acids under normal culture conditions, leading to reduced homeostatic mTORC1 activation.

Glutamine …

Analysis Of Biofilm Remediation Capacity For Octenyl Succinic Anhydride (Osa), A Bioactive Food Starch Modifier Compound, Matthew R. Borglin

Master's Theses

Matthew R. Borglin

This thesis demonstrates efficacy of Octenyl Succinic Anhydride (OSA), as a biofilm sanitizer. Biofilms allow bacteria to adhere to solid surfaces with the use of excreted polymeric compounds. For example, surfaces found in food production or processing facilities such as the interior of a raw milk holding tank, are some of the most susceptible to biofilm contamination. When present, biofilms can cause a variety of negative effects, which include; reduction of product shelf life, corrosion, and outbreaks of foodborne illnesses. The close association of biofilms with the majority of foodborne illness cases led the US Environmental Protection …

Odx: A Fitness Tracker-Based Device For Continuous Bacterial Growth Monitoring, Venkata V.B. Yallapragada, Uday Gowda, David Wong, Liam O'Faolain, Mark Tangney, Ganga C.R. Devarapu

Cappa Publications

Continuous monitoring of bacterial growth in aqueous media is a crucial process in academic research as well as in the biotechnology industry. Bacterial growth is usually monitored by measuring the optical density of bacteria in liquid media, using benchtop spectrophotometers. Due to the large form factor of the existing spectrophotometers, they cannot be used for live monitoring of the bacteria inside bacterial incubation chambers. Additionally, the use of benchtop spectrometers for continuous monitoring requires multiple samplings and is labour intensive. To overcome these challenges, we have developed an optical density measuring device (ODX) by modifying a generic fitness tracker. The …

Developing A Dissociative Nanocontainer For Peptide Drug Delivery, Michael Patrick Kelly

Dissertations, Theses, and Capstone Projects

The potency and specificity of bioactive peptides have propelled these agents to the forefront of pharmacological research. However, delivery of peptides to their molecular target in cells is a major obstacle to their widespread application. A Trojan Horse strategy of packaging a bioactive peptide within a modified protein cage to protect it during transport, and releasing it at the target site, is a promising delivery method. Recent work has demonstrated that the viral capsid of the P22 bacteriophage can be loaded with an arbitrary, genetically-encoded peptide, and externally decorated with a cell-penetrating peptide, such as HIV-Tat, to translocate across in …

Assessing The Potential Impact Of Nanotechnology On The Purification Of Water, Noor Khan

Quest

Independent Research Paper

Research in progress for PHYS 2426: University Physics II

Faculty Mentor: Raji Kannampuzha, Ph.D.

The following paper represents research work done by students in a University Physics 2426 class, the second half of a two-semester introductory course in physics. It is a calculus-based physics course, intended primarily for physics, chemistry, math, and engineering majors. Students are introduced to the concept of academic research by learning to ask research-focused questions and then use the library resources to pursue outside research to find answers. For this assignment, students are asked to investigate a physical science, biological science, or technology …

Supercharged Models Of Intrinsically Disordered Proteins And Their Utility In Sensing, Peter J. Schnatz

Dissertations, Theses, and Capstone Projects

In this thesis I show that greatly increasing the magnitude of a protein’s net charge using surface supercharging transforms that protein into a ligand-gated or counterion-gated conformational molecular switch. To demonstrate this I first modified the designed helical bundle hemoprotein H4 using simple molecular modeling, creating a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction by intrinsically disordered proteins in biology. Due to the high surface charge density, ligand binding to this protein is allosterically activated by low concentrations of divalent cations and the polyamine spermine. …

Developing Droplet Based 3d Cell Culture Methods To Enable Investigations Of The Chemical Tumor Microenvironment, Jacqueline Alexandria De Lora

Biomedical Sciences ETDs

Adaptation of cancer cells to changes in the biochemical microenvironment in an expanding tumor mass is a crucial aspect of malignant progression, tumor metabolism, and drug efficacy. In vitro, it is challenging to mimic the evolution of biochemical gradients and the cellular heterogeneity that characterizes cancer tissues found in vivo. It is well accepted that more realistic and controllable in vitro 3D model systems are required to improve the overall cancer research paradigm and thus improve on the translation of results, but multidisciplinary approaches are needed for these advances. This work develops such approaches and demonstrates that new droplet-based cell-encapsulation …

