Molecular, Cellular, and Tissue Engineering Commons^™

Development Of A Protocol To Measure Gene Expression In The Mouse Tibia, Daniel Hoover

Biomedical Engineering

Numerous molecular factors active in bone tissue direct fracture repair and remodeling which can be altered by disease conditions such as Peripheral Arterial Disease (PAD) and Osteoporosis. Methods of molecular biology are commonly applied to investigate the expression and role of these molecular factors. This project presents a robust three-step protocol for examining gene expression in the mouse tibia. The protocol begins with isolating RNA from a flash frozen tibia sample. The isolated RNA is reverse transcribed into cDNA. Finally, PCR is performed to indentify expressed genes. Establishing this protocol will allow further research into the mechanisms of bone remodeling …

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 …

Methamphetamine Administration Targets Multiple Immune Subsets And Induces Phenotypic Alterations Suggestive Of Immunosuppression., Robert Z. Harms, Brenda M. Morsey, Craig W. Boyer, Howard S. Fox, Nora E. Sarvetnick

Journal Articles: Regenerative Medicine

Methamphetamine (Meth) is a widely abused stimulant and its users are at increased risk for multiple infectious diseases. To determine the impact of meth on the immune system, we utilized a murine model that simulates the process of meth consumption in a typical addict. Our phenotypic analysis of leukocytes from this dose escalation model revealed that meth affected key immune subsets. Meth administration led to a decrease in abundance of natural killer (NK) cells and the remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear …

A Thousand Tiny Pieces: The Federal Circuit’S Fractured Myriad Ruling, Lessons To Be Learned, And The Way Forward, Jonathan R. K. Stroud

IP Theory

No abstract provided.

Mononuclear Phagocyte Intercellular Crosstalk Facilitates Transmission Of Cell-Targeted Nanoformulated Antiretroviral Drugs To Human Brain Endothelial Cells, Georgette D. Kanmogne, Sangya Singh, Upal Roy, Xinming Liu, Joellyn Mcmillan, Santhi Gorantla, Shantanu Balkundi, Nathan Smith, Yazen Alnouti, Nagsen Gautam, You Zhou, Larisa Poluektova, Alexander Kabanov, Tatiana Bronich, Howard Gendelman

Nebraska Center for Biotechnology: Faculty and Staff Publications

Despite the successes of antiretroviral therapy (ART), HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood–brain barrier (BBB) and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART) (atazanavir, ritonavir, indinavir, and efavirenz). We demonstrate that nanoART transfer from mononuclear phagocytes (MP) to human brain microvascular endothelial cells (HBMEC) can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP …

Differential Il-21 Signaling In Apcs Leads To Disparate Th17 Differentiation In Diabetes-Susceptible Nod And Diabetes-Resistant Nod.Idd3 Mice., Sue M. Liu, David H. Lee, Jenna M. Sullivan, Denise Chung, Anneli Jäger, Bennett O V. Shum, Nora E. Sarvetnick, Ana C. Anderson, Vijay K. Kuchroo

Journal Articles: Regenerative Medicine

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with …

Impaired Resistance Artery Reactivity Following Arteriogenesis, Michael (Mike) Machado

Biomedical Engineering

No abstract provided.

Determining A Method For Rendering Low Cost Cdse(Zns) Core(Shell) Quantum Dots Aqueous Soluble Via Amphiphilic Polymer Wrapping, Patrick Mcbride

Materials Engineering

Herein is described the procedure of two amphiphilic polymer wrapping techniques that may be employed for obtaining aqueous soluble quantum dots (QDs) for use in biological fluorescent imaging applications. The advent of QDs has led to new nanoscale fluorescent materials that exhibit unparalleled quantum yields (QYs), high resistance to photobleaching, tunable emissions, and
absorption over a large optical range. However, the QD synthesis employed here at Cal Poly to obtain bright, photostable CdSe(ZnS) core(shell) QDs involves the use of organic solvents and surfactants, leading to hydrophobic QDs. Since all of biology relies on aqueous solubility, this hydrophobicity creates a major …

Ischemia Impairs Vasodilation In Skeletal Muscle Resistance Artery, Kyle Remington Struthers

Master's Theses

Functional vasodilation in arterioles is impaired with chronic ischemia. We sought to examine the impact of chronic ischemia and age on skeletal muscle resistance artery function. To examine the impact of chronic ischemia, the femoral artery was resected from young (2-3mo) and adult (6-7mo) mice and the profunda femoris artery diameter was measured at rest and following gracilis muscle contraction 14 days later using intravital microscopy. Functional vasodilation was significantly impaired in ischemic mice (14.4±4.6% vs. 137.8±14.3%, p<0.0001 n=8) and non-ischemic adult mice (103.0±9.4% vs. 137.8±14.3%, p=0.05 n=10). In order to analyze the cellular mechanisms of the impairment, a protocol was developed to apply pharmacological agents to the experimental preparation while maintaining tissue homeostasis. Endothelial and smooth muscle dependent vasodilation were impaired with ischemia, 39.6 ± 13.6% vs. 80.5 ± 11.4% and 43.0 ± 11.7% vs. 85.1 ± 10.5%, respectively. From this data, it can be supported that smooth muscle dysfunction is the reason for the observed impairment in arterial vasodilation.

