Cell Biology Commons^™

Development Of A Long-Read Sequencing Protocol To Assess The Precision And Efficacy Of Gene Editing For Duchenne Muscular Dystrophy, Landon Andrew Burcham

Graduate Theses and Dissertations

This work establishes a method for assessing on-target precision due to CRISPR-Cas9 gene editing, especially within the context of exon skipping therapy for Duchenne Muscular Dystrophy. The proposed method utilizes an Oxford nanopore long-read sequencing approach to sequence amplified regions of DNA that have been edited using CRISPR-Cas9. NIH3T3 and C2C12 cell lines were treated with a dual-guide CRISPR-Cas9 system, that targets and deletes exon 23 from the DMD gene in mouse samples. Deletion PCR revealed deletion of exon 23 in both DNA and cDNA samples. Additionally, sequencing using Oxford Nanopore revealed targeted exon 23 deletion as the most prevalent …

Breaks In Longitudinal Elastic Fibers Of Human Femoropopliteal Arteries, Elham Zamani

UNO Student Research and Creative Activity Fair

Breaks in Longitudinal Elastic Fibers of Human Femoropopliteal Arteries

Elham Zamani1, Majid Jadidi1

1 Department of Biomechanics, University of Nebraska Omaha, Omaha, NE

Introduction: Elastin is a major protein in the body with half-life >50 years. It is thought that elastic fibers are formed before the postnatal period. In the femoropopliteal artery (FPA), the main artery in the leg, longitudinal elastic fibers are present in External Elastic Lamina (EEL). Our team has studied more than 1000 cadaveric human FPA and has noticed that there are big breaks in their longitudinal elastic fibers in some subjects. Our goal in this work …

An Antimicrobial Polydopamine Surface Coating To Reduce Biofouling On Telemetry Tags Used In Marine Conservation Practices, Ariana Smies

Dissertations, Master's Theses and Master's Reports

Satellite telemetry tags are used to track the migration patterns of large cetaceans. These tags penetrate the dermis and remain embedded in the underlying blubber tissue. As the dermis of cetaceans is host to a diverse microbiome, and it is impossible to clean the skin before implanting the devices, the potential for infection is increased when the tags penetrate through the skin. H₂O₂ is a potential antimicrobial agent that, in addition to showing broad-spectrum efficacy against gram-negative and gram-positive bacteria, can promote wound healing outcomes by promoting proliferative factors and peptides that protect against oxidative stress. However, …

Novel Approaches For Enhancing Cell Survival And Function In Vivo, Ou Wang

Department of Chemical and Biomolecular Engineering: Dissertations, Theses, and Student Research

FDA has approved several cell-based therapeutics and hundreds of cell therapy clinical trials are ongoing. Cells will be a significant type of medicine after small molecule and protein drugs. However, several obstacles need to be addressed to achieve the widespread use of cellular therapeutics. The first challenge is the low efficacy of cell transplantation due to low retention, survival, integration, and function of cells in vivo. The second challenge is producing a massive number of cells for clinical treatment with cost-effectively and reproducibly technologies.

In this thesis, we proposed and investigated two approaches to address these challenges. To begin …

Investigating Effects Of Microenvironmental Stress On Cell Metabolism Using Multiphoton Imaging, Lisa Rebello

Graduate Theses and Dissertations

Resistance to therapy in cancer is a major cause of poor prognosis in patients. Tumor hypoxia plays an active role in mediating treatment resistance and has been linked to metastases and metastatic potential in cancer. Our research focused on three objectives: i) To understand metabolic effects of chronic and intermittent hypoxia in murine breast cancer cells and its affiliation with metastatic potential ii) To identify the metabolic changes associated with radiation therapy in a panel of radiosensitive and radioresistant human head and neck cancer cells and iii) to monitor the changes in cell metabolism associated with gain of treatment resistance. …

Threshold Concentration And Random Collision Determine The Growth Of The Huntingtin Inclusion From A Stable Core, Sen Pei, Theresa C. Swayne, Jeffrey F. Morris, Lesley Emtage

Publications and Research

The processes underlying formation and growth of unfolded protein inclusions are relevant to neurodegenerative diseases but poorly characterized in living cells. In S. cerevisiae, inclusions formed by mutant huntingtin (mHtt) have some characteristics of biomolecular condensates but the physical nature and growth mechanisms of inclusion bodies remain unclear. We have probed the relationship between concentration and inclusion growth in vivo and find that growth of mHtt inclusions in living cells is triggered at a cytoplasmic threshold concentration, while reduction in cytoplasmic mHtt causes inclusions to shrink. The growth rate is consistent with incorporation of new material through collision and coalescence. …

