Browsing by Author "Olver, Christine, committee member"
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Item Open Access CD4+ T-cell derived IL-10 mitigates malarial anemia(Colorado State University. Libraries, 2013) Kiser, Patti, author; Avery, Anne, advisor; Callan, Robert, committee member; Olver, Christine, committee member; Quackenbush, Sandra, committee memberIndividuals living in malaria endemic areas develop effective anti-parasite immunity over several years of repeated exposure, but become resistant to severe disease after just one or two infections. This observation suggests that the acquired immune system plays a role in both processes, but may involve different mechanisms. Using the mouse model of malaria caused by non-lethal Plasmodium yoelii, we test the hypothesis that CD4+ T-cell derived IL-10 contributes to disease resistance by mitigating severe anemia. Here we show that IL-10 deficient mice develop significant anemia despite a very low parasite burden. Anemia in this model is mediated primarily by increased erythrocyte destruction and not from suppressed erythropoiesis. Wild type mice that have recovered from P. yoelii infection have an expanded population of IL-10 producing CD4 T cells, with the majority of these cells co-expressing IFN-γ and display a Th1 phenotype. In the absence of IL-10, there is an increase in IFN-γ+ T cells. We demonstrate that IL-10 competent CD4+ T cells protect athymic nude mice from anemia when compared to CD4 T cells taken from recovered IL-10 deficient mice. Utilizing an ex vivo system that tests the function of APCs in activating CD4+ T-cells, we also determined that APCs exposed to P. yoelii in vivo induced a greater population of CD4+ T-cells that express IL-10 compared to naïve APCs. We also demonstrate that IFN-γ is required, with the possible involvement of IL-10 and IL-12, for efficient IL-10 expression in CD4+ T-cells. Our findings suggest that one mechanism by which the acquired immune system contributes to resistance to severe anemia may be the development of CD4 T cells that co-express IL-10 and IFN-γ, thereby self-regulating IFN-γ levels, which then inhibits pro-inflammatory mediated destruction of naïve red blood cells.Item Open Access Coagulation abnormalities in Ehrlichia canis-infected dogs and detection and dynamics of anti-platelet antibodies in thrombocytopenic dogs(Colorado State University. Libraries, 2018) Shropshire, Sarah, author; Lappin, Michael, advisor; Dow, Steve, committee member; Olver, Christine, committee member; Webb, Craig, committee member; Ames, Marisa, committee memberVector-borne diseases affect millions of people and domestic animals worldwide resulting in significant morbidity and mortality rates. In dogs, vector-borne diseases such as Ehrlichia canis can cause a myriad of clinical signs (lethargy, weight loss, and epistaxis) and hematological abnormalities (hyperglobulinemia, thrombocytopenia, and anemia). It has been previously reported that E. canis results in systemic inflammation and vasculitis as well as the formation of immunoglobulin associated platelets or anti-platelet antibodies. It has been theorized that anti-platelet antibodies can deleteriously affect platelet function and if concurrent significant thrombocytopenia is present, signs of bleeding may manifest. However, anti-platelet antibodies and thrombocytopenia can occur in a variety of disease processes in the dog including other vector-borne diseases, neoplasia, and idiopathic primary immune syndromes. Thrombocytopenia is also one of the most common acquired hemostatic abnormality observed in dogs. Consequently, determining the underlying cause and mechanism for thrombocytopenia in dogs can represent a frequent diagnostic challenge. Additionally, inflammation is often present in dogs with thrombocytopenia due to various causes. Inflammation and immune system processes directly affect hemostasis which can lead to derangements in the coagulation system resulting in clinical signs of bleeding or thrombosis. The goals of the research described in this dissertation were to investigate the dynamic changes of anti-platelet antibodies in thrombocytopenic dogs and the changes that occur in the coagulation system during a vector-borne infection such as E. canis in dogs.Item Open Access Design and application of a droplet-digital PCR assay for detection of the STAT5BN642H mutation in feline T cell neoplasia(Colorado State University. Libraries, 2024) Bork, Sydney Bonnie, author; Avery, Anne, advisor; Olver, Christine, committee member; Webb, Craig, committee memberLymphoma