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Title: Investigating blood cell communication with novel sample handling and microfluidic technology
Creator: Anderson, Kari B.
Date: 2013
Collection: Electronic Theses & Dissertations
Description: ABSTRACTINVESTIGATING BLOOD CELL COMMUNICATION WITH NOVEL SAMPLE HANDLING AND MICROFLUIDIC TECHNOLOGYByKari B. AndersonPuringergic receptor signaling events in platelets is a major determinant in platelet function. ADP binding to the P2Y-type receptors on platelet membranes, and the subsequent pathways evoked from this type of binding, is well-established. However, an understanding activity of the ATP-sensitive P2X1 platelet receptor is incomplete due to a number of obstacles involved with...
Show moreABSTRACTINVESTIGATING BLOOD CELL COMMUNICATION WITH NOVEL SAMPLE HANDLING AND MICROFLUIDIC TECHNOLOGYByKari B. AndersonPuringergic receptor signaling events in platelets is a major determinant in platelet function. ADP binding to the P2Y-type receptors on platelet membranes, and the subsequent pathways evoked from this type of binding, is well-established. However, an understanding activity of the ATP-sensitive P2X1 platelet receptor is incomplete due to a number of obstacles involved with studying this receptor, in particular, the high concentrations of ATP already present in the washed platelet sample matrix. To circumvent this problem, most studies aimed at investigating P2X1 platelet activity require that the platelet samples contain apyrase in order to reduce already-existing levels of ATP and ready the P2X1 receptor for exogenously added ATP. Of course, one drawback to this method is that the apyrase will also rapidly degrade any added ATP. Here, we describe a method which, ironically, employs the reported P2X1 inhibitor NF449 to sensitize platelets in the absence of any added apyrase. Sensitization is verified by spectrofluorimetric determination of Ca2+ entry into the platelets after stimulation with concentrations of ATP ranging from 67 nM to 10 μM. Results suggest that sensitization of the P2X1 receptor by NF449 is not necessarily dependent upon the inhibitor concentration, but rather the ratio of the inhibitor and exogenously-added ATP concentrations. The sensitization by the NF449 was also found to be highly time-dependent. In order to study the platelet in the presence of red blood cells (RBCs) and endothelial cells a microfluidic approach was taken, but before vascular mimic could be created, some new microfluidic technologies were explored. A simple and inexpensive approach to fabricate polystyrene devices that is based upon molding polystyrene (PS) from Petri dishes against PDMS molds was developed. The ability to incorporate microchannels in polystyrene and integrate the resulting device with standard laboratory equipment such as an optical plate reader for analyte readout and pipets for fluid propulsion is described. A simple approach for sample and reagent delivery to the device channels using a standard, multi-channel micropipette and a PDMS-based injection block is detailed. Integration of the microfluidic device with these off-chip functions (sample delivery and readout) enables high-throughput screens and analyses. The device was sealed against a PDMS-base and compared against PDMS-based microchips in terms of their absorption of an anti-platelet drug, clopidogrel. Furthermore, these polystyrene devices were used to monitor two endothelial cell processes. One experiment involved the fluorescence measurement of nitric oxide (NO) produced within an endothelial cell line following stimulation with ATP. The result was a four-fold increase in NO production (as compared to a control), with this receptor-based mechanism of NO production verifying the maintenance of cell receptors following immobilization onto the PS substrate. The ability to monitor cellular uptake was also demonstrated by optical determination of Ca2+ into endothelial cells following stimulation with the Ca2+ ionophore A20317. The result was a significant increase (42%) in the calcium uptake in the presence of the ionophore, as compared to a control (17%) (p < 0.05). The ability to successfully culture cells on chip and measure analytes associated with that PS may be a useful material for microfluidic device fabrication. The PS device was further integrated with a polyester membrane in order to study drug transport across the membrane. However, significant absorption of clopidogrel still occurred so a device made only of PS was fabricated using an epoxy mold and hydraulic press to embed channels and seal devices. The result was a device with millimeter sized channels, but the ability to rapidly fabricate these devices was lost. Fluidic devices fabricated using conventional soft lithography are well suited as prototyping methods. Unfortunately, the prototypes tend to lack the ruggedness and reusability of methods associated with mass production techniques. 3 Dimensional (3D) printing, commonly used for producing design prototypes in industry, allows for one step production of devices. 3D printers build a device layer by layer based on 3D computer models. Here, a reusable, high throughput, 3D printed fluidic device was created that enables flow and incorporates a membrane above a channel in order to study drug transport and affect cells. The device contains 8 parallel channels, 3 mm wide 1.5 mm deep, connected to a syringe pump through standard, threaded fittings. The device was also printed to allow integration with commercially available cell inserts whose bottoms are constructed of a porous polycarbonate membrane; this insert enables molecular transport to occur from the channel to above the well. When concentrations of antibiotics, levofloxacin and linezolid, are pumped through the channels, approximately 18-21% of the drug migrates through the membrane. Finally, we show that mammalian cells cultured on this membrane can be affected by reagents flowing through the channels. Specifically, saponin was used to compromise cell membranes and a fluorescent label used to monitor the extent, resulting in a 4-fold increase in fluorescence for saponin treated cells.
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Title: Pancreatic secretions and their effect on circulatory and immune cells
Creator: Entwistle, Kristen
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia and insulin dysfunction, which often results in secondary complications associated with blood flow. Four major types of diabetes are defined by the American Diabetes Association: type 1 diabetes (T1D, insulin insufficiency), type 2 diabetes (T2D, insulin resistance), gestational diabetes (occurring during pregnancy), and other types, which includes cystic fibrosis related diabetes (CFRD). Although each of these...
Show moreDiabetes mellitus is a metabolic disease characterized by chronic hyperglycemia and insulin dysfunction, which often results in secondary complications associated with blood flow. Four major types of diabetes are defined by the American Diabetes Association: type 1 diabetes (T1D, insulin insufficiency), type 2 diabetes (T2D, insulin resistance), gestational diabetes (occurring during pregnancy), and other types, which includes cystic fibrosis related diabetes (CFRD). Although each of these types of diabetes is associated with insulin dysfunction, it is important to note that the treatment of oral medications or exogenous insulin, although sufficient to effectively manage blood glucose levels, is not sufficient to completely cure diabetes. Secondary complications resulting from diabetes include retinopathy, nephropathy and neuropathy, along with dysfunctions in immune response. In recent years, a surge in the research of C-peptide, the 31 amino acid peptide co-secreted with insulin, has revealed that C-peptide may actually help to ameliorate some of these secondary complications, including neuropathy and immune response. Although initial studies revealed promising results, a phase 2b clinical trial was shut down in 2014 due to indistinguishable results in placebo and treatment groups in T1D patients. To enhance our knowledge of these downstream problems, we here report the effect of C-peptide/zinc/albumin on RBCs and peripheral neutrophils (an immune cell) that mimic those from CF/CFRD patients using a chemical inhibitor (CFTRinh-172). C-peptide and zinc binding to RBCs confirms previously published data, and binding to CFTR-inhibited RBCs is increased from the control, however, the molar binding ratio of C-peptide to zinc remains 1:1. ATP release is increased in control and CFTR-inhibited cells only in the presence of the ensemble of C-peptide/zinc/albumin, but is abolished when any one of these components is missing. Similarly, in neutrophils, we observe a 1:1 molar binding ratio of C-peptide to zinc when albumin is present in both control and CFTR-inhibited cells. We also observed a significant increase in intracellular calcium, intracellular NO, extracellular NO, glucose uptake, and rate of chemotaxis in neutrophils that had been treated with the ensemble, but not when any one component was missing. In both RBCs and neutrophils, we observed an increase in membrane expression of glucose transporter 1 (GLUT1) only when C-peptide, zinc and albumin were present. Insulin did not have this effect. The results reported here are the first to indicate that C-peptide may be acting on cells that contain primarily GLUT1, and that the mechanism of action may be similar to the way that insulin acts in muscle and fat cells, by translocating GLUT4 to the plasma membrane. These results are also the first to suggest that C-peptide may directly be affecting peripheral neutrophils, which could have significant implications in the treatment of immune complications in all forms of diabetes. Additionally, these results are the first to examine the effect this peptide and its metal may have on patients who have CF, and may point to its use a potential therapy in both T2D and CFRD.
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Title: Investigating red blood cells in autoimmune diseases
Creator: Janes, Tiffany
Date: 2018
Collection: Electronic Theses & Dissertations
Description: "Work presented in this dissertation demonstrates that red blood cells (RBCs) from patients with multiple sclerosis (MS) or type 1 diabetes (T1D) have an altered cell metabolism compared to healthy RBCs. An overview of each disease, along with implications of increased or decreased RBC metabolism and downstream events are presented here. Additional experiments investigated methods to increase or decrease the RBC metabolism in an effort to alleviate complications of each disease. Glucose...