Human Ipsc Tissue-Engineered Cartilage For Disease Modeling Of Skeletal Dysplasia-Causing Trpv4 Mutations, Amanda R. Dicks

McKelvey School of Engineering Graduate Student Theses & Dissertations

Cartilage is essential to joint development and function. However, there is a variety of cartilage diseases, ranging from developmental (e.g., skeletal dysplasias) to degenerative (e.g., arthritis), in which treatments and therapeutics are lacking. For example, specific point mutations in the ion channel transient receptor potential vanilloid 4 (TRPV4) prevent proper joint development, leading to mild brachyolmia and severe, neonatally lethal metatropic dysplasia. Tissue-engineered cartilage offers an opportunity to elucidate the underlying mechanisms of these cartilage diseases for the development of treatments. Human induced pluripotent stem cells (hiPSCs) are an improved cell source option for cartilage tissue engineering given their minimal …

Synthetic Gene Circuits For Self-Regulating And Temporal Delivery Of Anti-Inflammatory Biologic Drugs In Engineered Tissues, Lara Pferdehirt

McKelvey School of Engineering Graduate Student Theses & Dissertations

The recent advances in the fields of synthetic biology and genome engineering open up new possibilities for creating cell-based therapies. We combined these tools to target repair of articular cartilage, a tissue that lacks a natural ability to regenerate, in the presence of arthritic diseases. To this end, we developed cell-based therapies that harness disease pathways and the unique properties of articular cartilage for prescribed, localized, and controlled delivery of biologics, creating the next generation of cell therapies and new classes of synthetic circuits. We created tissue engineered cartilage from murine induced pluripotent stem cells that had the ability to …

Impact Of Angiogenic And Osteogenic Factors In The Presence Of Biodegradable Piezoelectric Films, Jayla Millender

University Scholar Projects

One of the most common causes of bone graft rejection is lack of a vascular network connecting the graft to the existing native tissue – allowing for nutrient flow. Under current grafting techniques, the existing blood vessel network in the patient slowly invades the implant in order to supply the injured site with its necessary nutrients. The purpose of this research is to determine if a synthetic bone graft with a stable microvascular network can be developed in vitro. I hypothesize that the use of indirect angiogenic factors such as sonic hedgehog homolog and hypoxia-inducible factor-1 in combination with the …

U-251mg Spheroid Generation Using A Scaffold Based Method Protocol, Lara Carroll, Brijesh K. Tiwari, James F. Curtin, Janith Wanigasekara

Articles

3D cell culture is a technique that is used to grow cells in vitro that will mimic an in vivo environment. 3D cell models are a helpful learning tool for researchers to better understand disease mechanisms and to explore different therapeutic properties of drugs. 3D cell cultures can be developed using patient derived cancer cells. Once they have been grown, these 3D cells can be used to screen for small molecule drugs or for genetic modification in for analysis of disease pathways or to predict drug treatments toxicity or efficacy. 3D cell cultures are a big step towards the more …

Involvement Of The Ino80 Chromatin Remodeling Complex In Cell Division And Genomic Stability, Ethan Chen

Biomedical Engineering Undergraduate Honors Theses

Cell division is a vital biological process for growth and development in both single and multi-cellular organisms—whereby the cell must duplicate its organelles and genome in entirety and appropriately distribute the copied contents to the daughter cells. Cells undergo a cycle of two distinct phases: interphase and mitosis. During interphase, the cell replicates its genomic DNA (in the form of chromosomes) located within the nucleus. DNA replication is carried out in a euchromatin state, where the chromosome structure is loose and easily accessible by DNA polymerase and other replication enzymes. Upon the completion of replication, chromatin is condensed into highly …

Biomedical Applications And Syntheses Of Selected Anthraquinone Dyes, Richard Sirard

Senior Honors Theses

Anthraquinones are aromatic organic compounds that have multiple applications in the biomedical field. Some anthraquinone-based compounds are used as fluorophores to contrast cell nuclei while others act as chemotherapeutic agents. However, there are not many fluorescent anthraquinone cell stains currently available. In this study, commercially available anthraquinone dyes, in addition to other dye families and compounds, were reviewed for their unique properties, advantages, and drawbacks. The development and characterization of three novel anthraquinone fluorophores revealed promising photophysical characteristics, like large Stokes shifts. One of the compounds, RBS3, was chosen for fixed and live cell staining and exhibited desirable biomedical properties. …