Spontaneous Dna Damage To The Nuclear Genome Promotes Senescence, T Redox Imbalance And Aging, Andria R. Robinson, Matthew J. Yousefzadeh, Tania A. Rozgaja, Jin Wang, Xuesen Li, Jeremy S. Tilstra, Chelsea H. Feldman, Siobhan Q. Gregg, Caroline H. Johnson, Erin M. Skoda, Marie-Celine Frantz, Harris Bell-Temin, Hannah Pope-Varsalona, Aditi U. Gurkar, Luigi A. Nasto, Rena A.S. Robinson, Heike Fuhrmann-Stroissnigg, Jolanta Czerwinska, Sara J. Mcgowan, Nadiezhda Cantu-Madellin, Jamie B. Harris, Salony Maniar, Mark A. Ross, Christy E. Trussoni, Nicholas F. Larusso, Eugenia Cifuentes-Pagano, Patrick J. Pagano, Barbara Tudek, Nam V. Vo, Lora H. Rigatti, Patricia L. Opresko, Donna B. Stolz, Simon C. Watkins, Christin E. Burd, Claudette M. St, Croix, Gary Siuzdak, Nathan A. Yates, Paul D. Robbins, Yinsheng Wang, Peter Wipf, Eric E. Kelley, Laura J. Neidernhofer

Faculty & Staff Scholarship

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that …

Dielectric Properties Of Isolated Adrenal Chromaffin Cells Determined By Microfluidic Impedance Spectroscopy, A. C. Sabuncu, M. Stacey, G. L. Graviso, N. Semenova, P. T. Vernier, N. Leblanc, I. Chatterjee, J. Zaklit

Bioelectrics Publications

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1 kHz and 20 MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner …

Validation Of The Pre-B Cell Receptor As A Therapeutic Target In B Cell Precursor Acute Lymphoblastic Leukemia, Michael Frank Erasmus

Biomedical Sciences ETDs

This dissertation is built upon the fundamental idea that the pre-B cell receptor (pre-BCR) is important to leukemia cell survival and a logical therapeutic target in B cell precursor acute lymphoblastic leukemia (BCP-ALL). The pre-BCR is expressed early at a specific stage during B cell development where it plays a central role in survival of healthy B lymphocytes. This receptor is composed of the membrane heavy chain (mIgμ) associated with surrogate light chain components, 5 and VpreB. Through the use of advanced imaging modalities, in particular two-color single particle tracking (SPT), we showed that pre-BCRs formed transient, homotypic interactions. These …

Microfluidic Cantilever Detects Bacteria And Measures Their Susceptibility To Antibiotics In Small Confined Volumes, Hashem Etayash, M. F. Khan, Kamaljit Kaur, Thomas Thundat

Pharmacy Faculty Articles and Research

In the fight against drug-resistant bacteria, accurate and high-throughput detection is essential. Here, a bimaterial microcantilever with an embedded microfluidic channel with internal surfaces chemically or physically functionalized with receptors selectively captures the bacteria passing through the channel. Bacterial adsorption inside the cantilever results in changes in the resonance frequency (mass) and cantilever deflection (adsorption stress). The excitation of trapped bacteria using infrared radiation (IR) causes the cantilever to deflect in proportion to the infrared absorption of the bacteria, providing a nanomechanical infrared spectrum for selective identification. We demonstrate the in situ detection and discrimination of Listeria monocytogenes at a …

Measuring Radiation Exposure In Human Blood Using Gene Expression, Krystal Naranjo, Melissa Bentley, Harsha Konery, Matthew Coleman

STAR Program Research Presentations

Mammalian cells are known to express genes that are associated with repairing damaged DNA. The transcript CDKN1A is one of several cell cycle regulator genes expressed in response to cell damage by ionizing radiation (IR). In this study, male and female lymphocytes; previously exposed ex vivo to IR, were used to demonstrate linear gene expression responses that may vary between genders. We used qRT-PCR to generate response curves for CDKN1A. No differences were identified for the endogenous control gene GAPDH. CDKN1A expression demonstrated average fold changes well above three fold for three of the four healthy patient donors at 24 …