Show more"Work presented in this dissertation demonstrates that red blood cells (RBCs) from patients with multiple sclerosis (MS) or type 1 diabetes (T1D) have an altered cell metabolism compared to healthy RBCs. An overview of each disease, along with implications of increased or decreased RBC metabolism and downstream events are presented here. Additional experiments investigated methods to increase or decrease the RBC metabolism in an effort to alleviate complications of each disease. Glucose transporter 1 (GLUT1) is the main glucose transporter on RBCs, and all metabolism within the RBC involves glucose. RBC GLUT1 content was used as a measure of RBC metabolism, and elevated RBC GLUT1 content is proposed to be associated with increased cell metabolism. RBCs obtained from MS patients contained 23.3 ± 4.8% more GLUT1 than control RBCs, while T1D RBCs contained 23.6 ± 4.2% less GLUT1 than control RBCs. Further studies evaluated the effects of various therapies on RBCs with respect to MS and T1D. Steroids are commonly prescribed to MS patients to manage exacerbations of the disease; however, hyperglycemic conditions often result. Evidence presented here using measurements of RBC adenosine triphosphate (ATP) release, suggests that the steroids estriol and prednisolone decrease RBC metabolism. ATP is a product of glycolysis, which utilizes the glucose transported into the cell to produce energy. While MS RBCs released 104 ± 18 nM more ATP than control RBCs, treatment with estriol or prednisolone significantly decreased the RBC ATP release, thought to correlate to RBC metabolism. RBC ATP release stimulates endothelial nitric oxide (NO) production in vivo, which is detrimental at the high levels seen in MS due to its toxicity to the blood brain barrier. Results presented here also suggest that RBCs treated with physiological levels of estriol or prednisolone significantly decrease the downstream endothelial NO production up to 30%, thought to correlate to decreased blood brain barrier damage in MS patients. Shear stress is a main mechanism of RBC ATP release; however, chemical stimulus also leads to RBC ATP release. Further studies investigated the effect steroids had on C-peptide and Zn2+ binding to RBCs, a proven chemical stimulus of RBC ATP release. Results suggest that RBC treatment with prednisolone decreased the Zn2+ binding from 1.99 ± 0.12 nM to 0.18 ± 0.30 nM, and decreased the C-peptide binding from 2.02 ± 0.22 nM to -0.47 ± 0.27 nM. It follows that any effect that C-peptide and Zn2+ have on RBCs is attenuated as their binding to RBCs is attenuated, such as ATP release. Further studies investigated T1D RBCs and a proposed therapy involving a complex of C-peptide, Zn2+, and albumin. T1D patients suffer from insufficient blood flow, and this work suggests that the complex of C-peptide, Zn2+, and albumin may enhance blood flow and alleviate T1D complications. Evidence is presented here showing that an increase in RBC ATP release (34 ± 1.5 nM increase), RBC GLUT1 content (25.5 ± 1.3% increase), RBC C-peptide binding (1.95 ± 0.14 nM increase), and RBC Zn2+ binding (2.26 ± 0.20 nM increase) is only measured when the full complex of C-peptide, Zn2+ and albumin is present. While T1D RBCs have a lower RBC metabolism at basal level, treatment with this complex is suggested to enhance RBC metabolism and ATP release leading to enhanced blood flow, which will greatly help with the treatment of T1D."--Pages ii-iii.
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Title: EXPERIMENTAL EVIDENCE FOR A C-PEPTIDE COMPLEX RECEPTOR ON RED BLOOD CELLS
Creator: Geiger, Morgan
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Pancreatic β-cells secrete insulin and C-peptide, a 31-amino acid peptide, in a 1:1 mole ratio. People with type 1 diabetes (T1D) require exogenous insulin to survive due to damaged or destroyed β-cells. However, even with regular insulin treatments, patients develop complications such as retinopathy, neuropathy, and nephropathy, and it has been proposed that poor blood flow could be causing these complications. Prior research has shown that C peptide increases microvascular blood flow,...
Show morePancreatic β-cells secrete insulin and C-peptide, a 31-amino acid peptide, in a 1:1 mole ratio. People with type 1 diabetes (T1D) require exogenous insulin to survive due to damaged or destroyed β-cells. However, even with regular insulin treatments, patients develop complications such as retinopathy, neuropathy, and nephropathy, and it has been proposed that poor blood flow could be causing these complications. Prior research has shown that C peptide increases microvascular blood flow, therefore, it could be a useful therapy to prevent these complications. The unknown C peptide receptor and mechanism has been a major roadblock in utilizing C-peptide as a therapeutic. Our group has reported that approximately 1,800 C peptide molecules bind per red blood cell (RBC) in the presence of albumin, while there was no detectable C-peptide binding per RBC without albumin. Thus, it is hypothesized here that C-peptide binds to RBCs through an albumin/C-peptide complex receptor, as opposed to binding to the RBC alone. The work in this dissertation focuses on analyzing the binding of bovine serum albumin (BSA) to RBCs using a radiolabeling method to attach technetium-99m (Tc99m) to BSA for gamma decay detection. A binding saturation experiment was conducted to examine the BSA specific binding to RBCs with and without C-peptide. The specific binding curves revealed that albumin saturates at 14,021 (±1,489) BSA molecules/RBC with a Kd of 1.14 (±0.07) x 10-7 M. Whereas, in the presence of C-peptide and Zn2+, albumin saturates at 16,695 (±1,479) BSA molecules/RBC with a Kd of 2.00 (±0.05) x 10 7 M. At saturation, the additional 2,700 BSA molecules/RBC in the presence of C-peptide and Zn2+ indicates that not only does an albumin receptor exist on RBCs, but also a separate receptor for an albumin/C peptide complex. Due to its role in the delivery of C-peptide, the molecular state of albumin may be critical. Albumin is more glycated in individuals with T1D in comparison to healthy controls. This dissertation utilizes the radiolabeling saturation experiment to analyze the effects of glycation on BSA binding to RBCs. Varying percentage of glycation (11-48%) were analyzed with and without C-peptide and Zn2+. As the glycation percentage increased, the number of BSA molecules binding per RBC increased; however, the difference between samples containing C-peptide to those without C-peptide decreased. In fact, at the higher glycation levels, there is more albumin binding in the absence of C peptide. Importantly, the amount of RBC C-peptide uptake decreased when carried by glycated BSA compared to normal BSA. Abnormal albumin delivery may not only be a determinant of disease in people with T1D. Albumin delivery of C-peptide was also examined in people with multiple sclerosis (MS, an autoimmune disease). When comparing BSA binding to MS RBCs to healthy control RBCs, more BSA molecules bound per MS RBC. Previous research in our lab has shown that MS RBCs binds more C peptide and release higher concentrations of ATP than healthy controls. This suggests that in unhealthy conditions, the albumin/C peptide/Zn2+ complex is binding differently than in healthy conditions, which results in abnormal downstream effects.
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Title: Quantitative Analysis of New Key Factors of Immune Cells in Autoimmune Diseases
Creator: Bunn, Demarcus
Date: 2022
Collection: Electronic Theses & Dissertations
Description: AbstractExplorations of New Key Factors of Immune Cells in Autoimmune Diseases By Demarcus Bunn The work presented in this dissertation demonstrates an emerging role of immune cells in type one diabetes (T1D) and multiple sclerosis (MS). The immune cells that will be presented are neutrophils and T-cells. Presented work shows an altered cell metabolism in both disease states that leads to further secondary complications. An overview of the immunology, as well as each autoimmune disease, will...
Show moreAbstractExplorations of New Key Factors of Immune Cells in Autoimmune Diseases By Demarcus Bunn The work presented in this dissertation demonstrates an emerging role of immune cells in type one diabetes (T1D) and multiple sclerosis (MS). The immune cells that will be presented are neutrophils and T-cells. Presented work shows an altered cell metabolism in both disease states that leads to further secondary complications. An overview of the immunology, as well as each autoimmune disease, will be presented. Experimental efforts to increase or decrease cell metabolism in order to alleviate secondary complications will be shown. Furthermore, the use of 3D printed devices for in vitro models mimicking these disease states properties will be presented.Individuals T1D have a history of being more susceptible to infection. This section of the dissertation will demonstrate how a once forgotten pancreatic peptide, C-peptide, has a positive effect on raising immunity through improving immune cell energetics. Previous studies in the Spence lab have shown that C-peptide only binds to red blood cells (RBC) in the presence of albumin, but for the biological changes, Zn2+ is needed. Spence lab research has shown that the combination of C-peptide/Zn2+/albumin increases the metabolism of RBCs. This work shows novel data showing that C-peptide binds specifically to other cell types. Additionally, changes in cell metabolism will be investigated. This portion of the dissertation is important for alleviating reoccurring and persistent infections.MS is characterized by the destruction of the myelin sheath around the nerves. The cell type that does the damage is T lymphocytes. However, little research has been done investigating what makes the permeability of the blood-brain barrier increase. Here, we will introduce the potential role of NETosis, a form of programmed cell death, has on blood brain barrier permeability. There have been recent reports that exogenous adenosine triphosphate (ATP) increases the rate of NETosis production in vitro. Previous work in the Spence lab has shown that RBCs from individuals with MS secrete significantly more ATP than control red blood cells. Presented work will show that ATP derived directly from RBCs causes dysregulation of NETosis. The concluding section of the dissertation will be dedicated to 3D printing. An overview of the current state and future advancements of 3D printing will be presented. Presented work will show the use of 3D printing to provide more relevant conditions for in vitro experiments. Here, 3D printed models were used to investigate immune cell behaviors and changes in cell bioenergetics.
