Cell Biology Commons^™

Artificial Yeast Polarization Controlled By Chemical Gradient, James K. Nolan, Bernard Tao

The Summer Undergraduate Research Fellowship (SURF) Symposium

Engineering synthetic multicellular systems will lead to new synthetic biology technological platforms, inform developmental biology through recapitulation of natural systems and possibly unveil novel morphologies with practical applications not before reached throughout natural history (Maharbiz, 2012). Creating an exogenous molecular circuit that will polarize unicellular cells into “apical” and “basal” domains relative to a substrate plane would fulfill a missing component towards fully multicellular synthetic cellular communities (Maharbiz, 2012). To this end, a PIP₃ polarization network previously designed by Chau and associates (Chau, Walter, Gerardin, Tang, Lim 2012) was coupled to the specific activation by niacin of a recombinant …

New Types Of Morpho-Physiological Changes In Cells Exposed To Nanosecond Pulsed Electric Field, Mikhail A. Rassokhin

Theses and Dissertations in Biomedical Sciences

Exposure of cells to a pulsed electric field (PEF) is the basis of multiple techniques and treatments. Nanosecond pulsed electric field (nsPEF) poses unique characteristics to induce subtle cellular effects while preserving cell integrity. Improving understanding of the mechanisms triggered by nsPEF in cells inspires new applications for the nanosecond pulse technology. Although many effects of nsPEF remain unknown, they can be inferred from morpho-physiologic changes, or cell reshaping, that accompany nsPEF exposure. During the exposure cells undergo reshaping that is manifested in swelling and diffuse blebbing. Recently we identified two new distinct forms of reshaping, pseudopod-like blebbing and microvesiculation, …

Engineering Microenvironments To Modulate Calcium Information Processing In Neuronal Cells, Kinsey Cotton Kelly

Doctoral Dissertations

Tissue engineered microenvironments were constructed to test the effects glial cells have on calcium information processing, and to mimic conditions in vivo for tumor invasion and residual cancer after resection of tumor. Submaximal, nM, glutamate (GLU) stimuli were applied to the engineered environments, and the resulting calcium dynamic behavior of neuronal cells was measured to help predict and interpret chaotic systems in the experimental realm. Calcium is a key signaling ion which signals through the N-methyl-D-aspartate (NMDA) glutamate receptor on the neuronal membrane. GLU binding to the NMDA receptor (NMDAR) causes a large and dynamic increase in neuronal intracellular calcium. …

Recruitment Of The Intracellular Ca2+ By Ultrashort Electric Stimuli: The Impact Of Pulse Duration, Iurii Semenov, Shu Xiao, Olga N. Pakhomova, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca2⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We …

The Effects Of Hydrostatic Pressure On Early Endothelial Tubulogenic Processes, Ryan M. Underwood

Theses and Dissertations--Biomedical Engineering

The effects of mechanical forces on endothelial cell function and behavior are well documented, but have not been fully characterized. Specifically, fluid pressure has been shown to elicit physical and chemical responses known to be involved in the initiation and progression of endothelial cell-mediated vascularization. Central to the process of vascularization is the formation of tube-like structures. This process—tubulogenesis—is essential to both the physiological and pathological growth of tissues. Given the known effects of pressure on endothelial cells and its ubiquitous presence in the vasculature, we investigated pressure as a magnitude-dependent parameter for the regulation of endothelial tubulogenic activity. To …

Novel Copper (Ii) Mediated Transfection Nano-Systems And Biological Properties Of Pyridinium Surfactants, Sarit Pal

Open Access Theses & Dissertations

Efficacy of gene therapy is relied upon the improvement of gene delivery vectors into mammalian cells. Although viral-based polynucleotide carriers are still considered as the most common method for gene delivery but the cost effectiveness, nonspecific lethal immune response has evoked the renaissance of non-viral synthetic delivery systems. An initial discovery by Felgnar et al showed Cationic Liposomes (CLs) (having an overall positive charge) when mixed with DNA to form CL-DNA complexes can enhance the transfer of polynucleotides into cells followed by its expression. Presently, a flurry of experimental works is taking place for the better understanding of structural characterization …