Bacterial Killing By Dry Metallic Copper Surfaces, Christophe Espírito Santo, Ee Wen Lam, Christian Elowsky, Davide Quaranta, Dylan W. Domaille, Christopher J. Chang, Gregor Grass

Nebraska Center for Biotechnology: Faculty and Staff Publications

Metallic copper surfaces rapidly and efficiently kill bacteria. Cells exposed to copper surfaces accumulated large amounts of copper ions, and this copper uptake was faster from dry copper than from moist copper. Cells suffered extensive membrane damage within minutes of exposure to dry copper. Further, cells removed from copper showed loss of cell integrity. Acute contact with metallic copper surfaces did not result in increased mutation rates or DNA lesions. These findings are important first steps for revealing the molecular sensitive targets in cells lethally challenged by exposure to copper surfaces and provide a scientific explanation for the use of …

The Incidence Of Type-1 Diabetes In Nod Mice Is Modulated By Restricted Flora Not Germ-Free Conditions., Cecile King, Nora Sarvetnick

Journal Articles: Regenerative Medicine

In the NOD mouse, the incidence of type-1 diabetes is thought to be influenced by the degree of cleanliness of the mouse colony. Studies collectively demonstrate that exposure to bacterial antigen or infection in the neonatal period prevents diabetes [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], supporting the notion that immunostimulation can benefit the maturation of the postnatal immune system [11]. A widely accepted extrapolation from this data has been the notion that NOD mice maintained under germ-free conditions have an increased incidence of diabetes. However, evidence supporting this influential concept is surprisingly limited [12]. In this …

Fus Transgenic Rats Develop The Phenotypes Of Amyotrophic Lateral Sclerosis And Frontotemporal Lobar Degeneration, Cao Huang, Hongxia Zhou, Jianbin Tong, Han Chen, Yong-Jian Liu, Dian Wang, Xiaotao Wei, Xugang Xia

Nebraska Center for Biotechnology: Faculty and Staff Publications

Fused in Sarcoma (FUS) proteinopathy is a feature of frontotemporal lobar dementia (FTLD), and mutation of the fus gene segregates with FTLD and amyotrophic lateral sclerosis (ALS). To study the consequences of mutation in the fus gene, we created transgenic rats expressing the human fus gene with or without mutation. Overexpression of a mutant (R521C substitution), but not normal, human FUS induced progressive paralysis resembling ALS. Mutant FUS transgenic rats developed progressive paralysis secondary to degeneration of motor axons and displayed a substantial loss of neurons in the cortex and hippocampus. This neuronal loss was accompanied by ubiquitin aggregation and …

Comparative Manufacture And Cell-Based Delivery Of Antiretroviral Nanoformulations, Shantanu Balkundi, Ari S. Nowacek, Ram S. Veerubhotla, Han Chen, Andrea Martinez-Skinner, Upal Roy, R. Lee Mosley, Georgette Kanmogne, Xinming Liu, Alexander V. Kabanov, Tatiana Bronich, Joellyn Mcmillan, Howard E. Gendelman

Nebraska Center for Biotechnology: Faculty and Staff Publications

Nanoformulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz were manufactured by wet milling, homogenization or sonication with a variety of excipients. The chemical, biological, immune, virological, and toxicological properties of these formulations were compared using an established monocyte-derived macrophage scoring indicator system. Measurements of drug uptake, retention, release, and antiretroviral activity demonstrated differences amongst preparation methods. Interestingly, for drug cell targeting and antiretroviral responses the most significant difference among the particles was the drug itself. We posit that the choice of drug and formulation composition may ultimately affect clinical utility.