Cyclophilin D Is A Sensor Of Nano-Pulse Stimulation, Brittney Ruedlinger, Bani Hani Maisoun, Lucas Potter, Nicola Lai, Stephen J. Beebe

The Graduate School Posters

Nano-Pulse Stimulation (NPS), a pulsed power-derived technology, stimulates structural and functional changes in plasma membranes and cellular organelles. NPS induces a Ca²⁺ influx and opening of the mitochondrial permeability transition pore (mPTP) that dissipates the mitochondrial membrane potential (ΔΨ_m) and, when sustained, induces regulated cell death. Here we show that in rat cardiomyoblasts (H9C2) cyclophilin D (CypD) is a mitochondrial sensor for NPS as defined by observations that loss of ΔΨ_m is Ca²⁺ and mitochondrial reactive oxygen species (mROS) dependent and cyclosporin A (CsA)-sensitive, which are diagnostic qualities for effects on CypD and the mPTP. …

Trials And Tribulations Of Humanizing Mice For Cancer Research, Brittney Ruedlinger, Steven Warsof, Eric Feliberti, Mary Beth Hughes, Ayobami ‘Edwin’ Oshin, Chunqi Jiang, Brittany P. Lassiter, Siqi Guo, Stephen J. Beebe

The Graduate School Posters

Cancers are aggressive, evasive, and ruthless killers, claiming millions of lives every year. Cancers are heterogeneous and there is often no single, clearly defined problem as they harness and manipulate a multitude of fundamental mechanisms at the very essence of life. To investigate these mechanisms and vet potential interventive therapies, humanized mice offer a unique model as a prelude to the use of nanosecond pulse stimulation (NPS), a pulse power technology applying nanosecond duration, high electric field pulses, to ablate human tumors. Immunodeficient mouse strains, NSG and NSG-SGM3, were engrafted with human immune cells and human tumors, which would allow …

In Vivo Optical Metabolic Imaging Of Long-Chain Fatty Acid Uptake In Orthotopic Models Of Triple-Negative Breast Cancer, Megan C. Madonna, Joy E. Duer, Joyce V. Lee, Jeremy Williams, Baris Avsaroglu, Caigang Zhu, Riley Deutsch, Roujia Wang, Brian T. Crouch, Matthew D. Hirschey, Andrei Goga, Nirmala Ramanujam

Biomedical Engineering Faculty Publications

Targeting a tumor’s metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, …

Stobe Photography Mapping Of Cell Membrane Potential With Nanosecond Resolution, Allen S. Kiester, Bennett L. Ibey, Zachary N. Coker, Andrei G. Pakhomov, Joel N. Bixler

Bioelectrics Publications

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious …

Leveraging Chemical And Computational Biology To Probe The Cellulose Synthase Complex, B. Kirtley Amos

Theses and Dissertations--Plant and Soil Sciences

Cellular expansion in plants is a complex process driven by the constraint of internal cellular turgor pressure by an expansible cell wall. The main structural element of the cell wall is cellulose. Cellulose is vital to plant fitness and the protein complex that creates it is an excellent target for small molecule inhibition to create herbicides. In the following thesis many small molecules (SMs) from a diverse library were screened in search of new cellulose biosynthesis inhibitors (CBI). Loss of cellular expansion was the primary phenotype used to search for putative CBIs. As such, this was approached in a forward …

Regulation Of Osseous Healing By Macrophage-Derived Wnt Ligands, Jefferson Overlin Abaricia

Theses and Dissertations

Osseous healing is regulated by various classes of cells and signaling mechanisms. Of these, Wnt signaling has long been known to be critically important in directing the differentiation of mesenchymal stem cells (MSCs) into osteoblasts during bone healing. However, the source of these Wnt ligands is not known. Recently, a body of literature has arisen finding both beneficial and detrimental effects of macrophage-derived Wnt ligands on the regeneration of other tissues (liver, kidney, heart, and intestine), often by regulating inflammatory activation and stem cell behavior. As macrophages have been well-characterized as orchestrators of healing, their use of Wnt signaling to …

Impact Of Hemodynamic Vortex Spatial And Temporal Characteristics On Analysis Of Intracranial Aneurysms, Kevin W. Sunderland