is a commonly diagnosed hematopoietic neoplasm in cats. Small Cell T-cell Epitheliotropic Intestinal Lymphoma (SCL) is the most reported subtype of lymphoma in cats. Cats with SCL are presented with non-specific clinical signs such as chronic vomiting, diarrhea, and weight loss. Diagnostic work-up often includes collection of intestinal biopsies with histopathology for diagnosis. SCL is characterized by infiltration of neoplastic lymphocytes into the intestinal epithelium and lamina propria of the small intestines. Neoplastic cells are small to intermediate in size and of T-cell origin. Diagnosing SCL can be challenging for pathologists because cats also commonly develop a condition called inflammatory bowel disease (IBD), which has an almost identical clinical presentation and similar histopathologic patterns. However, in IBD, the lymphocytic infiltration is often heterogeneous (termed "lymphoplasmacytic enteritis"). When histopathology results are inconclusive, assessment of expression with immunohistochemistry markers can help further characterize the cell population. Additionally, advancements have been made with lymphocyte clonality testing by PARR (PCR for Antigen Receptor Rearrangement), a DNA-based assay that evaluates T-cell receptor (TCR) and Immunoglobulin (Ig) gene rearrangements. Cats diagnosed with SCL demonstrate a clonal TCR result, while cats with IBD demonstrate a polyclonal TCR result. Unfortunately, there are still cases where histopathology and PARR results are equivocal. Recent work in feline medicine has demonstrated that cats with SCL exhibit high expression of phosphorylated STAT5B with immunohistochemical staining on small intestinal biopsy samples compared to cats with IBD. Importantly, one group detected a STAT5BN642H mutation in cats diagnosed with SCL. In this study, 40% (17/42) of cats with intestinal lymphoma were classified as SCL by histopathology. A combination of Sanger sequencing and ARMS qPCR detected the STAT5BN642H mutation in 29.4% (5/17) of cats with SCL. This work correlates to a comparable disease entity in people, monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), which has reported the prevalence of the STAT5BN642H mutation to be 22-57%. Our group aimed to develop a droplet digital PCR (ddPCR) assay to detect wild-type and mutated STAT5B in cats. Our first aim was to design specific primers and locked-nucleic acid hydrolysis probes to detect and discriminate between wild-type and mutated STAT5B. The first step included analyzing data from control samples using wild-type DNA from cats without neoplasia and a positive control gene fragment block ("gBlock"). The second step included analyzing two cohorts of young cats (<6 years of age) without a diagnosis of lymphoid neoplasia to assess assay performance and determine if the mutated STAT5B could be considered a germ line polymorphism. The fractional abundance was calculated from the ddPCR data, estimating the percentage of mutated copies within a positive sample. The results of the first aim demonstrated that our ddPCR assay can distinguish between wild-type and mutated STAT5B with high sensitivity. Most young cats without a diagnosis of lymphoid neoplasia do not carry the STAT5BN642H mutation. Two cats with marked lymphoplasmacytic enteritis had detectable mutated STAT5BN642H. The second aim was to evaluate the prevalence of the STAT5BN642H mutation in cats with confirmed SCL. A sub-aim was to evaluate cats with CD4 T-cell leukemia to determine if this mutation could be found in other forms of T cell lymphoid neoplasia. The results from this aim demonstrate that cats with SCL frequently carry the STAT5B mutation (66.7%). We also discovered that this mutation is not exclusive to cats with SCL, as almost half of the cats with CD4 T-cell leukemia also carry this mutation (47.7%). These findings shed light on the prevalence of the STAT5BN642H mutation in cats with SCL and CD4 T-cell leukemia. This data suggests potential implications for ddPCR mutation detection to help further differentiate and diagnose cats with T cell neoplasia versus those with inflammatory conditions (such as SCL versus IBD), and investigate novel therapies (i.e., JAK/STAT inhibitors). Further research is warranted to investigate other JAK/STAT pathway mutations, particularly in cats where the STAT5BN642H mutation was not detected. Larger outcome studies should investigate the correlation of STAT5BN642H mutation status and the fractional abundance to evaluate disease risk, treatment response, and survival.Item Open Access Design and fabrication