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Title: The integration of computational methods and nonlinear multiphoton multimodal microscopy imaging for the analysis of unstained human and animal tissues
Creator: Murashova, Gabrielle Alyse
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Nonlinear multiphoton multimodal microscopy (NMMM) used in biological imaging is a technique that explores the combinatorial use of different multiphoton signals, or modalities, to achieve contrast in stained and unstained biological tissues. NMMM is a nonlinear laser-matter interaction (LMI), which utilizes multiple photons at once (multiphoton processes, MP). The statistical probability of multiple photons arriving at a focal point at the same time is dependent on the two-photon absorption ...
Show moreNonlinear multiphoton multimodal microscopy (NMMM) used in biological imaging is a technique that explores the combinatorial use of different multiphoton signals, or modalities, to achieve contrast in stained and unstained biological tissues. NMMM is a nonlinear laser-matter interaction (LMI), which utilizes multiple photons at once (multiphoton processes, MP). The statistical probability of multiple photons arriving at a focal point at the same time is dependent on the two-photon absorption (TPA) cross-section of the molecule being studied and is incredibly difficult to satisfy using typical incoherent light, say from a light bulb. Therefore, the stimulated emission of coherent photons by pulsed lasers are used for NMMM applications in biomedical imaging and diagnostics.In this dissertation, I hypothesized that due to the near-IR wavelength of the Ytterbium(Yb)-fiber laser (1070 nm), the four MP-two-photon excited fluorescence (2PEF), second harmonic generation (SHG), three-photon excited fluorescence (3PEF) and third harmonic generation (THG), generated by focusing this ultrafast laser, will provide contrast to unstained tissues sufficient for augmenting current histological staining methods used in disease diagnostics. Additionally, I hypothesized that these NMMM images (NMMMIs) can benefit from computational methods to accurately separate their overlapping endogenous MP signals, as well as train a neural network for image classification to detect neoplastic, inflammatory, and healthy regions in the human oral mucosa. Chapter II of this dissertation explores the use of NMMM to study the effects of storage on donated red blood cells (RBCs) using non-invasive 2PEF and THG without breaching the blood storage bag. Unlike the lack of RBC fluorescence previously reported, we show that with two-photon (2P) excitation from an 800 nm source, and three-photon (3P) excitation from a 1060 nm source, there was sufficient fluorescent signal from hemoglobin as well as other endogenous fluorophores. Chapter III employs NMMM to establish the endogenous MP signals present in healthy excised and unstained mouse and Cynomolgus monkey retinas using 2PEF, 3PEF, SHG, and THG. We show the first epi-direction detected cross-section and depth-resolved images of unstained isolated retinas obtained using NMMM with an ultrafast fiber laser centered at 1070 nm and a 303038 fs pulse. Two spectrally and temporally distinct regions were shown; one from the nerve fiber layer (NFL) to the inner receptor layer (IRL), and one from the retinal pigmented epithelium (RPE) and choroid. Chapter IV focuses on the use of minimal NMMM signals from a 1070 nm Yb-fiber laser to match and augment H&E-like contrast in human oral squamous cell carcinoma (OSCC) biopsies. In addition to performing depth-resolved (DR) imaging directly from the paraffin block and matching H&E-like contrast, we showed how the combination of characteristic inflammatory 2PEF signals undetectable in H&E stained tissues and SHG signals from stromal collagen can be used to analytical distinguish healthy, mild and severe inflammatory, and neoplastic regions and determine neoplastic margins in a three-dimensional (3D) manner. Chapter V focuses on the use of computational methods to solve an inverse problem of the overlapping endogenous fluorescent and harmonic signals within mouse retinas. The least-squares fitting algorithm was most effective at accurately assigning photons from the NMMMIs to their source. This work, unlike commercial software, permits using custom signal source reference spectra from endogenous molecules, not from fluorescent tags and stains. Finally, Chapter VI explores the use of the OSCC images to train a neural network image classifier to achieve the overall goal of classifying the NMMMIs into three categories-healthy, inflammatory, and neoplastic. This work determined that even with a small dataset (< 215 images), the features present in NMMMIs in combination with tiling, transfer learning can train an image classifier to classify healthy, inflammatory, and neoplastic OSCC regions with 70% accuracy.My research successfully shows the potential of using NMMM in tandem with computational methods to augment current diagnostic protocols used by the health care system with the potential to improve patient outcomes as well as decrease pathology departmental costs. These results should facilitate the continued study and development of NMMM so that in the future, NMMM can be used for clinical applications.
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Title: Development and applications of a microfluidic vascular mimic
Creator: Vogel, Paul A.
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Vascular wall biology and interactions with bloodstream components are areas of continued interest due to the high prevalence of cardiovascular disease and stroke. Cell types comprising the vascular wall, as well as those found in the bloodstream, are frequently isolated and cultured in vitro to determine their biological roles, including their involvement in disease progression. Recently, microfluidic systems have emerged as desirable platforms for mimicking biological systems, offering...
Show moreVascular wall biology and interactions with bloodstream components are areas of continued interest due to the high prevalence of cardiovascular disease and stroke. Cell types comprising the vascular wall, as well as those found in the bloodstream, are frequently isolated and cultured in vitro to determine their biological roles, including their involvement in disease progression. Recently, microfluidic systems have emerged as desirable platforms for mimicking biological systems, offering precise control of cellular environments. Here, work is presented that advances microfluidic technology and improves the utility of microfluidic systems for cell-based assays and cell culture. The endothelium is an important component of the vascular wall due to its role as a protective barrier, as well as its involvement in several important vascular functions. In vivo, the endothelium plays a vital role in the regulation of vascular tone by releasing several factors that cause vessel dilation, of which nitric oxide (NO) is one of the most potent. The endothelial cells comprising the endothelium are influenced by many environmental factors, such as shear stress generated by flowing blood and the degree of confluence of the endothelial monolayer. Presented here is the development of novel microfluidic system capable of mimicking the vasculature by incorporating microfluidic channels that enable blood flow past cultured endothelial monolayers. Importantly, this microfluidic vascular mimic is successfully integrated with a transendothelial electrical resistance (TEER) measurement system that allows for the monitoring of endothelial monolayer confluence and barrier integrity. This microfluidic system is used to highlight the impact of monolayer confluence on cellular behavior by showing that, in response to flowing red blood cells (RBCs), confluent endothelial monolayers produce significantly more NO than less confluent monolayers.C-peptide, a potential drug for type 1 diabetes, is known to improve blood flow by a previously unknown mechanism. This dissertation hypothesizes that C-peptide is capable of indirectly inducing vessel dilation by stimulating the release of adenosine triphosphate (ATP) from flowing RBCs, which can diffuse to the endothelium to bind purinergic receptors on the endothelial cell surface, ultimately resulting in endothelial NO production. To investigate this hypothesis, the microfluidic vascular mimic is utilized to monitor cell-cell communication, revealing C-peptide is capable of stimulating endothelial NO production by a mechanism mediated by the RBC which requires P2Y purinoreceptor activation by ATP. Importantly, it is shown that in order to observe this NO production, C-peptide must be prepared with Zn2+. This proposed mechanism describes how Zn2+/C-peptide may improve blood flow indirectly as a result of activity within the bloodstream. However, since C-peptide has never been observed exogenous of the bloodstream in vivo, it is unclear whether all C-peptide bioactivity results similarly, or if C-peptide can also escape the bloodstream to directly stimulate cells in surrounding tissue. To assess the ability of C-peptide to penetrate the vascular wall, the microfluidic TEER system is employed to determine the ability of C-peptide to permeate the endothelium, revealing that C-peptide can permeate confluent endothelial monolayers in vitro, suggesting that C-peptide may escape the bloodstream in vivo. Collectively, this work shows that C-peptide may have a more comprehensive biological role than is currently assumed.
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Title: Utilizing fluidic platforms for the development of in vitro pharmacokinetic/pharmacodynamic models
Creator: Lockwood, Sarah Yvonne
Date: 2014
Collection: Electronic Theses & Dissertations
Description: The pharmaceutical industry is constantly developing new therapies and treatments, while the cost of the drug discovery process currently is estimated at two billion dollars, spent over a 12-15 year period. Adding to the cost associated with bringing a drug to market is the high attrition rate, with only 1 in every 10,000 compounds being approved by the Food and Drug Administration. Interest in reassessing existing research procedures for improved efficiency has recently been garnering...
