Biochemistry, Biophysics, and Structural Biology Commons^™

Microfluidic Biopsy Trapping Device For The Real-Time Monitoring Of The Tumor Microenvironment, Angela Holton

USF Tampa Graduate Theses and Dissertations

The tumor microenvironment is composed of cellular and stromal components such as tumor cells, mesenchymal cells, immune cells, cancer associated fibroblasts and the supporting extracellular matrix. The tumor microenvironment provides crucial support for growth and progression of tumor cells and affects tumor response to therapeutic interventions. To better understand tumor biology and to develop effective cancer therapeutic agents it is important to develop preclinical platforms that can faithfully recapitulate the tumor microenvironment and the complex interaction between the tumor and its surrounding stromal elements. Drug studies performed in vitro with conventional two-dimensional cancer cell line models do not optimally represent …

Activation Of The Phospholipid Scramblase Tmem16f By Nanosecond Pulsed Electric Field (Nspef) Facilitates Its Diverse Cytophysiological Effects, Claudia Muratori, Andrei G. Pakhomov, Elena Gianulis, Jade Meads, Maura Casciola, Peter A. Mollica, Olga N. Pakhomova

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca²⁺-dependent phospholipid scramblase and Ca²⁺-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, …

Mutagenesis Of Human Alpha-Galactosidase A For The Treatment Of Fabry Disease, Erin Stokes

Dissertations, Theses, and Capstone Projects

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40-60 years of age. There is currently only one FDA approved treatment (Fabrazyme) classified as an enzyme replacement therapy. However, approximately 88% of patients experience a severe immune response that, rarely, can be fatal and is a huge cost burden at average $250,000 a year per patient. The structure of α-galactosidase A has been previously determined to be a …

Improving The Thermal Stability Of Cellobiohydrolase Cel7a From Hypocrea Jecorina By Directed Evolution, Frits Goedegebuur, Lydia Dankmeyer, Peter Gualfetti, Saeid Karkehabadi, Henrik Hansson, Suvamay Jana, Vicky Huynh, Bradley R. Kelemen, Paulien Kruithof, Edmund A. Larenas, Pauline J. M. Teunissen, Jerry Ståhlberg, Christina M. Payne, Colin Mitchinson, Mats Sandgren

Chemical and Materials Engineering Faculty Publications

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and …

Fret Biosensors: Engineering Fluorescent Proteins As Biological Tools For Studying Parkinson’S Disease, Nathan J. Leroy, Jacob R. Norley, Saranya Radhakrishnan, Mathew Tantama

The Summer Undergraduate Research Fellowship (SURF) Symposium

Parkinson’s Disease (PD) is a common neurodegenerative disease with over 200,000 new cases each year. In general, the cause of the disease is unknown, but oxidative stress inside of neurons has been associated with the disease’s pathology for some time. Currently, techniques to study the onset of PD inside of neurons are limited. This makes treatments and causes difficult to discover. One solution to this has been fluorescent protein biosensors. In short, these proteins can be engineered to glow when a certain state is achieved inside a cell. The present research discusses the engineering of a genetically-encoded fluorescent protein (FP) …

Bacterial Motility And Its Role In Biofilm Formation, Clayton J. Culp, Arezoo M. Ardekani, Adib Ahmadzadegan

The Summer Undergraduate Research Fellowship (SURF) Symposium

Bacterial biofilms are known to cause millions of dollars in damage in the medical industry per year via infection of central venous catheters, urinary catheters, and mechanical heart valves. Unfortunately, there are some characteristics of biofilm formation that are yet to be fully understood. Recently much work has been done to investigate the motility characteristics of bacteria with hopes of better understanding the phenomena of biofilm formation. Still, one of the least understood stages is bacterial attachment or adhesion, a process designed to anchor bacteria in an advantageous environment. Providing a better understanding of bacterial motility near solid interfaces will …

Membrane Surface Engineering For Biochemical Applications, Anh T. Vu

Graduate Theses and Dissertations

Synthetic membranes have been frequently used for many fields, such as, the food and beverage, biopharmaceutical, and biofuel industries. In the beer industry, microfiltration frequently suffers from fouling due to the interaction between different species. It is shown that polyphenols can form cross-links with protein molecules, forming insoluble aggregates. However, by adding an optimal amount of polysaccharides these aggregates can be disrupted thus reducing fouling by the aggregates. Confocal laser scanning microscopy (CLSM) is a powerful technique to locate the foulants inside the wet membrane in order to understand more about the behavior of fouling in microfiltration.