Evaluations Of A Mechanistic Hypothesis For The Influence Of Extracellular Ions On Electroporation Due To High-Intensity, Nanosecond Pulsing, V. Sridhara, R. P. Joshi

Electrical & Computer Engineering Faculty Publications

The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na+ in this case) and the carbonyl oxygen of …

The Geometry And Sensitivity Of Ion-Beam Sculpted Nanopores For Single Molecule Dna Analysis, Ryan Connor Rollings

Graduate Theses and Dissertations

In this dissertation, the relationship between the geometry of ion-beam sculpted solid-state nanopores and their ability to analyze single DNA molecules using resistive pulse sensing is investigated. To accomplish this, the three dimensional shape of the nanopore is determined using energy filtered and tomographic transmission electron microscopy. It is shown that this information enables the prediction of the ionic current passing through a voltage biased nanopore and improves the prediction of the magnitude of current drop signals when the nanopore interacts with single DNA molecules. The dimensional stability of nanopores in solution is monitored using this information and is improved …

Response To "Sodium Current Inhibition By Nanosecond Pulsed Electric Field (Nspef) - Fact Or Artifact?" By Verkerk Et Al, Andrei G. Pakhomov

Bioelectrics Publications

It was nice to learn that our studies of nanosecond pulsed electric field (nsPEF) effects on membrane currents [Nesin et al., 2012; Nesin and Pakhomov, 2012] gained the attention of scientists outside the immediate field of bioelectromagnetics.

Real-Time In Vivo Imaging Of Size-Dependent Transport And Toxicity Of Gold Nanoparticles In Zebrafish Embryos Using Single Nanoparticle Plasmonic Spectroscopy, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Noble metal nanoparticles (NPs) show distinctive plasmonic optical properties and superior photostability, enabling them to serve as photostable multicoloured optical molecular probes and sensors for real-time in vivo imaging. To effectively study biological functions in vivo, it is essential that the NP probes are biocompatible and can be delivered into living organisms non-invasively. In this study, we have synthesized, purified and characterized stable (non-aggregated) gold (Au) NPs (86.2 +/- 10.8 nm). We have developed dark-field single NP plasmonic microscopy and spectroscopy to study their transport into early developing zebrafish embryos (cleavage stage) and their effects on embryonic development …

Energy-Saving Hypothermia Reduces Flight Ability In Mourning Doves, Jennie M. Carr, Steven L. Lima

Symposium 2012

Overwintering birds are frequently exposed to thermal challenges that can quickly decrease energy reserves, thus leading to an increase in the risk of starvation. During these periods of energetic hardship, many avian species use nocturnal hypothermia to conserve energy that would otherwise be lost remaining warm throughout the night (McKechnie & Lovegrove 2002). However, a cool body temperature (Tb) may limit a bird’s ability to monitor the environment and may slow their response to a potential threat. Thus, birds likely trade-off between the benefits of energy-saving hypothermia and the potential costs of reduced behavioral responsiveness to predators (Welton et al. …

Microfluidic Impedance Spectroscopy As A Tool For Quantitative Biology And Biotechnology, Ahmet C. Sabuncu, Jie Zhuang, Juergen F. Kolb, Ali Beskok

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to …

Plasma Membrane Voltage Changes During Nanosecond Pulsed Electric Field Exposure, W. Frey, R. O. Price, P. F. Blackmore, R. P. Joshi, R. Nuccitelli, S. J. Beebe, K. H. Schoenbach, J. F. Kolb

Bioelectrics Publications

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of similar to 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat …

Selective Field Effects On Intracellular Vacuoles And Vesicle Membranes With Nanosecond Electric Pulses, Ephrem Tekle, Hammou Oubrahim, Sergey M. Dzekunov, Juergen F. Kolb, Karl H. Schoenbach

Bioelectrics Publications

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of …

The Effects Of Intense Submicrosecond Electrical Pulses On Cells, Jingdong Deng, Karl H. Schoenbach, E. Stephen Buescher, Pamela S. Hair, Paula M. Fox, Stephen J. Beebe

Bioelectrics Publications

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the submicrosecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 μs– 60 ns, electric field intensities 3–150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of …