Modulation Of Cell-Matrix Interaction For Cryopreservation Of Engineered Tissue, Angela Christine Seawright

Open Access Theses

Long term preservation of functional engineered tissues can significantly advance tissue engineering industry and regenerative medicine. Several preservation techniques have been proposed and investigated for this purpose, and cryopreservation is a leading candidate. While tissues are cryopreserved, ice forms in both the extracellular and intracellular spaces and causes freezing-induced spatiotemporal deformation of the tissue. During this process the cells undergo dehydration by the freezing-induced osmotic pressure difference and mechanical deformation, transmitted through cell-extracellular matrix adhesions. However, the significance and interaction of these cellular level transport and mechanics processes are not well understood. Therefore, this study aims to establish mechanistic understanding …

Real-Time Analysis Of Brain Tumor Cell Dynamics: Novel Thermoelectric Detection Of L-Glutamate And Cell Metabolism Using Microfluidics, Siva Mahesh Tangutooru

Doctoral Dissertations

This study describes the design, fabrication and applications of a novel thermoelectric microfluidic bio-sensor. The bio-sensor is used for real time detection of the L-glutamate (L-glu) dynamics and metabolism for brain tumor cells immobilized in a microfluidic device. The microfluidic device is fabricated using a polymer/glass laminating technique (Xurography). An antimony-bismuth thin-film thermopile (primary sensing element) is integrated to the microfluidic device. The brain tumor cells are immobilized over the thermopile covering measuring and reference junctions of the thermopile using a poly-l-lysine coating layer. L-glutamate oxidase (L-GLOD) is immobilized over the measuring junctions of the thermopile prior to the immobilization …

Electrospun Polycaprolactone Nanofiber Scaffolds For Tissue Engineering, Andreas Haukas

Graduate Theses and Dissertations

Stem cell and tissue engineering offer us with a unique opportunity to research and develop new therapies for treating various diseases that are otherwise incurable using traditional medicines. However, development of these new therapies replies upon the establishment of in vitro cell culture and differentiation systems that mimic in vivo microenvironments required for cell-cell and cell-ECM interaction. The development of these cell culture systems depends upon the identification of appropriate biomaterials and cell sources. Biomaterials should be carefully selected and fabricated into scaffolds for supporting cell growth and differentiation. In this study, we explored the fabrication of 3D electrospun nanofiber …

Nonosecond Pulsed Electric Field Induced Changes In Dielectric Properties Of Biological Cells, Jie Zhuang

Electrical & Computer Engineering Theses & Dissertations

Nanosecond pulsed electric field induced biological effects have been a focus of research interests since the new millennium. Promising biomedical applications, e.g. tumor treatment and wound healing, are emerging based on this principle. Although the exact mechanisms behind the nanosecond pulse-cell interactions are not completely understood yet, it is generally believed that charging along the cell membranes (including intracellular membranes) and formation of membrane pores trigger subsequent biological responses, and the number and quality of pores are responsible for the cell fate. The immediate charging response of a biological cell to a nanosecond pulsed electric field exposure relies on the …

Oxidative Effects Of Nanosecond Pulsed Electric Field Exposure In Cells And Cell-Free Media, Olga N. Pakhomova, Vera A. Khorokhorina, Angela M. Bowman, Raminta Rodaitė-Riševičienė, Gintautas Saulis, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′, 7′-dichlorodihydrofluoresein (H₂DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H₂DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat …

A Preliminary Study On The Potential Of Manuka Honey And Platelet-Rich Plasma In Wound Healing, Scott A. Sell, Patricia S. Wolfe, Andrew J. Spence, Isaac A. Rodriguez, Jennifer M. Mccoll, Rebecca L. Petrella, Koyal Garg, Jeffery J. Ericksen, Gary L. Bowlin