of bioactive coatings to catalytically generate nitric oxide on the surfaces of extracorporeal circuits(Colorado State University. Libraries, 2022) Wick, Tracey V., author; Reynolds, Melissa M., advisor; Kipper, Matthew, committee member; Olver, Christine, committee memberBlood-contacting medical devices suffer from biofouling caused by proteins, platelets and other cells adhering to the surface which often leads to severe complications and eventual device failure. In particular, extracorporeal membrane oxygenation (ECMO) is a life support treatment that is highly prone to coagulation issues due to a large blood-contacting surface area and turbulent blood flow. The ECMO circuits are constructed from catheters, tubing, and an oxygenator which all come into contact with blood and have several connections that alter the blood flow. The standard therapy to decrease thrombotic complications is to administer a systemic anticoagulant, usually unfractionated heparin. While this reduces clotting, harmful and potentially fatal hemorrhagic complications arise. Researchers have looked to nitric oxide (NO), a common biomolecule produced by the endothelium, as an alternative to locally inhibit clotting. Previous work has shown a reduction in thrombotic activity using NO-releasing substances, but these substances only last for a short period of time. An approach explored herein takes advantage of a catalytic mechanism to generate NO from endogenous NO-donors, S-nitrosothiols (RSNOs). RSNOs have been shown to catalytically generate NO through copper catalysis and in particular, with a copper-based metal-organic framework, H3[(Cu4Cl)3(BTTri)8-(H2O)12]·72H2O where H3BTTri = 1,3,5-tris(1H-1,2,3-triazole-5-yl)benzene] (CuBTTri). Importantly, CuBTTri has been shown to be stable under biological conditions and compatible with human cells; therefore, it is a promising candidate for biomedical applications. This report explores the addition of CuBTTri on the surfaces of ECMO. In Chapter 2, a CuBTTri-doped composite is coated onto the extracorporeal circuitry tubing. The fabrication method to apply CuBTTri to the tubing is reported, and the coating was shown to actively generate NO when exposed to a RSNO and no adverse effects were noted during hemocompatibility testing. In Chapter 3, CuBTTri is immobilized on the surface of an ECMO oxygenator using polydopamine. The morphology of the coating was evaluated and the CuBTTri on the surface of the oxygenator was catalytically active, generating NO when exposed to a RSNO. The incorporation of CuBTTri on the surfaces of these components could improve the hemocompatibility of the device, providing a safer and more effective life support system.Item Open Access Determining the cancer risks presented by space radiation: genomic mapping in outbred mice reveals overlap in genetic susceptibility for HZE ion and γ-ray induced tumors(Colorado State University. Libraries, 2016) Edmondson, Elijah F., author; VandeWoude, Sue, advisor; Weil, Michael, advisor; Thamm, Douglas, committee member; Olver, Christine, committee member; Kamstock, Debra, committee memberTo view the abstract, please see the full text of the document.Item Embargo Evidence for acute inflammation in the pathogenesis of different mouse models of post-traumatic osteoarthritis(Colorado State University. Libraries, 2022) Timkovich, Ariel E., author; Santangelo, Kelly, advisor; Olver, Christine, committee member; Moreno, Julie, committee member; Goodrich, Laurie, committee memberPost-traumatic osteoarthritis (PTOA) is a debilitating, degenerative condition affecting more than 5.6 million people in the United States. Following injury to the knee, a person is 4.2 times more likely to develop PTOA in that joint, compared to an uninjured joint. PTOA is characterized by clinical symptoms such as pain, swelling, and decreased range of motion of the knee or affected joint. As a leading cause of pain and disability, PTOA is a major contributor to a decreased quality of life. Notably, loss of mobility associated with PTOA can increase the risk of a multitude of other ailments including heart disease and diabetes. PTOA due to traumatic joint injury can result in damage to intra-articular structures, such as menisci, ligaments, cartilage, and subchondral bone. Despite current treatment options, many of these joint injuries progress to end-stage PTOA within 10 to 20 years. Rupture of the most commonly injured knee ligament, the anterior cruciate ligament (ACL), typically occurs in athletes and people who are physically active, with the majority of ACL tears