Membrane surface modification …

Membranes For Food And Bioproduct Processing, Alexandru Marius Avram

Graduate Theses and Dissertations

Modified membranes for process intensification in biomass hydrolysis

Production of biofuels and chemicals from lignocellulosic biomass is one of the leading candidates for replacement of petroleum based fuels and chemicals. However, conversion of lignocellulosic biomass into fuels and chemicals is not cost effective compared to the production of fuels and chemicals from crude oil reserves. Some novel and economically feasible approaches involve the use of ionic liquids as solvents or co-solvents, since these show improved solvation capability of cellulose over simple aqueous systems. Membranes offer unique opportunities for process intensification which involves fractionation of the resulting biomass hydrolysate leading to …

Removal Of Endocrine Disrupting Compounds Using Membrane Bioreactor, Mohanad Ali Abdulsahib Kamaz

Graduate Theses and Dissertations

The presence of endocrine disrupting compounds (EDCs) and pharmaceutically active compounds (PhAC) such as pesticides, personal care products, antibiotics and pharmaceutical compounds, in sewage, industrial, and domestic waters has extensively become the major concern for health and environmental organizations. These compounds have the ability to interact with mammalian endocrine system and disrupting their functions. The traditional activated sludge processes are designed to degrade solids, organic carbon and nitrogen loading. Although several treatment steps in a wastewater treatment plant can contribute to partial removal of EDCs, effective removal has been a challenge due to their resistant chemical and biological degradation and …

Experiment-Based Quantitative Modeling For The Antibacterial Activity Of Silver Nanoparticles, Mohammad Aminul Haque

Graduate Theses and Dissertations

Silver (Ag) has been well known for its antimicrobial activity for a long time. Recent research showed the potential of Ag nanoparticles as emerging antimicrobial agents. However, little quantitative analysis has been performed so far to decipher the mechanism of interaction between nanoparticles and bacteria. Here, a detailed analysis based on kinetic growth assay and colony forming unit assay has been carried out to study the antimicrobial effect of Ag nanoparticles against Escherichia coli (E. coli) bacteria. It was observed that the presence of Ag nanoparticles increased the lag time of bacterial growth while not affecting the maximum growth rate …

Real-Time Sensing Of Single-Ligand Delivery With Nanoaperture-Integrated Microfluidic Devices, W. Elliott Martin, Ning Ge, Bernadeta R. Srijanto, Emily Furnish, C. Patrick Collier, Christine A. Trinkle, Christopher I. Richards

Chemistry Faculty Publications

The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, …

Perspective: The Physics, Diagnostics, And Applications Of Atmospheric Pressure Low Temperature Plasma Sources Used In Plasma Medicine, M. Laroussi

Electrical & Computer Engineering Faculty Publications

Low temperature plasmas have been used in various plasma processing applications for several decades. But it is only in the last thirty years or so that sources generating such plasmas at atmospheric pressure in reliable and stable ways have become more prevalent. First, in the late 1980s, the dielectric barrier discharge was used to generate relatively large volume diffuse plasmas at atmospheric pressure. Then, in the early 2000s, plasma jets that can launch cold plasma plumes in ambient air were developed. Extensive experimental and modeling work was carried out on both methods and much of the physics governing such sources …

The Impact Of The Mitochondrial Metabolism Of Induced Pluripotent Stem Cells Upon Differentiation, Stefanie T. Shahan