Ellmer School of Nursing Faculty Publications

Aim. The purpose of this study was to determine the in vitro response of cells critical to the wound healing process in culture media supplemented with a lyophilized preparation rich in growth factors (PRGF) and Manuka honey. Materials and Methods. This study utilized cell culture media supplemented with PRGF, as well as whole Manuka honey and the medical-grade Medihoney (MH), a Manuka honey product. The response of human fibroblasts (hDF), macrophages, and endothelial cells (hPMEC) was evaluated, with respect to cell proliferation, chemotaxis, collagen matrix production, and angiogenic potential, when subjected to culture with media containing PRGF, MH, Manuka honey, …

Impact Of Collateral Enlargement On Smooth Muscle Phenotype, Alexander Jerome Bynum

Master's Theses

Peripheral Artery Disease is a very serious disease characterized by an arterial occlusion due to atherosclerotic plaques. In response to an arterial occlusion, arteriogenesis occurs, causing smooth muscle cells to transition from a contractile to synthetic state. Also following an arterial occlusion, functional impairment was seen in the collateral circuit. An immunofluorescence protocol was developed in order to assess the impact of collateral enlargement (arteriogenesis) on smooth muscle phenotype at various time points. Smooth muscle α-actin was used to mark all smooth muscle cells, Ki-67 was used to label proliferating smooth muscle cells, and a fluorescent nuclear stain was used …

An Apoptosis Targeted Stimulus With Nanosecond Pulsed Electric Fields (Nspefs) In E4 Squamous Cell Carcinoma, Wei Ren, Stephen J. Beebe

Bioelectrics Publications

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was [95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. …

Regulation Of Cell Adhesion Strength By Spatial Organization Of Focal Adhesions, Kranthi Kumar Elineni

USF Tampa Graduate Theses and Dissertations

Cell adhesion to extracellular matrix (ECM) is critical to various cellular processes like cell spreading, migration, growth and apoptosis. At the tissue level, cell adhesion is important in the pathological and physiological processes that regulate the tissue morphogenesis. Cell adhesion to the ECM is primarily mediated by the integrin family of receptors. The receptors that are recruited to the surface are reinforced by structural and signaling proteins at the adhesive sites forming focal adhesions that connect the cytoskeleton to further stabilize the adhesions. The functional roles of these focal adhesions extend beyond stabilizing adhesions and transduce mechanical signals at the …

Dispersion Of Cytotoxic Properties Of Multi-Walled Carbon Nanotubes Suspended In Biological Solutions With Tween 80: Their Role In Enhancing Killing Effects Of Nanosecond Pulse Electric Fields On Tumor Cell Lines, Bhargava S. Kalluri

Biological Sciences Theses & Dissertations

The objective of this study was to determine whether multi-walled carbon nanotubes (MWCNTs) suspended in the surfactant Tween 80 give an additive killing effect on tumor cells when exposed to nsPEFs. In this study, MWCNTs were suspended in DMEM and RPMI with or without T80 (surfactant). The size distribution of MWCNTs suspended in these solutions was evaluated with a Delsa™ Nano Zeta potential and sub micro particle Size Analyzer and confirmed with microscopy. The cytotoxicity of MWCNTs dispersed in different concentrations of T80 was evaluated in PANC1 (Human pancreatic cancer cell line) and Jurkat cell lines (Human T-cell lymphoblast cell …

Identification Of An Archaeal Presenilin-Like Intramembrane Protease, Celia Torres-Arancivia, Carolyn M. Ross, Jose Chavez, Zahra Assur, Georgia Dolios, Filippo Mancia, Iban Ubarretxena-Belandia

Publications and Research

Background: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the c-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of b-amyloid peptides (Ab) implicated in Alzheimer’s disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea.

Methodology and Principal Findings: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, …

Human Osteoblast Proliferation In Culture Following A Nanosecond Pulsed Electric Field (Nspef), Leonard Joseph Carinci Jr.