Show moreThe pharmaceutical industry is constantly developing new therapies and treatments, while the cost of the drug discovery process currently is estimated at two billion dollars, spent over a 12-15 year period. Adding to the cost associated with bringing a drug to market is the high attrition rate, with only 1 in every 10,000 compounds being approved by the Food and Drug Administration. Interest in reassessing existing research procedures for improved efficiency has recently been garnering attention. Specifically, pharmacology studies, which utilize in vivo studies to obtain pharmacokinetic (PK) and pharmacodynamic (PD) information during the preclinical stage of the drug discovery process, have been a focal point. By complimenting the in vivo studies with in vitro models, an increase in efficiency is able to be realized by a reduction in consumed materials. In this dissertation, a diffusion-based dynamic in vitro (DDIV) PK model, fabricated on a microfluidic polydimethyl siloxane (PDMS) platform, was used to characterize the loading and elimination of a PK profile. However, challenges traditionally associated with the microfluidic devices, such as the fragility of the membrane due to device flexibility, reusability, and lack of automation make long-term PK studies incredibly difficult to perform, as well as reproduce. DDIV models fabricated on a rigid three-dimensional (3D) printed platform are rugged, reusable, and amenable to automation when integrated with a disposable cell culture insert. The 3D printed DDIV PK/PD device was characterized using fluorescein (332.31 g/mol) and validated using the antibiotic levofloxacin (361.37 g/mol). The loading profiles were achieved by flowing concentrated analyte through the device channels while adding buffer to the membrane insert to create a concentration gradient across the porous membrane, thereby allowing diffusion from the channel into the insert. Parameters related to the loading portion of a PK curve, such as loading time, flow rate, volume of the insert, and initial concentration in the channel were characterized. The profiles obtained during the characterization of the initial concentrations (7.5, 15, 30 μM) in the channel yielded a prediction model for both the concentration along the loading profile and the maximum concentration (Cmax) at a given loading time. Elimination of analyte from the membrane insert was proven to undergo first order rate kinetics. The elimination profile, and the resulting elimination rate constant are used to obtain the half-life. Ultimately, a prediction model for the half-life will be crucial to the characterization of the DDIV model, however preliminary gradient studies highlighted the importance of a correction factor pertaining to the amount of analyte absorbed by the device. Upon complete characterization, the reusable 3D printed DDIV PK/PD millifluidic device will allow researchers to mimic in vivo dosing regimens on an in vitro platform, resulting in a useful tool to be used in tandem with animal models.
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Title: Modeling the vascular system with microfluidic technology
Creator: Halpin, Stephen T.
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Here are presented several technologies advancing the state of microfluidic modeling of the vascular system through the integration of high throughput analysis equipment with microfluidic systems, allowing for the monitoring of cell-cell communication between the red blood cell (RBC) and endothelial cells. A review of the prior knowledge of the role of adenosine triphosphate (ATP) and nitric oxide (NO) release from the RBC is presented, along with the current understanding of the role these...
Show moreHere are presented several technologies advancing the state of microfluidic modeling of the vascular system through the integration of high throughput analysis equipment with microfluidic systems, allowing for the monitoring of cell-cell communication between the red blood cell (RBC) and endothelial cells. A review of the prior knowledge of the role of adenosine triphosphate (ATP) and nitric oxide (NO) release from the RBC is presented, along with the current understanding of the role these molecules play in vasodilation along with the endothelium. Here, this dissertation hypothesizes that hypoxic vasodilation of blood vessels requires ATP release from the RBC that stimulates NO synthesis in the endothelium, resulting in vasodilation. To investigate this hypothesis, a microfluidic model capable of quantitatively determining NO and culturing endothelial cells near a flowing RBC channel is fabricated. Techniques for fabrication of microfluidic devices are reviewed, along with detection and cell culture systems integrated in microfluidic systems. First presented is a novel system for analysis of NO released from RBCs under hypoxic conditions that uses a polydimethylsiloxane (PDMS) based microfluidic device incorporating a polycarbonate membrane. This membrane separates a flowing RBC sample from fluorogenic probe, DAF-FM, which when reacted with NO, has a larger fluorescence emission. This device was designed to be integrated into a fluorescence plate reader to obtain readout, which was a significant improvement over prior systems requiring custom detection platforms or microscopes for readout. Using this technology, a significant increase in NO release from RBCs under hypoxic conditions was observed.Next endothelial cell culture was incorporated onto the membrane of the device, and inhibition studies performed to investigate the origin of NO which reaches beyond an endothelial layer. In this model system, NO measured above an endothelial layer is representative of that available to the smooth muscle to induce vasodilation. Using this system, which integrated novel approaches of using the DAF-FM probe for NO in an extracellular manner and plate reader detection, it was shown that under hypoxic conditions, an increase in NO detected above the endothelium only is observed when RBC ATP release function and endothelial NO synthesis function is present, suggesting a pathway of hypoxic vasodilation requiring RBC ATP release and endothelial NO productionAlso, technology was developed enabling electrochemical oxygen detection within a flowing channel on a microfluidic device using epoxy-immobilized gold and silver wires as working and reference electrodes, respectively. This presents an easily reusable and low cost platform to potentially vary then detect the oxygen concentration in a flowing cell sample prior to other analysis. This would allow the investigation of hypoxic systems at oxygen concentrations other than completely oxygenated, or completely deoxygenated. Lastly, in efforts to explore other applications of the polycarbonate membrane based microfluidic devices, in particular their potential utility in drug screening and development. To this end, transport of several selected pharmaceutical molecules with the membrane based devices was investigated. In this process, it was discovered that some of the more hydrophilic molecules can absorb into PDMS based devices. To remove this limitation, techniques were developed which use the available fabrication equipment to produce PDMS devices to fabricate polystyrene devices. As polystyrene is frequently used in cell culture applications, this should allow for future work to more readily transfer cell culture protocols and techniques to microfluidic systems.
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Title: LEPTIN DELIVERS β-CELL-DERIVED C-PEPTIDE TO ERYTHROCYTES
Creator: Meints, Lisa
Date: 2021
Collection: Electronic Theses & Dissertations
Description: ABSTRACTLEPTIN DELIVERS β-CELL-DERIVED C-PEPTIDE TO ERYTHROCYTES By Lisa Meints Diabetes mellitus, a global health concern, is a disorder of chronic hyperglycemia associated with deficiency or resistance to insulin. Although manageable with treatments aimed at increasing the body’s utilization of insulin, often, complications associated with poor microvascular blood flow persist. Erythrocytes play an important role in regulation of microvascular blood flow by their ability to release ATP, a...
Show moreABSTRACTLEPTIN DELIVERS β-CELL-DERIVED C-PEPTIDE TO ERYTHROCYTES By Lisa Meints Diabetes mellitus, a global health concern, is a disorder of chronic hyperglycemia associated with deficiency or resistance to insulin. Although manageable with treatments aimed at increasing the body’s utilization of insulin, often, complications associated with poor microvascular blood flow persist. Erythrocytes play an important role in regulation of microvascular blood flow by their ability to release ATP, a known stimulus of NO. Although insulin produced by the pancreatic β-cells acts on GLUT4, erythrocytes and most other bloodstream cells possess GLUT1, suggesting that a different molecular secretion may stimulate GLUT1 trafficking to the cell membrane. Fewer microvascular complications are observed for diabetic patients who possess residual pancreatic β-cell activity, suggesting that other pancreatic secretions are important. C-peptide, the 31 amino acid peptide that connects the alpha and beta chains of insulin during its synthesis, is released into the bloodstream in equimolar quantities to insulin after its cleavage from proinsulin. The Spence group, and others, have shown that C-peptide increases erythrocyte-derived ATP release and downstream NO production. However, unlike all other groups, the Spence group has only observed cellular activity of C-peptide in the presence a carrier protein and transition metal. In vivo, the candidate metal is likely Zn2+, due to its high concentrations in the pancreatic β-cells. In 2015, it was verified by ITC that HSA can carry C-peptide, with a Ka of 1.75  0.64 x 10-5 M-1 and binding stoichiometry of two C-peptide to a single HSA. Due to the long half-life of HSA (~12-21 days) it is subject to the slow process of non-enzymatic glycation, impacting its ability to act as a carrier molecule, which is particularly problematic in conditions of persistent hyperglycemia. For this reason, Spence group hypothesized that other molecules be able to carry C-peptide and Zn2+ to the erythrocyte. Leptin, a recently discovered adipocyte-derived hormone with a well-known role in metabolism, and a half-life of ~25 minutes, has been implicated in a variety of physiological processes, including glucose regulation, nitric oxide release and blood flow. It was confirmed that leptin, like albumin, can carry C-peptide and Zn2+, increase erythrocyte-derived ATP release and stimulate GLUT1 trafficking. This dissertation investigates of leptin’s binding interactions with erythrocytes and C-peptide and how they are impacted by Zn2+, albumin, and hyperglycemia. The results reported herein demonstrate that leptin has a saturable and specific binding site on the erythrocyte with Kd = (1.79 ± 0.46) x 10-7 M and a binding affinity in the presence of 20 nM C-peptide with Kd = (2.05 ± 0.20) x 10-7 M. Specific binding between C-peptide and leptin has been confirmed by SPR, with a Kd of 2.40 x 10-6 M. The data presented herein provide important information toward understanding the mechanism by which C-peptide and leptin are involved in erythrocyte energetics and will aid in creation of more targeted therapeutics to address the microvascular complications of diabetes.