Dissertations, Master's Theses and Master's Reports

Subarachnoid hemorrhage is a potentially devastating pathological condition in which bleeding occurs into the space surrounding the brain. One of the prominent sources of subarachnoid hemorrhage are intracranial aneurysms (IA): degenerative, irregular expansions of area(s) of the cerebral vasculature. In the event of IA rupture, the resultant subarachnoid hemorrhage ends in patient mortality occurring in ~50% of cases, with survivors enduring significant neurological damage with physical or cognitive impairment. The seriousness of IA rupture drives a degree of clinical interest in understanding these conditions that promote both the development and possible rupture of the vascular malformations. Current metrics for the …

A Mechanism Behind The Mechanotransduction Of Surface Characteristics In Osteoblasts, Otto J. Juhl Iv

Theses and Dissertations

Biomaterials for use in bone regeneration and healing range from metal and metal alloy implants to hydrogel-based solutions. These materials can be optimized to increase bone healing and integration by improving the mechanical and biological properties. Regardless of the material itself, the cell-substrate interaction is key to the success of the biomaterial once implanted. Substrate surface characteristics such as roughness, wettability, and particle density are well-known contributors to a substrate’s overall osteogenic potential, and therefore the substrate's overall success. Unfortunately, it is still unknown how these substrate surface characteristics are transduced into intracellular signals by cells, preventing specific tailoring of …

Investigating The Antitumor Effects Of A Dsrna-Nanoparticle Complex In An In Vitro Ovarian Cancer Model, Aaron Lewis

Theses and Dissertations (Comprehensive)

An estimated 1 in 70 women will be diagnosed with ovarian cancer in their lifetime. Despite advanced detection and treatment methods, it remains a silent killer with an expected survival rate of 50%. A developing method in cancer treatment is the use of compounds that stimulate the immune system to aid in the body's fight against the disease. This project focused on the use of the potent immune stimulant double-stranded RNA (dsRNA), commercially available as polyinosinic:polycytidylic acid, poly(I:C), to induce cytotoxicity in two ovarian cancer cell lines; SKOV-3 and OVCAR-3. Some challenges exist with the delivery of dsRNA due to …

Modulation Of Ros In Nanosecond-Pulsed Plasma-Activated Media For Dosage-Dependent Cancer Cell Inactivation In Vitro, Chunqi Jiang, Esin Bengisu Sozer, Shutong Song, Nicola Lai, P. Thomas Vernier, Sigi Guo

Bioelectrics Publications

Dosage control of reactive oxygen and nitrogen species (RONS) is critical to low-temperature plasma applications in cancer therapy. Production of RONS by atmospheric pressure, nonequilibrium plasmas in contact with liquid may be modulated via plasma conditions including plasma treatment time and pulse voltage and repetition frequency. In this study, a terephthalic acid-based probe was used to measure hydroxyl radicals [OH_aq] in water exposed to plasma and to demonstrate that the OH_ag concentration increases linearly with treatment time. Fluorometric measurements of hydrogen peroxide concentration in plasma-activated water show a linear relationship between the H₂O₂ production …

Biomechanical And Biophysical Properties Of Breast Cancer Cells Under Varying Glycemic Regimens, Diganta Dutta, Xavier-Lewis Palmer, Jose Ortega-Rodas, Vasundhara Balraj, Indrani Ghosh Dastider, Surabhi Chandra

Electrical & Computer Engineering Faculty Publications

Diabetes accelerates cancer cell proliferation and metastasis, particularly for cancers of the pancreas, liver, breast, colon, and skin. While pathways linking the 2 disease conditions have been explored extensively, there is a lack of information on whether there could be cytoarchitectural changes induced by glucose which predispose cancer cells to aggressive phenotypes. It was thus hypothesized that exposure to diabetes/high glucose alters the biomechanical and biophysical properties of cancer cells more than the normal cells, which aids in advancing the cancer. For this study, atomic force microscopy indentation was used through microscale probing of multiple human breast cancer cells (MCF-7, …

Sonoporation-Mediated Loading Of Trehalose In Cells For Cryopreservation., Charles W. Shaffer Iv, David F. Grimm, Michael A. Menze, Jonathan A. Kopechek

Undergraduate Research Events

Trehalose, a non-reducing disaccharide, is present in many microorganisms and metazoans. In these organisms, trehalose acts as a stress protectant and helps preserve lipid membranes of cells during states of desiccation and freezing. Trehalose is required on both sides of the cell membrane to achieve a significant cryoprotective effect. Specific loading methods for trehalose are required since this sugar is impermeant to mammalian cells. Trehalose loading in mammalian cells has been achieved by fluid-phase endocytosis and genetic modification for the expression of trehalose transporters, however cryoprotective outcomes are unable to compete with established methods of cryopreservation for mammalian cells. Sonoporation …

Secretion Of Proteins And Antibody Fragments From Transiently Transfected Endothelial Progenitor Cells, Loree Heller, Reynald Thinard, Melanie Chevalier, Sezgi Arpag, Yu Jing, Ruth Greferath, Richard Heller, Claude Nicolau

Bioelectrics Publications

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization.