occurring in patients who are less than 30 years old. The detrimental pathways driving PTOA remain loosely defined and little progress has been made in predicting, preventing, and treating the disease. The goal of this work was to further our understanding of PTOA mouse models through both development and characterization of models, as well as through the testing of therapeutics in pre-established PTOA mouse models. In the first part of this work (Chapter 2), we created and characterized a novel full and partial ACL rupture model. This work is the first published partial ACL rupture model to date and provides a novel platform to use as a preclinical model for testing of potential therapeutics and to further our understanding of PTOA following ACL rupture. Chapter 3 evaluates the therapeutic potential of a toll-like receptor 4 (TLR4) antagonist on a full depth PTOA model. We found that short term treatment with a systemic TLR4 antagonist significantly improved relevant gait parameters, and improved cartilage structural metrics and modified Mankin PTOA scores, implying improvement of clinical signs. Chapter 4 addresses the therapeutic potential of intra-articular bone marrow aspirate concentrate (BMAC), for treatment of PTOA in a well characterized meniscus degeneration model, compared to red blood cell (RBC) depleted BMAC or no injection controls. These results suggest that RBC-depleted BMAC improved treatment of PTOA in an animal model and may have translational therapeutic potential for humans suffering from similar disease processes. Finally, Chapter 5 sought to: (1) Identify transcriptional sex differences in knee joint tissue from naïve animals; and (2) Identify compensatory gene expression changes related to sex that are specific to animals receiving DMM surgery as compared to a naïve animal when taking into account surgery changes (i.e., sham surgery). The work described in this dissertation has improved our understanding of the mouse knee joint during PTOA progression and potential therapeutics and targets for clinical treatment of PTOA.Item Open Access Feline chronic kidney disease: novel approaches to etiology, specific therapy and supportive care(Colorado State University. Libraries, 2012) Quimby, Jessica M., author; Dow, Steve, advisor; Lappin, Michael, advisor; Lunn, Katharine, committee member; Olver, Christine, committee memberChronic kidney disease is one of the leading causes of morbidity and mortality in geriatric cats, affecting conservatively 30% of the population; an estimated 24 million cats nationwide in the United States. Despite the common nature of the disease, its etiology is yet unknown, and there is no definitive cure short of renal transplantation. The goals of the research described in this dissertation were to explore possible etiologies of chronic kidney disease and to develop novel treatment strategies to help cats afflicted with this disease. The first part of this project investigated a possible etiology for CKD; renal aging as manifested by telomere shortening and cellular senescence. In these studies telomere length and cellular senescence were assessed in cats with CKD in comparison to young healthy and geriatric healthy controls. Using a TELI-FISH assay to measure telomere length in specific renal cell populations, significantly shorter telomeres were found in the renal proximal and distal tubular cell population of CKD cats compared to young normal or geriatric normal cats. There was no difference between CKD cats and normal cats when liver or skin telomere length was measured. Additionally, β-galactosidase assay revealed increased cellular senescence in the kidneys of CKD cats in comparison to young normal. CKD cats tended to have increased β-galactosidase staining in comparison to normal geriatric cats, but this did not reach statistical significance. Neither telomere length nor cellular senescence were correlated with age, but the normal geriatric population available for assessment was small. It was concluded that telomere shortening and cellular senescence are present in feline CKD; future studies will be necessary to determine cause and effect aspects of this relationship. Demonstration of an association between telomere shortening, cellular senescence and feline CKD could be the foundation of new treatment strategies. Cats with CKD frequently have poor appetites and nutritional management of these patients is important. Mirtazapine is an appetite stimulant and anti-nausea medication that has recently gained popularity in veterinary medicine and anecdotally appears to be