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Title: Investigating blood flow and antibiotic dosing using traditional microfluidics and novel 3D printed devices
Creator: Meisel, Jayda Erkal
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Over the last 25 years, it has been established that the red blood cell (RBC) is a major determinant in blood flow, which it can modulate through release of adenosine triphosphate (ATP). Although RBCs store intracellular ATP in mM concentrations, measurements indicate that the cells release nM concentrations when stimulated by deformation, hypoxia (lowered oxygen tension), or incubation with pharmacological stimuli such as hydroxyurea (HU), which is the only approved drug for treatment of...
Show moreOver the last 25 years, it has been established that the red blood cell (RBC) is a major determinant in blood flow, which it can modulate through release of adenosine triphosphate (ATP). Although RBCs store intracellular ATP in mM concentrations, measurements indicate that the cells release nM concentrations when stimulated by deformation, hypoxia (lowered oxygen tension), or incubation with pharmacological stimuli such as hydroxyurea (HU), which is the only approved drug for treatment of sickle cell disease. Upon release, RBC-derived ATP can induce vessel dilation via activation of endothelial cell nitric oxide synthase (eNOS) to produce nitric oxide (NO). To probe the fate of increased ATP release from human RBCs incubated with the drug hydroxyurea, a traditional soft polymer platform was utilized to facilitate measurement of cell-to-cell communication between RBCs and a cultured endothelium. This device contained an array of micron-scale channels through which RBC samples were pumped. The sample flow was separated from a detection well by a porous polycarbonate membrane. Stimulated ATP released from the RBCs diffused across the membrane to the detection wells and was measured using the luciferin-luciferase chemiluminescence assay, integrated with a plate reader for detection. RBCs incubated with 100 uM of HU released on average 2.06 ± 0.37 times more ATP relative to the control sample. Through the use of various inhibitors, this increase in ATP release was subsequently demonstrated to depend on RBC deformability, RBC NOS activity, and the cystic fibrosis transmembrane conductance regulator protein (CFTR). The fate of the measured RBC-derived ATP was also investigated by probing ATP signalling to an adjacent cultured endothelium.ATP release from RBCs increases in response to hypoxia, or lowered oxygen tension; however, the dependence of RBC ATP release on oxygen tension has not been investigated. To enable measurement of RBC ATP release and oxygen tension in a flowing stream of RBCs, a 3D printed device was designed to accomodate commercial transwell inserts for ATP measurements, as well as threaded Clark-type electrodes for amperometric oxygen measurements. The device consisted of a channel 2 mm wide and 0.5 mm in height with two ports for analyte detection and one threaded port for an electrode. The Clark-type electrode was fabricated from gold and silver wires secured into a finger tight fitting. Oxygen standards and RBC samples were prepared using air and argon purged buffers. Using the 3D printed device, RBC ATP release and oxygen tension were measured simultaneously from prepared RBC samples. Relative to controls, RBC ATP release increased significantly in response to systematically lowered oxygen tension with a maximum increase of 2.38 ± 0.43 fold more ATP when exposed to 5.35 ± 0.12 ppm oxygen. ATP release saturated, i.e., was not significantly different, at lower oxygen tensions. This increase in ATP release was inhibited by incubating RBCs with the cell stiffening agent, diamide. The dependence of hypoxic RBC ATP release on the conformation of heme in hemoglobin (Hb) is demonstrated by converting measured oxygen tensions to Hb saturation.The 3D printed platforms presented herein were also utilized as in vitro tools to model pharmacokinetic dosing profiles, specifically with applications for studying antibiotic resistance. The World Health Organization, Centers for Disease Control, and the White House have issued reports that outline strategies to combat antibiotic resistance. The Spence lab has developed a 3D printed device to mimic in vivo drug dosing profiles on an in vitro platform for applications in drug discovery. This 3D printed diffusion-based dynamic dosing device mimics the dosing capabilities of the hollow fiber chamber reactor (HFCR). The in vitro 3D printed device contains 6 ports to house commercial polyester transwell membrane inserts (0.4 micron) and in house fabricated 0.2 micron pore size inserts (polyester), which can be loaded with a sample of Escherichia coli. Chemically competent, kanamycin resistant E. coli were dosed with the DNA gyrase and topoisomerase IV inhibitor levofloxacin, which reached a maximum concentration (Cmax) of 21.0 ± 5.7 uM (0.4 micron pore size) and 68.0 ± 7.1 uM (0.2 micron pore size) of levofloxacin in approximately 1 hour. After dosing, the viability of the bacteria samples was measured using standard plating methods.
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Title: A rapid assay to detect antibiotic resistance with novel 3D printed pharmacokinetic/pharmacodynamic technologies
Creator: Heller, Andrew A.
Date: 2019
Collection: Electronic Theses & Dissertations
Description: "The over-prescription and misuse of antibiotics has led some bacterial strains to become resistant to one, multiple, or all currently available antibiotics. In order to treat an antibiotic resistant bacterial infection, a novel antibiotic or therapeutic is required. Up until the last few years, there had been a trend of fewer new antibiotics due to pharmaceutical companies not pursuing antibiotic development. The continuous threat of antibiotic resistance and the lack of antibiotic research...
Show more"The over-prescription and misuse of antibiotics has led some bacterial strains to become resistant to one, multiple, or all currently available antibiotics. In order to treat an antibiotic resistant bacterial infection, a novel antibiotic or therapeutic is required. Up until the last few years, there had been a trend of fewer new antibiotics due to pharmaceutical companies not pursuing antibiotic development. The continuous threat of antibiotic resistance and the lack of antibiotic research led to the National Action Plan, which called for novel, rapid diagnostic tools and new therapeutics to combat antibiotic resistance. The main reason for the lack of interest in antibiotic development is that drug development now cost a pharmaceutical greater than $2.5 billion and can take over 10 years. In addition, only 10.4% of drugs that enter clinical trials eventually are approved. One of the main causes of this low success rate is that the drugs do not have the same pharmacokinetics (PK) or pharmacodynamics (PD) as the drugs did in in vitro and in vivo animal models. These differences in PK/PD can lead to safety and efficacy concerns in humans. In this dissertation, this issue is combated with new technologies for antibiotic resistance identification. A rapid, static susceptibility assay was created in order monitor the growth of a bacterial culture by measuring the extracellular ATP/OD600, which in a healthy culture should increase to a maximum during early logarithmic growth phase and then decrease. Adding an antibiotic to a growing culture after this ATP/OD600 maximum led to an increase in the ATP/OD600, while a healthy culture decreased leading a statistical difference (alpha = 0.05) in 20--60 minutes after adding the antibiotic. This increase in the ATP/OD600 was due to the antibiotic's ability to effectively kill the bacteria by lysing leading to the OD600 remaining stable and extracellular ATP levels to increase. This trend was not seen when an antibiotic that the bacteria were resistant to was added. This procedure could also determine which antibiotic is killing the most bacteria in a mixed bacterial culture. The above procedure was adapted to be dynamic in order to expose the bacteria to a PK curve similar to that seen in a human so more clinically-relevant PD data could be measured. This was achieved by creating a fluidic, two compartment model that was 3D printed, which utilized porous membrane inserts that were created by novel 3D printed procedures to incorporate the membranes into the 3D printing structure. The device was characterized using fluorescein (332.31 g/mol) due to having similar properties to the antibiotic, levofloxacin (361.37 g/mol). The devices were impervious to leaking and were successful in replicating PK curves for an oral, intermittent intravenous (IV), and continuous IV administration. Replacing the fluorescein solution with a levofloxacin solution in growth media, bacteria were able to be exposed to an oral levofloxacin PK curve (C max = 12.4 +/- 3.0 microM; tmax = 1 hour; half-life = 5.2 +/- 0.5 hours). A kanamycin-resistant strain of Escherichia coli was determined to have a statistical difference in the ATP/OD600 when exposed to a levofloxacin concentration of 3.5 +/- 1.3 microM in the secondary compartment while a chloramphenicol-resistant strain of Bacillus subtilis showed a statistical difference at a concentration of 4.8 +/- 1.8 microM."--Pages ii-iii.
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Title: 3D-printed in vitro analytical devices for diabetes therapeutics and blood banking studies
Creator: Chen, Chengpeng
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Erythrocytes (ERYs) play an important role in regulating blood flow via a pathway involving ERY-derived adenosine triphosphate (ATP) and endothelium-derived nitric oxide (NO). Impaired ATP release from ERYs of diabetic patients is potentially responsible for diabetic complications, thereby therapies involving stimulation of ERY-derived ATP release might limit such complications. This work explores 3D-printing to fabricate a novel microfluidic device to mimic the physiology of ERYs. 3D...