Biological Sciences Theses & Dissertations

Osteoblasts are mononucleate bone forming cells responsible for the deposition of new bone. Application of mechanical stress on bone reveals its ability to produce and release electric potentials across the cell membrane called piezoelectricity. The electric potentials produced in response to mechanical stress may have a direct correlation on osseous cells and the signaling pathways that regulate proliferation. Nanosecond pulsed electric fields (nsPEFs) are high intensity, ultrashort pulses which have the ability to maintain the integrity of the cell membrane by avoiding traditional electroporation. We delivered 8 nsPEFs (0.5 Hz) of a 25 kV/cm or 35 kV/cm electric field strength …

An Algorithm For Identifying Novel Targets Of Transcription Factor Families: Application To Hypoxia-Inducible Factor 1 Targets, Yue Jiang, Bojan Cukic, Donald A. Adjeroh, Heath D. Skinner, Jie Lin, Qingxi J. Shen, Bing-Hua Jiang

Electrical & Computer Engineering Faculty Research

Efficient and effective analysis of the growing genomic databases requires the development of adequate computational tools. We introduce a fast method based on the suffix tree data structure for predicting novel targets of hypoxia-inducible factor 1 (HIF-1) from huge genome databases. The suffix tree data structure has two powerful applications here: one is to extract unknown patterns from multiple strings/sequences in linear time; the other is to search multiple strings/sequences using multiple patterns in linear time. Using 15 known HIF-1 target gene sequences as a training set, we extracted 105 common patterns that all occur in the 15 training genes …

Stochastic Calculations For Computation Of Radiation Effects And Cell Survivability Under Voltage Pulsing, Madhuri Ganapathiraju

Electrical & Computer Engineering Theses & Dissertations

Statistical computations are an important tool for the analysis of stochastic phenomena and processes that are characterized by variability. Biological systems (e.g., cells, tissues etc.) are perfect examples wherein response to a given external stimulus can be varied and needs to be adequately considered. The Monte Carlo method of analysis has now been recognized as the most effective way of treating stochastic variability.

This thesis uses Monte Carlo based simulations to probe two problems that require the quantification and modeling of effects caused by energy deposition onto biological matter from external sources. One problem involves the probabilistic study of the …

Self-Consistent Analyses For Potential Conduction Block In Nerves By An Ultrashort High-Intensity Electric Pulse, R. P. Joshi, A. Mishra, Q. Hu, K. H. Schoenbach, A. Pakhomov

Bioelectrics Publications

Simulation studies are presented that probe the possibility of using high-field (>100kV ∕ cm), short-duration (∼50ns) electrical pulses for nonthermal and reversible cessation of biological electrical signaling pathways. This would have obvious applications in neurophysiology, clinical research, neuromuscular stimulation therapies, and even nonlethal bioweapons development. The concept is based on the creation of a sufficiently high density of pores on the nerve membrane by an electric pulse. This modulates membrane conductance and presents an effective "electrical short" to an incident voltage wave traveling across a nerve. Net blocking of action potential propagation can then result. A continuum approach based …

Development Of Self-Assembled Monolayer-Based Cell Culture Platform Towards Fabrication Of A Three-Dimensional Bioreactor, Rajendra Kandoor Aithal

Doctoral Dissertations

The extracellular matrix (ECM) plays an important role in regulating a number of cellular properties and functions like cell differentiation, cell synthesis and degradation, cell viability and proliferation, cell function, and cell aging. Surface modification of planar substrates with self-assembled monolayers (SAMs) is a promising technique to achieve stable ECMs.

In this work, substrates such as silicon (Si), gallium arsenide (GaAs) and indium tin oxide (ITO) substrates were modified with SAMS containing amino (-NH2), methyl (-CH3), thiol (-SH) and carboxylic (-COOH) end groups and characterized using contact angle measurements, surface infrared (IR) spectroscopy and atomic force microscopy (AFM). Different cell …

Diverse Effects Of Nanosecond Pulsed Electric Fields On Mammalian Cells, Jody Anne White

Theses and Dissertations in Biomedical Sciences

The continuing effort to manipulate cell-signaling pathways for therapeutic benefit has lead to the exploration of electric field effects on cells. Current electric field applications include electroporation of the plasma membrane for introduction of drugs, genes, or other macromolecules into cells. Modeling of how these pulsed electric fields affect cells depicts the cell as an excitable circuit. In this model, the electric fields, administered in short pulses to a cell, charge the plasma and internal membranes, which act as dielectric layers, and between these the cytoplasm acts as a conductive medium. The pulse lengths of this treatment are traditionally in …