helpful for the management of appetite. However, no pharmacokinetic or pharmacodynamic information exists on the drug in cats. The aims of the second part of these studies were a) the assessment of the pharmacokinetics and pharmacodynamics of commonly prescribed doses of mirtazapine in normal cats, elderly cats and cats with CKD, and b) a placebo-controlled blinded crossover clinical trial to assess the efficacy of mirtazapine in CKD cats. These studies demonstrated that there are differences in the metabolism of mirtazapine between young normal cats, geriatric normal cats and CKD cats. Based on the pharmacokinetic studies, young cats could receive daily mirtazapine at a low dose without significant likelihood of drug accumulation whereas CKD cats should receive the drug every other day due to delayed clearance. In a subsequent clinical trial, mirtazapine significantly increased appetite, activity and weight in CKD cats when administered at a low dose every other day for three weeks. Additionally, a significant decrease in vomiting was noted. This demonstrated that mirtazapine does have significant appetite stimulating and anti-nausea effects in CKD cats. The information gathered in this body of work will help clinicians prescribe mirtazapine more effectively with a decreased incidence of unwanted drug side effects. Most importantly, it will help improve the quality of life and potentially prognosis of cats suffering from CKD. Most treatments for CKD are palliative in nature and do not directly address the underlying pathology. CKD is characterized by tubulointerstitial inflammation, fibrosis and progressive loss of renal function. Mesenchymal stem cell (MSC) therapy is thought to be anti-inflammatory, and has the potential to improve or stabilize renal function in animals with renal failure, based on evidence from rodent model studies of induced renal disease. At present, there is little published work regarding the use of MSC for treatment of naturally occurring CKD. The last section of this body of work focuses on the evaluation of MSC therapy as a novel treatment strategy for cats with CKD. A series of pilot studies was performed; a pilot study of intrarenal injection of autologous stem cells and two pilot studies of intravenously injected allogeneic cryopreserved MSC. Urinary cytokines were measured to assess intra-renal inflammation, fibrosis and vascular health and the possible effects of MSC injection on these factors. We determined that MSC could be successfully harvested and cultured from bone marrow and adipose sources, but the latter was preferred for ease of collection, expansion and superior yield. Intrarenal injection did not induce immediate or longer-term adverse effects. Two CKD cats that received intrarenal adipose-derived MSC experienced modest improvement in GFR and a mild decrease in serum creatinine concentration. In the allogeneic cryopreserved intravenous study, six cats received 2 x 106 MSC per injection and experienced a significant decrease in serum creatinine with negligible side effects. Five cats received 4 x 106 MSC per injection and side effects included vomiting during infusion and increased respiratory rate. Variable decreases in serum creatinine, increases in GFR by iohexol clearance and changes in urinary cytokines were seen. Despite the mild improvement in creatinine seen in some of the cats, none had improvement to the extent described in rodent models. While MSC therapy potentially holds promise for palliation of CKD, additional work is necessary to determine if this therapy can be manipulated to increase its efficacy. The work described in this dissertation has increased our knowledge of the biology of renal aging and its relationship to CKD. In addition it has assessed the effect of two novel treatment strategies on cats with CKD. This information will directly improve the lives of cats with CKD as well as providing a strong foundation for further research in this area.Item Open Access Mycobacterium tuberculosis – mediated modulation of host macrophage metabolism in the granuloma microenvironment(Colorado State University. Libraries, 2020) Kiran, Dilara, author; Basaraba, Randall, advisor; Podell, Brendan, committee member; Obregon-Henao, Andres, committee member; Olver, Christine, committee member; Chicco, Adam, committee memberMycobacterium tuberculosis (Mtb) is the leading cause of death by an infectious agent, and tuberculosis (TB) disease continues to be a prominent global health concern. Infection with Mtb incites granulomatous inflammation, chronic antigen stimulation, and