Show moreErythrocytes (ERYs) play an important role in regulating blood flow via a pathway involving ERY-derived adenosine triphosphate (ATP) and endothelium-derived nitric oxide (NO). Impaired ATP release from ERYs of diabetic patients is potentially responsible for diabetic complications, thereby therapies involving stimulation of ERY-derived ATP release might limit such complications. This work explores 3D-printing to fabricate a novel microfluidic device to mimic the physiology of ERYs. 3D-printing enables fabrication of the device following a standard 96-well plate geometry for efficient and high throughput readout with a plate reader. This 3D-printed rugged device was reusable after simple rinsing, which enables the detection of a batch of samples on the same device during a long-term experiment. This 3D-printed fluidic device facilitated the investigation of the efficacy of C-peptide on stimulating ERY-derived ATP. Without albumin, C-peptide and Zn2+ cannot increase ERY-derived ATP, suggesting the indispensable role of albumin in the process. The glutamic acid at the 27 position of C-peptide participated in the binding to albumin. Collectively, the ensemble of albumin, C-peptide, and Zn2+ enhances ERY-derived ATP, which may reduce diabetes complications.To make the results more physiologically conclusive, an Organs-on-a-Chip platform that combined pancreatic β-cells, ERYs and endothelial cells as a blood barrier mimic was developed. The secretion profiles of the β-cells on the device simulate the physiological secreting process well. Subsequent cell-cell communication investigations showed that β-cell secretions do not affect the endothelial cells but increase ATP release from ERYs, which in turn, exerts a downstream effect on endothelial cells by stimulating NO production. Currently approved hyperglycemic ERY storage solutions impair ATP release from ERYs. This work continues to investigate the reversibility of ATP release from stored ERYs and shows that 15 days of storage is a turning point, after which the ATP release is no longer reversible. This result is consistent with the clinical reports that blood stored longer than 2 weeks is more likely to result in transfusion complications. The mechanism by which hyperglycemia impairs ATP release was also explored by studying ERY deformability using a 3D-printed demand-based cell filter, finding that hyperglycemia permanently alters the deformability of ERYs after 5 days of storage. A 3D-printed intravenous device was developed to mimic a transfusion process in vitro. Addition of β-cells on the platform showed that hyperglycemia-stored ERYs failed to respond to the endocrine cells.Summarily, 3D-printing yields reusable, robust and reproducible microfluidic devices, and demand-based devices. The ensemble of albumin, C-peptide and Zn2+ can be a potential therapy for diabetes complications, and the current ERY storage protocol adversely alter the physiology of stored ERYs. A normoglycemic alternative may avoid this problem.
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Title: Delivery of zinc to red blood cells and the downstream effects in multiple sclerosis
Creator: Letourneau, Suzanne
Date: 2013
Collection: Electronic Theses & Dissertations
Description: The research presented here shows that zinc is delivered to the red blood cell (RBC) and investigates the effects of this in multiple sclerosis. A review of MS, as well as prior research investigating RBC-derived adenosine triphosphate (ATP) and the stimulation of nitric oxide (NO) are presented here. This dissertation hypothesizes that the increase in RBC-derived ATP seen in MS patients may be the result of increased zinc levels and leads to increased levels of NO, a molecule known to...
Show moreThe research presented here shows that zinc is delivered to the red blood cell (RBC) and investigates the effects of this in multiple sclerosis. A review of MS, as well as prior research investigating RBC-derived adenosine triphosphate (ATP) and the stimulation of nitric oxide (NO) are presented here. This dissertation hypothesizes that the increase in RBC-derived ATP seen in MS patients may be the result of increased zinc levels and leads to increased levels of NO, a molecule known to increase the permeability of the blood brain barrier (BBB), which is a precursor to lesion formation. C-peptide, a biologically active byproduct in the formation of insulin, also increases the release of ATP from RBCs, but only when bound to Zn2+. Evidence is presented here showing that C-peptide can deliver Zn2+ to RBCs. When bound to C-peptide, 2.54 ± 0.23 pmol of Zn2+ were delivered to RBCs, compared to 0.09 ± 0.21 pmol when Zn2+ alone was introduced RBCs. The significance of this in diabetes mellitus will be discussed.Because of these findings, C-peptide was used to deliver Zn2+ to the RBCs of MS patients. Significantly more Zn2+ is delivered to the RBCs of MS patients, at a value of 3.61 ± 0.22 pmol, than to those of healthy controls, at a value of 2.26 ± 0.24 pmol. Additionally, the basal level of Zn2+ in the RBCs of MS patients and those of healthy controls was measured. The RBCs of MS patients were found to have 41.8 ± 1.7 μg of Zn2+/g Hb where the RBCs of healthy controls only had 32.9 ± 2.2 μg Zn2+/g Hb.To further the research into the increase ATP release from the RBCs of MS patients, this was measured in a flow system that mimics the shear stress experienced by the RBCs in vivo. It was found that the RBCs of MS patients release significantly more ATP, at a value of 344.7 ± 46.8 nM, than those of healthy controls, at a value of 132.1 ± 14.1 nM. Glybenclamide, an inhibitor of ATP release from the RBC, decreased this value to 65.3 ± 11.6 nM in the RBCs of MS patients, showing that this is indeed the result of increase release of ATP, as opposed to cell lysis. The glucose uptake into these cells that may be leading to the increased ATP release is also discussed.Finally, reports have shown that estrogens have a protective effect in MS. Here, the effect of estradiol and estriol on the RBC-derived ATP was measured. Estradiol and estriol reduced the ATP release from healthy RBCs to 74 ± 4% and 70 ± 11% that of healthy controls, respectively. Through the use of a microfluidic device, the effects of estradiol on RBC-derived ATP and the subsequent endothelial cell NO production were investigated. When these RBCs were incubated estradiol, the NO production from the endothelial cells was attenuated to a value that was only 59 ± 7% of RBCs in the absence of estradiol. The ability of estrogens to decrease the ATP release from RBCs and subsequently the NO production of endothelial cells has major implications in the treatment of MS.
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Title: Effects of hyperglycemia on red blood cell storage lesion in transfusion medicine
Creator: Wang, Yimeng
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Red blood cell (RBC) transfusions are an important component of critical healthcare. RBCs can be donated, processed, and stored in a blood bank for future transfusion. A review of RBC storage development, as well as RBC storage lesion, is presented here. Whole blood is collected into an anticoagulant-nutrient solution such as citrate phosphate dextrose (CPD). The RBCs separated from the plasma and platelets, are then added into an additive solution, such as AS-1, which supports the nutrient...
Show moreRed blood cell (RBC) transfusions are an important component of critical healthcare. RBCs can be donated, processed, and stored in a blood bank for future transfusion. A review of RBC storage development, as well as RBC storage lesion, is presented here. Whole blood is collected into an anticoagulant-nutrient solution such as citrate phosphate dextrose (CPD). The RBCs separated from the plasma and platelets, are then added into an additive solution, such as AS-1, which supports the nutrient needs of the RBCs in storage. The glucose concentrations in CPD and AS-1 are 129 mM and 111 mM, respectively. Thus, the glucose level in this system (estimated > 40 mM) is much higher than the healthy glucose level in vivo (4-6 mM). This dissertation hypothesizes that the hyperglycemic conditions in the current storage system result in some changes in RBCs and thereby have adverse effects on vascular function.In addition to the primary function of oxygen delivery, RBCs can function as a regulator of vascular tone. It is known that adenosine triphosphate (ATP) is released from intact RBCs in response to several stimuli and further stimulates nitric oxide (NO) production in the endothelium lining the blood vessels. This NO functions to relax the smooth muscle cells surrounding circulatory vessels, thereby increasing blood flow and oxygen delivery to the tissues. In order to investigate the effects of hyperglycemia, RBCs were processed and stored in hyperglycemic and normoglycemic conditions. The vascular function and other properties of these cells were then studied.In an in vitro microflow system, the RBCs stored in hyperglycemic conditions resulted in significantly less RBC-derived ATP for 4 weeks than the RBCs in normoglycemic conditions. During the same storage duration, microfluidic technologies enabled measurements of endothelium-derived NO that was stimulated by the ATP release from the stored RBCs. In comparison to normoglycemic solutions, the NO release decreased by more than 25% in the presence of the RBCs stored in the hyperglycemic conditions. Control experiments using inhibitors of ATP release from the RBCs, or ATP binding to the endothelium, strongly suggest that the decreased NO release by the endothelium is directly related to the ability of the stored RBCs to release ATP. Furthermore, the mechanisms behind the effect of hyperglycemia on the ability of RBCs to release ATP were investigated and discussed. It was found that an osmotic imbalance was formed in RBCs in hyperglycemic conditions, which thereby reversibly impaired the ATP release from RBCs. In addition, longer hyperglycemic storage resulted in sorbitol accumulation within RBCs and cell membrane damage in terms of lipid peroxidation, which irreversibly impaired ability of the RBCs to release ATP. Therefore, the transfusion of those stored RBCs would result in inappropriate vasodilation, less blood flow, and insufficient oxygen delivery, which are often associated with post-transfusion complications. If the RBCs could be stored and maintained in normoglycemic conditions, it may be possible to reduce these complications to some extent and hopefully improve RBC transfusion efficacy.
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Title: 3D printing in the biosciences : applications for diabetic complications
Creator: Castiaux, Andre, II
Date: 2018
Collection: Electronic Theses & Dissertations
Description: Work presented in this dissertation demonstrates the utility of 3D printers in scientific research with specific applications to diabetic research. An overview of 3D printing techniques is discussed with special emphasis on PolyJet 3D printing. This printing technique is utilized to explore Cell-to-Cell communication in relationship to diabetes, binding of protein- ligand complexes under diabetic conditions, and teaching and research applications tangentially related to diabetes. PolyJet 3D...