Transgenic Rat Model Of Neurodegeneration Caused By Mutation In The Tdp Gene, Hongxia Zhou, Cao Huang, Han Chen, Dian Wang, Carlisle P. Landel, Pedro Yuxing Xia, Robert Bowser, Yong-Jian Liu, Xu Gang Xia

Nebraska Center for Biotechnology: Faculty and Staff Publications

TDP-43 proteinopathies have been observed in a wide range of neurodegenerative diseases. Mutations in the gene encoding TDP-43 (i.e., TDP) have been identified in amyotrophic lateral sclerosis (ALS) and in frontotemporal lobe degeneration associated with motor neuron disease. To study the consequences of TDP mutation in an intact system, we created transgenic rats expressing normal human TDP or a mutant form of human TDP with a M337V substitution. Overexpression of mutant, but not normal, TDP caused widespread neurodegeneration that predominantly affected the motor system. TDP mutation reproduced ALS phenotypes in transgenic rats, as seen by progressive degeneration of motor neurons …

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 …

Generation Of Recombinant Mouse Embryonic Stem Cell Lines And Theirapplication For In Vivo Bioluminiscence Imaging In The Heart, Ramana Kammili

Electronic Theses and Dissertations

Cardiovascular disease is the major cause of death in the United States, with 80 million people suffering from some form of heart disease each year. One major limitation is the inability of the heart to repair the damaged tissue. Stem cell therapy holds enormous promise to repair and regenerate the damaged myocardium, but there are many technical difficulties that must first be overcome. One such difficulty is the present lack of ability to track and assess transplanted stem cells over time in vivo. The central hypothesis of this thesis is that in vivo bioluminescence imaging is a safe and useful …

Il-21 Limits Peripheral Lymphocyte Numbers Through T Cell Homeostatic Mechanisms., Shrimati Datta, Nora E. Sarvetnick

Journal Articles: Regenerative Medicine

BACKGROUND: IL-21, a member of the common gamma-chain utilizing family of cytokines, participates in immune and inflammatory processes. In addition, the cytokine has been linked to autoimmunity in humans and rodents.

METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanism whereby IL-21 affects the immune system, we investigated its role in T cell homeostasis and autoimmunity in both non-autoimmune C57BL/6 and autoimmune NOD mice. Our data indicate that IL-21R knockout C57BL/6 and NOD mice show increased size of their lymphocyte population and decreased homeostatic proliferation. In addition, our experimental results demonstrate that IL-21 inhibits T cell survival. These data suggest that IL-21 acts …

The Highly Similar Arabidopsis Homologs Of Trithorax Atx1 And Atx2 Encode Proteins With Divergent Biochemical Functions, Abdelaty Saleh, Raul Alvarez-Venegas, Mehtap Yilmaz, Oahn-Le, Guichuan Hou, Monther Sadder, Ayed Al-Abdallat, Yuannan Xia, Guoqinq Lu, Istvan Ladunga, Zoya Avramova

Nebraska Center for Biotechnology: Faculty and Staff Publications

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different …

Resistance Of The Target Islet Tissue To Autoimmune Destruction Contributes To Genetic Susceptibility In Type 1 Diabetes., Natasha J. Hill, Aleksandr Stotland, Michelle Solomon, Patrick Secrest, Elizabeth Getzoff, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Type 1 diabetes occurs when self-reactive T lymphocytes destroy the insulin-producing islet beta cells of the pancreas. The defects causing this disease have often been assumed to occur exclusively in the immune system. We present evidence that genetic variation at the Idd9 diabetes susceptibility locus determines the resilience of the targets of autoimmunity, the islets, to destruction. Susceptible islets exhibit hyper-responsiveness to inflammatory cytokines resulting in enhanced cell death and increased expression of the death receptor Fas. Fas upregulation in beta cells is mediated by TNFR2, and colocalization of TNFR2 with the adaptor TRAF2 in NOD beta cells is altered. …

Desulfovibrio Desulfuricans G20 Tetraheme Cytochrome Structure At 1.5 A˚ And Cytochrome Interaction With Metal Complexes, Mrunalini Pattarkine, J J. Tanner, C A. Bottoms, Y H. Lee, Judy D. Wall

Faculty Works

The structure of the type I tetraheme cytochrome c3 from Desulfovibrio desulfuricans G20 was determined to 1.5 A˚ by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated …

Nanoelectropulse-Driven Membrane Perturbation And Small Molecule Permeabilization, P. Thomas Vernier, Yinghua Sun, Martin A. Gundersen

Bioelectrics Publications

Background
Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization.

Results
In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to …

Identification And Expansion Of Pancreatic Stem/Progenitor Cells., You-Qing Zhang, Marcie Kritzik, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Pancreatic islet transplantation represents an attractive approach for the treatment of diabetes. However, the limited availability of donor islets has largely hampered this approach. In this respect, the use of alternative sources of islets such as the ex vivo expansion and differentiation of functional endocrine cells for treating diabetes has become the major focus of diabetes research. Adult pancreatic stem cells /progenitor cells have yet to be recognized because limited markers exist for their identification. While the pancreas has the capacity to regenerate under certain circumstances, questions where adult pancreatic stem/progenitor cells are localized, how they are regulated, and even …

Stimulation Of Capacitative Calcium Entry In Hl-60 Cells By Nanosecond Pulsed Electric Fields, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200-700 nM, respectively, above basal levels (similar to100 nM), while the uptake of …