the development of granuloma lesions. These lesions compress tissue architecture in a way that reduces blood supply and creates central regions of hypoxia. Complex lesion pathology, multi-drug resistance of Mtb, co-morbidities with other endemic diseases, the lack of an effective vaccine, and slow drug development pipelines have hindered progress in the field. Researchers have worked to combat these difficulties through their exploration of host-directed therapeutic strategies, which aim to better equip the host immune system to respond to Mtb infection, with a focus on immunometabolism as a target pathway. The metabolism of host macrophages plays a role in modulating disease pathogenesis, with a metabolic switch from oxidative phosphorylation to glycolysis characterizing Mtb infected macrophages. This metabolic switch is primarily regulated at the transcriptional level by hypoxia inducible factor-1α (HIF-1α), which regulates the cellular response to hypoxic stressors encountered within the chronic granuloma lesion microenvironment. Downstream impacts of HIF-1α activation include increased glycolysis, increased lactate production, and increased lactate transport. HIF-1α becomes stable and undergoes its transcriptional activity in conditions of low oxygen, as a result of prolyl hydroxylase (PHD) inhibition. Additionally, hypoxia-independent factors interfere with PHD leading to HIF-1α stabilization, including iron chelation. Bacteria, such as Mtb, have developed iron chelating siderophores to sequester iron from host cells, and knocking-out these iron chelators has been demonstrated to reduce stable HIF-1α activation. As a result, we hypothesized that the Mtb siderophore, mycobactin, plays a role in driving stabilization of HIF-1α during early infection, prior to the development of hypoxic lesion microenvironments. This would serve as a pathogen-driven mechanism that would support macrophage adaptation to hypoxia later during disease progression, and thus develop an Mtb survival niche. Using purified iron chelators deferoxamine (DFO) and mycobactin J (MbtJ), we demonstrated that treated CD1 mouse bone marrow derived macrophages (BMDMs) increase HIF-1α via Western Blot and potently increase glycolytic metabolism as demonstrated by Seahorse Extracellular Flux Analysis. Additionally, the use of mycobactin synthase K (mbtK) knock-out, complement, or wild-type H37Rv strains of Mtb demonstrated the role that mycobactin plays in the metabolic response of macrophages in vitro, having a significant impact on oxidative metabolism. Hypoxia-independent mechanisms of HIF-1α activation by mycobactin may be a critical pathway through which Mtb drives macrophages toward a phenotype conducive for bacterial survival. Lactate produced as a result of increased glycolytic metabolism during infection may also play an important role as a metabolic intermediate and as a signaling molecule during Mtb infection. Metabolic symbioses exist in multiple systems between highly glycolytic, hypoxic cells and more oxidative, normoxic cells, wherein glycolytic cells uptake glucose, convert glucose to lactate via lactate dehydrogenase (LDHA) and export lactate in large amounts via monocarboxylate transporter 4 (MCT4). Normoxic cells import lactate via monocarboxylate transporter 1 (MCT1) and convert it back to pyruvate via lactate dehydrogenase B (LDHB) and utilize lactate-derived pyruvate to fuel mitochondrial respiration. This preserves glucose for hypoxic cells which rely heavily on glycolysis for metabolic survival. While this type of lactate shuttle has been demonstrated to regulate the tumor microenvironment, it has yet to be explored within the context of the similar TB granuloma microenvironment. As a result, we explored the role of a lactate shuttle within Mtb infection by detecting lactate in guinea pig plasma, detecting lactate shuttle components within guinea pig granuloma lesions, and by using the LDHA inhibitor sodium oxamate and the MCT1 inhibitor α-Cyano-4-hydroxycinnamic acid (α-CHC), both of which are commercially available. We showed that Mtb infection significantly increases lactate on both a systemic and cellular level. We successfully demonstrated that LDH and MCT inhibition augments metabolism in macrophages by blocking glycolysis and decreasing mitochondrial spare capacity. Through in vitro Mtb infection models, we were able to show that inhibitor treatment can reduce the amount of lactate accumulated. These studies demonstrated proof of concept for the role of a lactate shuttle in modulating macrophage metabolism during Mtb