Show moreWork presented in this dissertation demonstrates the utility of 3D printers in scientific research with specific applications to diabetic research. An overview of 3D printing techniques is discussed with special emphasis on PolyJet 3D printing. This printing technique is utilized to explore Cell-to-Cell communication in relationship to diabetes, binding of protein- ligand complexes under diabetic conditions, and teaching and research applications tangentially related to diabetes. PolyJet 3D printing technology is a recent technique to the 3D printing field. It works by utilizing a liquid photocurable resin that can be sprayed onto a substrate layer by layer and cured into a final desirable three-dimensional object. With this printer multiple materials and colors can be incorporated into a single model. By incorporating multiple materials into a device, researchers can use the rubber like properties to imbed and seal various non-printable components into a rigid plastic device. One such non-printable component of interest is membranes for size exclusion of molecules up through cells.Diabetes is characterized by the bodies inability to produce insulin (Type 1) or the bodies inability to effectively utilize the insulin produced (Type 2) leading to elevated glucose levels within the body. With this definition, there is an implication made that insulin is the only important molecule in relation to this disease. However, C-peptide is co-secreted with insulin and is suspected to play an important role in the health of the microvasculature. The ability to monitor the communication between cells types would lend to a better understanding of the role of C-peptide under diabetic conditions. Specifically, looking at the communication of pancreatic β-cells, where C-peptide and insulin are synthesized, with red blood cells and endothelial cells, would allow researchers to understand the potential beneficial effects of C-peptide. Proposed within is a new ex vivo 3D printed platform that can selectively capture cells secretions while monitoring the effects on various cell types under both heathy and diseased states.In addition to understanding the role of molecules no longer present or effective under diabetic conditions, it is important to understand the role of molecules synthesized under diabetic conditions. Specifically, the increased glucose concentration leads to the non-enzymatic addition of glucose (glycation) to long lived molecules in the body or Advanced Glycation End Products (AGEs) and can alter the behavior of molecules in vivo. A 3D printed device was developed to study the effect of glycation on albumins ability to bind zinc. It was determined that under healthy condition human serum albumin (HSA) will bind a maximum of 2 molecules of zinc per molecule of HSA whereas under diabetic or glycated conditions there is 1 molecule of zinc per 2 molecules of HSA.In addition to research related to diabetic conditions, applications for 3D printing exist in labware and teaching aids. Utilizing 3D printers, cheap disposable pumps can be employed in hazardous conditions such as handling of radioactive materials. Data presented here demonstrates the development of a multichannel 3D printed peristaltic pump. The majority of the components are 3D printed and the pump is powered by a microcontroller and a continuous rotation servo motor. All in this pump can be assembled for under $150 and can achieve flow rates from 0.4 to 1.4 mL/ min.
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Title: NOVEL ANALYTICAL TOOLS FOR STUDYING A POTENTIAL TYPE-1 DIABETES THERAPY
Creator: Pinger, Cody
Date: 2018
Collection: Electronic Theses & Dissertations
Description: ABSTRACTRed blood cells (RBCs) are known to play an important role in regulating microvascular circulation by releasing the signaling molecule adenosine triphosphate (ATP). Extracellular ATP in the bloodstream reacts with endothelial cells lining the blood vessel via a P2Y purinergic receptor, causing the endothelial cells to create and release nitric oxide, a known vasodilator. Interestingly, RBCs from Type-1 diabetic (T1D) subjects release significantly less ATP than RBCs from non-diabetic...
Show moreABSTRACTRed blood cells (RBCs) are known to play an important role in regulating microvascular circulation by releasing the signaling molecule adenosine triphosphate (ATP). Extracellular ATP in the bloodstream reacts with endothelial cells lining the blood vessel via a P2Y purinergic receptor, causing the endothelial cells to create and release nitric oxide, a known vasodilator. Interestingly, RBCs from Type-1 diabetic (T1D) subjects release significantly less ATP than RBCs from non-diabetic subjects. This effect is believed to impair microvascular blood flow and be a potential cause of the prevalent downstream microvascular complications seen in T1D. In T1D, the immune system destroys the pancreatic b-cells, therefore creating a deficiency of the hormones normally secreted by these cells. Since the early 1920’s, T1D patients have been administering the pancreatic b-cell secretion insulin to manage their blood sugar and stay alive. However, insulin treatment alone with diet and exercise does not prevent the microvascular complications seen in the disease. C-peptide is a 31-amino acid peptide that is co-secreted with insulin from the pancreatic b-cells along with Zn2+. Researchers have found that treating T1D RBCs with a combination of C-peptide and Zn2+ in the presence of albumin, a prevalent bloodstream protein, can significantly increase the amount of ATP released from the cells. However, RBCs are unaffected when treated with C-peptide and Zn2+ in the absence of albumin. Researchers have shown that albumin from diabetic patients has different properties than albumin from non-diabetic subjects. Therefore, the hypothesis of the work in this thesis is that diabetic albumin interferes with the interaction of RBCs with C-peptide and Zn2+.The work in this dissertation is focused on the role of albumin and its interactions with C-peptide, Zn2+, and RBCs. 3D-printing technology was used in order to create novel devices to study interactions between albumin and C-peptide and Zn2+ under diabetic conditions. A 3D-printed equilibrium dialysis device was used to measure the binding affinity between human serum albumin (HSA) and Zn2+ (Kd= 562 ± 93 nanomolar). It was found that the affinity of this interaction was decreased by glycation of albumin, but not by immediate addition of glucose. The device was also used to measure that C-peptide does not alter the affinity of Zn2+to albumin. The dialysis device is compatible with plate-reader technology and enables automated and direct measurements of certain analyte ligands. Further, a 3D-printed ultrafiltration device was created in order to measure the binding affinity between HSA and C-peptide (Kd= 2.4 ± 0.3 micromolar). The affinity of this interaction was not altered by Zn2+, glucose, or glycation of albumin. Novel 3D-printing techniques were developed in order to create these devices, such as the Print-Pause-Print method for integrating membranes directly into 3D-printed devices, and a support-free Polyjet printing technique.Lastly, the interaction of RBCs with C-peptide and Zn2+ was measured in the presence of normal and glycated albumin. The ability of RBCs to release ATP under flow conditions was increased significantly in the presence of C-peptide/Zn2+ and normal albumin (p<0.05); whereas the RBCs treated with C-peptide/Zn2+ and glycated albumin did not release statistically more ATP than the control (n=4 blood draws). The ability of C-peptide to bind to RBCs in the presence of normal and glycated albumin was also measured. Approximately 1.8 ± 0.1 picomoles of C-peptide bound to RBCs in the presence of normal albumin, whereas 0.8 ± 0.1 picomoles bound in the presence of glycated albumin (n=3 blood draws, p<0.05). Therefore, the ability of C-peptide to bind and affect RBCs was affected by the presence of glycated albumin, such as seen in diabetes. The results of the studies reported in this thesis should be taken into consideration by those studying C-peptide as a replacement therapy in T1D.
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Title: Mechanistic and functional studies of zinc (II) activation of C-peptide and its effect on red blood cell metabolism
Creator: Medawala, Wathsala
Date: 2011
Collection: Electronic Theses & Dissertations
Description: ABSTRACT MECHANISTIC AND FUNCTIONAL STUDIES OF ZINC (II) ACTIVATION OF C-PEPTIDE AND ITS EFFECT ON RED BLOOD CELL METABOLISM By Wathsala Medawala C-peptide, a 31 amino acid peptide co-secreted with insulin from β-cells in the pancreas, has long been considered a by-product of insulin synthesis. Later it was discovered that C-peptide could ameliorate diabetic complications such as neuropathy, nephropathy, retinopathy and microvascular disease. However, the molecular mechanism behind the...