McKelvey School of Engineering Graduate Student Theses & Dissertations

Induced pluripotent stem cells (iPSCs) can be differentiated into any cell type found in the body. The derivation of a stem cell derived β cell (SC-β) capable of responding to glucose by secreting insulin was hugely significant for diabetes research and opened up the possibility of cell replacement therapy to combat this widespread disease (Pagliuca et al. 2014). The optimization of differentiation procedures such as this could improve yield, function, cost, and efficiency of a stem cell-derived product. Current approaches to improve differentiation are primarily focused on signal transduction pathways, while the metabolic state of the cells has received little …

Desulfination By 2′-Hydroxybiphenyl-2-Sulfinate Desulfinase Proceeds Via Electrophilic Aromatic Substitution By The Cysteine-27 Proton, Inacrist Geronimo, Shawn R. Nigam, Christina M. Payne

Chemical and Materials Engineering Faculty Publications

Biodesulfurization is an attractive option for enzymatically removing sulfur from the recalcitrant thiophenic derivatives that comprise the majority of organosulfur compounds remaining in hydrotreated petroleum products. Desulfurization in the bacteria Rhodococcus erythropolis follows a four-step pathway culminating in C–S bond cleavage in the 2′-hydroxybiphenyl-2-sulfinate (HBPS) intermediate to yield 2-hydroxybiphenyl and bisulfite. The reaction, catalyzed by 2′-hydroxybiphenyl-2-sulfinate desulfinase (DszB), is the rate-limiting step and also the least understood, as experimental evidence points to a mechanism unlike that of other desulfinases. On the basis of structural and biochemical evidence, two possible mechanisms have been proposed: nucleophilic addition and electrophilic aromatic substitution. Density …

Mri Applications In Tissue Engineering, Shadi Othman

Science Seminar Series

Shadi Othman of the School of Engineering and Computer Science Bioengineering Program, will speak on his research on MRI applications in tissue engineering.

Oxidation Of Substituted Catechols At The Air-Water Interface: Production Of Carboxylic Acids, Quinones, And Polyphenols, Elizabeth A. Pillar, Marcelo I. Guzman

Chemistry Faculty Publications

Anthropogenic activities contribute benzene, toluene, and anisole to the environment, which in the atmosphere are converted into the respective phenols, cresols, and methoxyphenols by fast gas-phase reaction with hydroxyl radicals (HO(•)). Further processing of the latter species by HO(•) decreases their vapor pressure as a second hydroxyl group is incorporated to accelerate their oxidative aging at interfaces and in aqueous particles. This work shows how catechol, pyrogallol, 3-methylcatechol, 4-methylcatechol, and 3-methoxycatechol (all proxies for oxygenated aromatics derived from benzene, toluene, and anisole) react at the air-water interface with increasing O3(g) during τc ≈ 1 μs contact time and contrasts their …

Testing Effectiveness Of Aqueous Phases From Struvite And Hydroxyapatite Mineral Precipitation As Algal Nutrient Sources, Aaron Lyons

Virginias Collegiate Honors Council Conference

As interest in algal biofuel grows, the need to explore diverse nutrient sources for growing algae becomes apparent. Struvite and Hydroxyapatite (HAP) were precipitated after flash hydrolysis. The leftover aqueous phase (AP) could serve as nutrients source for algae cultivation and the potential reuse of this waste stream is promising. To test this, 12 bottles of Scenedemus sp., four of Struvite, four of HAP, and four control AM-14 synthetic media were cultivated. 10% of the Phosphorus in the synthetic media was replaced with phosphorous from the struvite AP, and 10% of the nitrogen in the synthetic media was replaced …

Computing Spatiotemporal Heat Maps Of Lipid Electropore Formation: A Statistical Approach, Willy Wriggers, Frederica Castellani, Julio A. Kovacs, P. Thomas Vernier

Mechanical & Aerospace Engineering Faculty Publications

We extend the multiscale spatiotemporal heat map strategies originally developed for interpreting molecular dynamics simulations of well-structured proteins to liquids such as lipid bilayers and solvents. Our analysis informs the experimental and theoretical investigation of electroporation, that is, the externally imposed breaching of the cell membrane under the influence of an electric field of sufficient magnitude. To understand the nanoscale architecture of electroporation, we transform time domain data of the coarse-grained interaction networks of lipids and solvents into spatial heat maps of the most relevant constituent molecules. The application takes advantage of our earlier graph-based activity functions by accounting for …