infection and maintaining infection dynamics within the granuloma microenvironment. Overall, the research presented herein seeks to understand the ways in which Mtb infection drives host macrophages to alter their metabolic phenotype in a way that promotes Mtb survival and contributes to disease pathogenesis. A better understanding of the interactions which occur at the host-pathogen interface will provide important insight for the development of host-directed therapeutic strategies which will better equip host cells to combat Mtb infection.Item Open Access The development of hyaluronan enhanced expanded polytetrafluoroethylene and linear low density polyethylene for blood contacting applications(Colorado State University. Libraries, 2019) Bui, Hieu T., author; James, Susan, advisor; Reynolds, Melissa, committee member; Popat, Ketul, committee member; Olver, Christine, committee memberCardiovascular disease is the number one cause of death in high income, industrialized countries. Designing cardiovascular implants from synthetic polymers is a cost-effective solution to the growing demand for medical treatments such as heart valve replacements and cardiovascular bypass procedures. Synthetic polymers are often known for their tunability, durability, and low production cost. Unfortunately, these materials are also prone to induce thrombosis. Therefore, improving the blood compatibility of these polymers is still a major challenge in the biomedical field. This dissertation discusses the alteration of two synthetic polymers, linear low density polyethylene (LLDPE) and expanded polytetrafluoroethylene (ePTFE), using hyaluronan (HA) to improve their blood compatibility. HA, a naturally occurring polysaccharide in the human body, is known for its wound healing and anticoagulant properties. In this work, two unique methods were developed for HA enhancement of ePTFE (HA-ePTFE) and LLDPE (HA-LLDPE). This was a process driven research that aimed at designing HA-ePTFE and HA-LLDPE by analyzing the effect of different treatment parameters on the properties of the resultant materials. In the case of ePTFE, it was demonstrated that HA can be incorporated into vascular ePTFE grafts by exploiting the micro pores of the polymer and adjusting the spraying treatment. In the HA-LLDPE fabrication process, its parameters were varied to assess their effects on the interpenetrating polymer network (IPN) formation. Surface characterization such as water contact angle goniometry, infrared spectroscopy, and toluidine blue O (TBO) staining prove that HA treatment successfully changed the surface chemistry and increased the hydrophilicity of ePTFE and LLDPE. Thermal analysis and gas chromatography-mass spectrometry were used to quantify the effects of different treatment conditions on material properties. Tensile properties such as elastic modulus, tensile strength, yield stress and ultimate strain are unchanged by HA enhancement for both polymers. The biological results reveal that HA-ePTFE and HA-LLDPE are not cytotoxic and result in less blood clotting and platelet activation than ePTFE and LLDPE.Item Open Access The roles of iron, the infrapatellar fat pad, and dietary factors in the Hartley guinea pig model of spontaneous osteoarthritis(Colorado State University. Libraries, 2018) Radakovich, Lauren, author; Santangelo, Kelly, advisor; Olver, Christine, committee member; MacNeill, Amy, committee member; Foster, Michelle, committee member; Pagliassotti, Michael, committee memberOsteoarthritis (OA) is the most prevalent musculoskeletal disorder across the world, affecting close to 300 million people. The disease manifests as degeneration and loss of articular cartilage, synovial hyperplasia, formation of osteophytes, subchondral bone remodeling, and joint space narrowing. These changes result in decreased range of motion as well as painful mobility in affected individuals. The knee joint is the most commonly afflicted joint. Osteoarthritic changes may develop secondary to a localized injury, referred to as post-traumatic OA. Degenerative changes can also develop without an inciting cause, which is referred to as spontaneous, or primary OA. Spontaneous OA is an insidious disease that is associated with aging, and, more recently, with obesity. The mechanisms contributing to disease development are not yet fully characterized, which has impeded implementation of successful treatment options. Currently, there are no treatments that are able to restore degraded cartilage. Thus, most patients with symptomatic knee OA undergo costly total knee joint