Show moreABSTRACT MECHANISTIC AND FUNCTIONAL STUDIES OF ZINC (II) ACTIVATION OF C-PEPTIDE AND ITS EFFECT ON RED BLOOD CELL METABOLISM By Wathsala Medawala C-peptide, a 31 amino acid peptide co-secreted with insulin from β-cells in the pancreas, has long been considered a by-product of insulin synthesis. Later it was discovered that C-peptide could ameliorate diabetic complications such as neuropathy, nephropathy, retinopathy and microvascular disease. However, the molecular mechanism behind the effects of C-peptide is not yet completely understood. The effect of C-peptide on the microcirculation is important, because problems in the circulation have been linked to other diabetic complications. C-peptide improves the microcirculation by increasing endothelial-derived nitric oxide (NO), red blood cell (RBC) deformability and Na+/K+-ATPase activity. C-peptide increases glucose utilization and ATP release from RBCs and endothelial cells. ATP is a stimulus for the production of endothelial-derived NO, which is a known vasodilator. Therefore, the C-peptide mediated increase in RBC-derived ATP release can lead to the improvement of blood flow.C-peptide alone did not increase RBC-derived ATP release. The presence of a metal ion such as Zn2+ was needed to elicit this response. However, the role of Zn2+ in C-peptide activity is not fully understood. A fluorescence based study was used to investigate the binding of Zn2+ to C-peptide. Results indicate that C-peptide binds one Zn2+ ion and has a binding constant of 1.02 x 107 M-1 with Zn2+ at pH 5.5, which is the pH inside a mature β-cell granule. At physiological pH of 7.4, C-peptide binds with two Zn2+ ions and has a binding constant of 7.99 x 106 M-1. This indicates that C-peptide may bind with Zn2+ inside the β-cell granule and release Zn2+ upon entering the blood stream. Circular dichroism studies suggest that a 1:1 Zn2+ to C-peptide ratio elicits a decrease in the randomness of the peptide chain, which is lost at higher Zn2+ to C-peptide ratios.Five single amino acid peptide mutants of C-peptide were used to study the effect of the acidic amino acid residues on Zn2+ binding. Substitution of glutamate residues at positions 27, 11 and 3 decreased the Zn2+ binding to C-peptide by ~50%, agreeing with the activity of these mutants in other bio-assays. Several studies have indicated the necessity of insulin for C-peptide activity. The Zn2+ that available with insulin hexamers may be responsible for activation of C-peptide. Using a RBC-derived ATP assay, it was shown that Zn2+ added in the form of Zn-insulin activates C-peptide, as observed by the increase in ATP release. The C-peptide interaction with RBCs was studied using an ELISA. Zn2+-activated C-peptide bound to RBCs in a dose dependent manner, 2 pmol of Zn2+-activated C-peptide binding to 1 mL of 7% red blood cell sample at saturation (~1500 molecules/ cell). The interaction did not change significantly even in the absence of Zn2+. However, Zn2+ uptake in RBCs was observed only in the presence of C-peptide, indicating a possible role for C-peptide as a Zn2+ carrier. One argument against C-peptide being a potential medication is that patients with type 2 diabetes develop the complications of the disease despite having circulating C-peptide. Red blood cells incubated in high glucose (>10 mM), showed lower interaction with Zn2+-activated C-peptide and lower ATP release, showing that patients with uncontrolled blood glucose levels might show resistance to the action of the peptide. Also, the exposure of Zn2+-activated C-peptide to serum albumin and mM levels of other cations and anions decreases its activity. Taken together, the work presented in this dissertation explains the role of Zn2+ in C-peptide action and how reproducible results may be obtained in C-peptide clinical trials.
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Title: Quantitative investigation of the benefits from storing red blood cells under normoglycemic conditions
Creator: Mu, Ruipeng
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Red blood cell (RBC) transfusion has become a highly organized and life-saving component of critical healthcare. Approximately 40,000 units of RBCs are transfused every day in the US, therefore the safety and efficiency of RBC transfusions are essential to patients' health. However, post-transfusion complications still exist, therefore remaining a threat to the patients' life. It is well-known that stored RBCs experience metabolic and physical changes during the storage period, collectively...
Show more"Red blood cell (RBC) transfusion has become a highly organized and life-saving component of critical healthcare. Approximately 40,000 units of RBCs are transfused every day in the US, therefore the safety and efficiency of RBC transfusions are essential to patients' health. However, post-transfusion complications still exist, therefore remaining a threat to the patients' life. It is well-known that stored RBCs experience metabolic and physical changes during the storage period, collectively known as the storage lesion, which may cause adverse effects after transfusion. The Spence group hypothesizes that the high concentration of glucose present in the blood storage solutions plays an important role in the development of the storage lesion. In order to improve the quality of stored RBCs, normoglycemic versions for the current FDA approved blood storage solutions have been proposed, where the glucose concentrations were modified to 5.5 mM. The benefits of normoglycemic storage of RBCs were quantitatively evaluated through a variety of experiments. First, flow-induced ATP released from stored RBCs was studied using a 3D-printed fluidic device. It is well-known that the primary function of the RBC is to deliver oxygen to tissues. Besides that, the RBC acts as a blood flow regulator by releasing adenosine triphosphate (ATP) into the blood stream. RBC-derived ATP release can stimulate nitric oxide (NO), a known vasodilator, production within endothelial cells, increasing blood flow. RBC derived-ATP release decreases approximately 50% when cells are stored in hyperglycemic environments, compared to normoglycemic storage. This result indicates that RBCs stored in normoglycemic conditions may have improved blood flow during and after transfusion. Next, the mechanism of impaired ATP release was explored by studying cell deformability. This work was done by applying a 3D-printed, multi-port membrane based device accompanied with flow cytometry for cell counting. It is shown that RBCs stored in hyperglycemia lost 15 - 20% of their deformability. One possible element causing cell deformability loss is oxidative damage triggered by the massive production of intracellular sorbitol from RBCs stored in standard storage solutions. In order to further discover the mechanisms of concentrated glucose damage to stored RBCs, cell membrane phosphatidylethanolamine (PE) glycation was analyzed by a high resolution/accurate mass spectrometry. The results showed that the glycated product of PE, Amadori-PE, to normal PE ratio on the RBC membrane decreases as a function of time during the storage period when cells are stored in physiological levels of glucose. In summary, the work here demonstrates that the excess glucose present in storage solutions might be the main contributor for the development of storage lesions. Alternatively, storing RBCs in normoglycemic conditions can reduce the severity of those deleterious effects, therefore having high potential benefits in the clinic."--Pages ii-iii.
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Title: Microfluidic and 3D printing technologies for the development of an in vitro thrombus mimic
Creator: Gross, Bethany
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Platelets from individuals with diabetes, cystic fibrosis, multiple sclerosis, hypertension, and sickle cell anemia are hyperactive or more likely to aggregate and form a blood clot or thrombus. Furthermore, each of these diseases exhibits abnormal red blood cell (RBC) adenosine triphosphate (ATP) release, an agonist of platelet activation. RBC-derived ATP release is also a proven factor in the regulation of vascular tone through a signal cascade that results in nitric oxide release from...
Show morePlatelets from individuals with diabetes, cystic fibrosis, multiple sclerosis, hypertension, and sickle cell anemia are hyperactive or more likely to aggregate and form a blood clot or thrombus. Furthermore, each of these diseases exhibits abnormal red blood cell (RBC) adenosine triphosphate (ATP) release, an agonist of platelet activation. RBC-derived ATP release is also a proven factor in the regulation of vascular tone through a signal cascade that results in nitric oxide release from endothelial cells and relaxation of smooth muscle cells. This relaxation leads to vessel dilation and a localized increase in blood flow. The presence of hyperactive platelets in conjunction with altered RBC-derived ATP release results in an impaired ability to dilate local resistance vessels, and ultimately puts individuals with these diseases at higher risk for deleterious thrombus formation. The work detailed in this dissertation outlines the development of microfluidic and 3D printed in vitro models of in vivo circulation, capable of inducing an injury to a localized region of the endothelium. Specifically, chemical and electrical lysis of endothelial cells will be demonstrated through the use of embedded or removable electrodes or with laser irradiation of a photochemical dye. The fabricated devices mimic flow seen in blood vessels, facilitate the study of platelet adhesion to sub-endothelial collagen, and allow for the study of thrombus formation in stored blood samples showing altered ATP release from RBCs. With a more rapid fabrication process, reusability of the final device, and possibility of standardization via open software sharing, 3D printing offers a more attractive method to develop and utilize an in vitro thrombus mimic compared to more widely employed soft lithographic techniques. Channels of 3D printed devices featured a stenosis region (0.8 mm height, 2 mm length, and 1 mm width) and wide regions (device 1: 5; device 2: 3.83 mm width). Surface modification of channels with either polydimethylsiloxane (PDMS) or polystyrene (PS) was necessary to promote endothelial cell adherence. Thicknesses of PDMS and PS channel coatings were determined using scanning electron microscopy. The PDMS coating varied in thickness from 3 μm to 100 μm. Multiple PS coatings were required to form a 100 μm thick coating. Cells remained viable on the devices for five days (98% viable), though cell coverage decreased after day four with static media delivery. Optimal lysis conditions (applied electrical potential and duration) were determined for the two different geometries of the 3D printed devices to ensure localized endothelial cell clearance. Selective cell lysis was achieved with efficiencies of 94% (device 1) and 96% (device 2). FDA approved blood storage solutions expose RBCs to hyperglycemic amounts of glucose. As seen with RBCs from individuals with diabetes, ATP release from stored RBCs is significantly decreased compared to control RBCs. The effect of this decreased ATP release on thrombus formation was evaluated when hyper and normoglycemic stored RBCs were reincorporated with platelet rich plasma by measuring percent transmittance through the device, where increased thrombus coverage corresponded to a decrease in transmittance. Within the same storage period, RBCs stored in hyper and normoglycemic conditions showed no significant difference in hemoglobin absorption indicative of cell adherence. However, a significant decrease in cell adherence between day 1 and week 3 hyper and normoglycemic samples was observed (normo: p = 0.005, n ≥ 3; hyper: p ˂ 0.003, n ≥ 3).
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