The Stromal Cell-Derived Factor-1alpha/Cxcr4 Ligand-Receptor Axis Is Critical For Progenitor Survival And Migration In The Pancreas., Ayse G. Kayali, Kurt Van Gunst, Iain L. Campbell, Aleksandr Stotland, Marcie Kritzik, Guoxun Liu, Malin Flodström-Tullberg, You-Qing Zhang, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking …

Ccr4-Bearing T Cells Participate In Autoimmune Diabetes., Soon H. Kim, Mary M. Cleary, Howard S. Fox, Icos Coporation, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Chemokine receptor expression is exquisitely regulated on T cell subsets during the course of their migration to inflammatory sites. In the present study we demonstrate that CCR4 expression marks a pathogenic population of autoimmune T cells. CCR4 was found exclusively on memory CD4(+) T cells during the progression of disease in NOD mice. Cells expressing the CCR4 ligand TARC (thymus- and activation-regulated chemokine) were detected within infiltrated islets from prediabetic mice. Interestingly, neutralization of macrophage-derived chemokine (MDC) with Ab caused a significant reduction of CCR4-positive T cells within the pancreatic infiltrates and inhibited the development of insulitis and diabetes. Furthermore, …

Presented Antigen From Damaged Pancreatic Beta Cells Activates Autoreactive T Cells In Virus-Mediated Autoimmune Diabetes., Marc S. Horwitz, Alex Ilic, Cody Fine, Enrique Rodriguez, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The induction of autoimmunity by viruses has been attributed to numerous mechanisms. In mice, coxsackievirus B4 (CB4) induces insulin-dependent diabetes mellitus (IDDM) resembling the final step of disease progression in humans. The immune response following the viral insult clearly precipitates IDDM. However, the molecular pathway between viral infection and the subsequent activation of T cells specific for islet antigen has not been elucidated. These T cells could become activated through exposure to sequestered antigens released by damaged beta cells, or they could have responded to factors secreted by the inflammatory response itself. To distinguish between these possibilities, we treated mice …

Studies On The Formation Of Dna-Cationic Lipid Composite Films And Dna Hybridization In The Composites, Murali Sastry, Vidya Ramakrishnan, Mrunalini Pattarkine, Krishna N. Ganesh

Faculty Works

The formation of composite films of double-stranded DNA and cationic lipid molecules (octadecylamine, ODA) and the hybridization of complementary single-stranded DNA molecules in such composite films are demonstrated. The immobilization of DNA is accomplished by simple immersion of a thermally evaporated ODA film in the DNA solution at close to physiological pH. The entrapment of the DNA molecules in the cationic lipid film is dominated by attractive electrostatic interaction between the negatively charged phosphate backbone of the DNA molecules and the protonated amine molecules in the thermally evaporated film and has been quantified using quartz crystal microgravimetry (QCM). Fluorescence studies …

Construction Of A Derivative Of Agrobacterium Tumefaciens C58 That Does Not Mutate To Tetracycline Resistance, Zhao-Qing Luo, Thomas E. Clemente, Stephen K. Farrand

Nebraska Center for Biotechnology: Faculty and Staff Publications

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tet_C58, that is present in the genome of this strain. By deleting the tet_C58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

Cationic Surfactant Mediated Hybridization And Hydrophobization Of Dna Molecules At The Liquid/Liquid Interface And Their Phase Transfer, Murali Sastry, Ashavani Kumar, Mrunalini Pattarkine, Vidya Ramakrishnan, Krishna N. Ganesh

Faculty Works

Hybridization of complementary oligonucleotides mediated by a cationic surfactant at the water/hexane interface leads to hydrophobic, double-helical DNA which may be readily phase transferred to the organic phase and cast into thin films on solid substrates.

Hybridization Of Dna By Sequential Immobilization Of Oligonucleotides At The Air-Water Interface, Murali Sastry, Vidya Ramakrishnan, Mrunalini Pattarkine, Anand Gole, K. N. Ganesh

Faculty Works

The hybridization of DNA by sequential electrostatic and hydrogen-bonding immobilization of single-stranded complementary oligonucleotides at the air-water interface with cationic Langmuir monolayers is demonstrated. The complexation of the single-stranded DNA molecules with octadecylamine (ODA) Langmuir monolayers was followed in time by monitoring the pressure-area isotherms. A large (and slow) expansion of the ODA monolayer was observed during each stage of complexation in the following sequence: primary single-stranded DNA followed by complementary single-stranded DNA followed by the intercalator, ethidium bromide. Langmuir-Blodgett (LB) films of the ODA-DNA complex were formed on different substrates and characterized using quartz-crystal microgravimetry (QCM), Fourier transform infrared …