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 …

Improved Energy Model For Membrane Electroporation In Biological Cells Subjected To Electrical Pulses, R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

A self-consistent model analysis of electroporation in biological cells has been carried out based on an improved energy model. The simple energy model used in the literature is somewhat incorrect and unphysical for a variety of reasons. Our model for the pore formation energy E(r) includes a dependence on pore population and density. It also allows for variable surface tension, incorporates the effects of finite conductivity on the electrostatic correction term, and is dynamic in nature. Self-consistent calculations, based on a coupled scheme involving the Smoluchowski equation and the improved energy model, are presented. It is shown that E(r) becomes …

Theoretical Predictions Of Electromechanical Deformation Of Cells Subjected To High Voltages For Membrane Electroporation, R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

An electromechanical analysis based on thin-shell theory is presented to analyze cell shape changes in response to external electric fields. This approach can be extended to include osmotic-pressure changes. Our calculations demonstrate that at large fields, the spherical cell geometry can be significantly modified, and even ellipsoidal forms would be inappropriate to account for the deformation. Values of the surface forces obtained from our calculations are in very good agreement with the 1–10 mN/m range for membrane rupture reported in the literature. The results, in keeping with reports in the literature, demonstrate that the final shape depends on membrane thickness. …

Mechanism For Membrane Electroporation Irreversibility Under High-Intensity, Ultrashort Electrical Pulse Conditions, R. P. Joshi, K. H. Schoenbach

Bioelectrics Publications

An improved electroporation model is used to address membrane irreversibility under ultrashort electric pulse conditions. It is shown that membranes can survive a strong electric pulse and recover provided the pore distribution has a relatively large spread. If, however, the population consists predominantly of larger radii pores, then irreversibility can result. Physically, such a distribution could arise if pores at adjacent sites coalesce. The requirement of close proximity among the pore sites is more easily satisfied in smaller organelles than in outer cell membranes. Model predictions are in keeping with recent observations of cell damage to intracellular organelles (e.g., mitochondria), …

Self-Consistent Simulations Of Electroporation Dynamics In Biological Cells Subjected To Ultrashort Electrical Pulses, R. P. Joshi, Q. Hu, R. Aly, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

The temporal dynamics of electroporation of cells subjected to ultrashort voltage pulses are studied based on a coupled scheme involving the Laplace, Nernst-Plank, and Smoluchowski equations. A pore radius dependent energy barrier for ionic transport, accounts for cellular variations. It is shown that a finite time delay exists in pore formation, and leads to a transient overshoot of the transmembrane potential V_mem beyond 1.0 V. Pore resealing is shown to consist of an initial fast process, a 10⁻⁴s delay, followed by a much slower closing at a time constant of about 10 ⁻¹s. This establishes a …

Characterization Of The Biological Activities Of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (Gfp/Hzp3), Zhiyong Lin

Theses and Dissertations in Biomedical Sciences

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).

This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell …

Retention Of Cellular Viability And Growth Potential In Cryopreserved Bovine Arterial Tissue: Implications For Clinical Use Of Cryopreserved Vascular Allografts, Kip G. Gardner

Biological Sciences Theses & Dissertations

Segments of bovine calf aortas and pulmonary arteries were cryopreserved at -196° C for two weeks to 18 months and subsequently thawed. Cellular morphology, viability, and growth potential in these segments were then compared with the same attributes in segments from fresh (control) arteries. Scanning electron microscopy revealed no disruption of intimal surfaces attributable to cryopreservation. Endothelial cells were successfully cultured from all cryopreserved arterial segments. Endothelial monolayers grown from cryopreserved arteries had a cobblestone appearance and expressed Factor VIII antigen but not smooth muscle alpha-actin. Plating efficiency in primary cultures of endothelial cells was lower for cryopreserved arteries than …