A Balanced Approach To Adaptive Probability Density Estimation, Julio Kovacs, Cailee Helmick, Willy Wriggers

Mechanical & Aerospace Engineering Faculty Publications

Our development of a Fast (Mutual) Information Matching (FIM) of molecular dynamics time series data led us to the general problem of how to accurately estimate the probability density function of a random variable, especially in cases of very uneven samples. Here, we propose a novel Balanced Adaptive Density Estimation (BADE) method that effectively optimizes the amount of smoothing at each point. To do this, BADE relies on an efficient nearest-neighbor search which results in good scaling for large data sizes. Our tests on simulated data show that BADE exhibits equal or better accuracy than existing methods, and visual tests …

Quantifying Cyanide Inhibition Of Nitrification And Developing Cost-Effective Treatment Processes, Germano M. Salazar-Benites

Civil & Environmental Engineering Theses & Dissertations

All wastewater treatment plants that operate multiple hearth furnaces (MHF) and are required to nitrify must manage the inhibitory effects of free cyanide (HCN, CN-) in the scrubber return flows due to inhibitory impacts on nitrifying bacteria.

HRSD Boat Harbor Treatment Plant (BHTP) a 25 MGD facility consisting of primary and secondary treatment, employs an anoxic selector process for nitrification and partial denitrification and operates a MHF. There is a desire to improve TN removal performance at BHTP due to an annual mass-based bubble permit limit on a combined discharge from seven HRSD plants, and there are no discharge limitations …

Quantitative Limits On Small Molecule Transport Via The Electropermeome - Measuring And Modeling Single Nanosecond Perturbations, Esin B. Sözer, Zachary A. Levine, P. Thomas Vernier

Bioelectrics Publications

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated …

Characterization And Analysis Of Real-Time Capillary Convective Pcr Toward Commercialization, Xianbo Qiu, Shiyin Zhang, Lanju Mei, Di Wu, Ke Li, Shengxiang Ge, Xiangzhong Ye, Ningshao Xia, Michael G. Mauk

Mechanical & Aerospace Engineering Faculty Publications

Almost all the reported capillary convective polymerase chain reaction (CCPCR) systems to date are still limited to research use stemming from unresolved issues related to repeatability, reliability, convenience, and sensitivity. To move CCPCR technology forward toward commercialization, a couple of critical strategies and innovations are discussed here. First, single- and dual-end heating strategies are analyzed and compared between each other. Especially, different solutions for dual-end heating are proposed and discussed, and the heat transfer and fluid flow inside the capillary tube with an optimized dual-end heating strategy are analyzed and modeled. Second, real-time CCPCR is implemented with light-emitting diode and …

Engineering Fret Biosensors For Microrna Presence/Absence Analysis, Nicholas E. Larkey, Sean M. Burrows

Biomedical Engineering Western Regional Conference

No abstract provided.

Biophysical Tools To Study Cellular Mechanotransduction, Ismeel Muhamed, Farhan Chowdhury, Venkat Maruthamuthu

Mechanical & Aerospace Engineering Faculty Publications

The cell membrane is the interface that volumetrically isolates cellular components from the cell's environment. Proteins embedded within and on the membrane have varied biological functions: reception of external biochemical signals, as membrane channels, amplification and regulation of chemical signals through secondary messenger molecules, controlled exocytosis, endocytosis, phagocytosis, organized recruitment and sequestration of cytosolic complex proteins, cell division processes, organization of the cytoskeleton and more. The membrane's bioelectrical role is enabled by the physiologically controlled release and accumulation of electrochemical potential modulating molecules across the membrane through specialized ion channels (e.g., Na^⁺, Ca²⁺, K^⁺ channels). …

Effects Of Exposure To Quantum Dots In The External And Internal Environments Of Organisms, Kenton Layne Leigh