Metabolic Network Analysis Of Filamentous Cyanobacteria, Daniel Alexis Norena-Caro

LSU Doctoral Dissertations

Cyanobacteria were the first organisms to use oxygenic photosynthesis, converting CO₂ into useful organic chemicals. However, the chemical industry has historically relied on fossil raw materials to produce organic precursors, which has contributed to global warming. Thus, cyanobacteria have emerged as sustainable stakeholders for biotechnological production. The filamentous cyanobacterium Anabaena sp. UTEX 2576 can metabolize multiple sources of Nitrogen and was studied as a platform for biotechnological production of high-value chemicals (i.e., pigments, antioxidants, vitamins and secondary metabolites). From a Chemical engineering perspective, the biomass generation in this organism was thoroughly studied by interpreting the cell as a microbial …

A Micro-Optic Stalk (Muos) System To Model The Collective Migration Of Retinal Neuroblasts, Stephanie Zhang, Miles Markey, Caroline D. Pena, Tadmiri Venkatesh, Maribel Vasquez

Publications and Research

Contemporary regenerative therapies have introduced stem-like cells to replace damaged neurons in the visual system by recapitulating critical processes of eye development. The collective migration of neural stem cells is fundamental to retinogenesis and has been exceptionally well-studied using the fruit fly model of Drosophila Melanogaster. However, the migratory behavior of its retinal neuroblasts (RNBs) has been surprisingly understudied, despite being critical to retinal development in this invertebrate model. The current project developed a new microfluidic system to examine the collective migration of RNBs extracted from the developing visual system of Drosophila as a model for the collective motile processes …

Higher Tensile Forces Across Cellular Junctions And An Intact Nuclear Linc Complex Is Required For Epithelial Function And Stability, Fnu Vani Narayanan

Theses and Dissertations

Recent advances in three-dimensional (3D) cell culture systems have provided key insights into the understanding of biochemical and physiological states of native tissue. A significant progress in the field of mechanobiology involves measuring cellular traction forces in a more native 3D environment. However, the effects of mechanical forces exerted across cellular junctions and the nuclear LINC complex, in an organized 3D system has not been investigated thus far. Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen, when grown in 3D matrices. The size of the lumen is dependent on apical secretion …

The Impact Of Aging And Mechanical Injury On Alveolar Epithelial And Macrophage Responses In Acute Lung Injury And Inflammation, Michael S. Valentine

Theses and Dissertations

Patients with severe lung pathologies, such as Acute Respiratory Distress Syndrome (ARDS), often require mechanical ventilation as a clinical intervention; however, this procedure frequently exacerbates the original pulmonary issue and produces an exaggerated inflammatory response that potentially leads to sepsis, multisystem organ failure, and mortality. This acute lung injury (ALI) condition has been termed Ventilator-Induced Lung Injury (VILI). Alveolar overdistension, cyclic atelectasis, and biotrauma are the primary injury mechanisms in VILI that lead to the loss of alveolar barrier integrity and pulmonary inflammation. Stress and strains during mechanical ventilation are believed to initiate alveolar epithelial mechanotransduction signaling mechanisms that contribute …

In Vivo Metabolic And Vascular Response To Hypoxia In Twist Knockdown Murine Breast Cancer, Brandon Sturgill

Graduate Theses and Dissertations

Twist transcription factor is often overexpressed in aggressive tumors. Although needed in early embryonic development for organogenesis, Twist is known to induce an epithelial to mesenchymal transition in cells. In cancer, epithelial to mesenchymal transitions can lead to increased motility and invasiveness. It has also been linked to metabolic reprogramming and increased metastatic risk. Furthermore, metabolic preferences can increase proliferation, enhance metastatic potential, and influence the site of metastasis. We hypothesize that Twist directly affects the metabolism of cancer cells. We expect to see in vivo what we have seen in vitro; Twist overexpression should promote a shift away from …