Student Theses and Dissertations

The use of nanotechnology in research and consumer products is increasing, but there are few studies regarding the safety of nanoparticles (NPs). NPs’ small size and increased surface area allow for unique physical and chemical properties not seen in bulk counterparts of the same material. Thus, NP interactions within environmental and biological matrices are not well understood. Specifically, quantum dots (QDs) have garnered increased attention due to their superior optical properties. However, little research on the effects of QDs on the environment is available. The studies described herein provide data regarding interactions of QDs in aquatic and terrestrial environments using …

Engineering A Fluorescent Protease Sensor For In Vivo Protein Detection, Thomas C. Kinard

Honors Scholar Theses

This report details the results of an ongoing project to engineer a mutant form of Red Fluorescent Protein (RFP) variant mCherry that acts as a real-time in vivo protease sensor. The sought-after mutant only becomes fluorescent when exposed to Tobacco Etch Virus (TEV) Protease, this system’s model protease. This will be accomplished via the insertion of the TEV Protease Recognition Site (TEV-PRS) in such a position that, before cleavage, will prevent the protein from folding to fluorescent conformation, but upon cleavage, will allow for fluorescent conformation to occur. The cylindrical structure of the protein, composed of beta-pleated sheets, contains “loops” …

Inhibition Of Mammalian Glycoprotein Ykl-40 Identification Of The Physiological Ligand, Abhishek A. Kognole, Christina M. Payne

Chemical and Materials Engineering Faculty Publications

YKL-40 is a mammalian glycoprotein associated with progression, severity, and prognosis of chronic inflammatory diseases and a multitude of cancers. Despite this well documented association, identification of the lectin′s physiological ligand and, accordingly, biological function has proven experimentally difficult. YKL-40 has been shown to bind chito-oligosaccharides; however, the production of chitin by the human body has not yet been documented. Possible alternative ligands include proteoglycans, polysaccharides, and fibers like collagen, all of which makeup the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles …

Beneficial Aerodynamic Effect Of Wing Scales On The Climbing Flight Of Butterflies, Nathan Slegers, Michael Heilman, Jacob Cranford, Amy Lang, John Yoder, Maria Laura Habegger

Faculty Publications - Biomedical, Mechanical, and Civil Engineering

It is hypothesized that butterfly wing scale geometry and surface patterning may function to improve aerodynamic efficiency. In order to investigate this hypothesis, a method to measure butterfly flapping kinematics optically over long uninhibited flapping sequences was developed. Statistical results for the climbing flight flapping kinematics of 11 butterflies, based on a total of 236 individual flights, both with and without their wing scales, are presented. Results show, that for each of the 11 butterflies, the mean climbing efficiency decreased after scales were removed. Data was reduced to a single set of differences of climbing efficiency using are paired t …

Pretreatment, Enzymatic Hydrolysis, And Fermentation To Ethanol Using A Lignocellulosic Feedstock And Subsequent Recovery Of A Value Added Co-Product: Pure Crystalline Cellulose, Ryan J. Bouza

Electronic Theses and Dissertations

As more demand for alternatives to petroleum and the industrial world’s love of cars increase, cellulosic ethanol will become more important. The ethanol can, of course, be used in the transportation fuel sector, but there is also a potential for co-products to be developed out of the cellulose to ethanol process. Some of these co-products have the potential to replace current petrol products. These co-products may provide the extra revenue generation needed for further investment and development of this industry. This would not only provide better energy independence, but in the United States, it would better satisfy the cellulosic ethanol …

The Synthetic Biology Of N2-Fixing Cyanobacteria For Photosynthetic Terpenoid Production, Charles T. Halfmann

Electronic Theses and Dissertations

In the last few decades, concerns over global climate change, energy security, and environmental pollution have been rising. To overcome these challenges, the concept of “-n^th generation” biofuels has emerged as a strategy to convert solar radiation into fuels and bulk industrial chemicals for societal use, while decreasing our consumption of nonrenewable energy sources. Nitrogen-fixing cyanobacteria hold a distinct advantage in biofuel production over plants, given their ability to convert sunlight, air (CO₂ and N₂), and mineralized water to energy-dense carbon molecules, as well as fix atmospheric nitrogen gas into ammonia for metabolism. Engineered cyanobacteria with …