replacement surgeries. The aims of this dissertation were to explore the roles that aging-associated iron accumulation, the infrapatellar fat pad, and calorie restriction with various diets may play in OA development. These studies were performed in the Hartley guinea pig, one of the only small animal models of spontaneous OA. One study was performed in Strain 13 guinea pigs, a strain that is OA-resistant. Iron is an element that acts as a double-edged sword. It is essential for oxidative phosphorylation and heme synthesis, yet its redox potential means it has the capacity to incite oxidant damage when present in excess. As there is no direct excretion mechanism for iron, it tends to accumulate slowly within tissues over time. This cellular iron accrual has been implicated in many degenerative diseases associated with aging, but its potential role in spontaneous OA has not been well-studied. In our first studies, we demonstrated that systemic iron levels have an influence on OA. We were able to induce bony and cartilage lesions in OA-resistant Strain 13 guinea pigs by systemic administration of iron dextran. Immunohistochemistry (IHC) indicated higher levels of lipid peroxidation in cartilage, menisci, the infrapatellar fat pad (IFP), and synovium in the iron overload animals. Special stains revealed that iron content was significantly higher in the IFP in these animals, which we propose serves as a local depot of oxidant damage to the knee joint. In a parallel study, we fed OA-prone Hartley guinea pigs an iron deficient diet to determine if reducing systemic iron levels may have a protective effect on the knee joint. Cartilage lesions were significantly lower in the iron deficient diet group compared to controls. Likewise, IHC for lipid peroxidation revealed less oxidant damage in the iron deficient pigs. However, no differences were noted in knee joint iron content, so the exact mechanisms for the lessened OA remain unclear. Because the iron overload study pointed to the IFP as a potential iron depot, we wanted to further characterize how this adipose tissue contributes to overall knee joint homeostasis. First, we demonstrated that quantitative iron content in the IFP was increased in aged, osteoarthritic guinea pigs compared to young, healthy animals. Gene expression data collected suggested that dysregulated iron trafficking, particularly increased expression of ZIP14 – which has been linked to pathologic iron uptake in other conditions – may be contributing to this aging-associated increase of iron in the IFP. Because of our suspicion that the IFP may be inciting local oxidant damage to the knee, we surgically removed it from a set of young Hartley guinea pigs. An identical sham procedure was performed in the contralateral limb. Four months post-surgery, animals were collected to evaluate OA in both limbs. Both cartilage and bony OA scores were markedly decreased in the IFP removal limb compared to the sham surgery limb. It is possible that removal of the IFP removed a source of local inflammatory mediators and iron, which resulted in lessened OA. As the IFP was replaced by a thick band of fibrous connective tissue, increased joint stability was also considered a contributing factor. Future studies will more closely examined contributions of biomechanical factors that may be at play. Finally, we aimed to determine how dietary manipulations may influence early OA, as previous studies suggest that calorie restriction may improve end-stage OA. Additionally, many studies have shown high fat diet (HFD)-induced obesity plays a role in OA development due to the inflammatory nature of excess adipose tissue. In our study, we demonstrated that calorie restriction with a low fat regular chow diet, but not a calorie restricted HFD, delayed onset of OA in Hartley guinea pigs. In fact, the HFD group had higher levels of systemic inflammation than the restricted regular chow group. The HFD group had similar levels of inflammation and OA scores as obese animals. Thus, we concluded that the pro-inflammatory nature of a HFD supersedes any positive effects of calorie restriction in the onset of spontaneous OA.Item Open Access Understanding and leveraging mechanical forces in haemostasis(Colorado State University. Libraries, 2023) Macleod Briongos, Iain, author; Bark, David, advisor; Olver, Christine, committee member; Popat, Ketul, committee member; Henry, Charles, committee memberCardiovascular disease accounts for one third of deaths worldwide, of which over 75% are in low- and middle-income countries. Platelets and von Willebrand Factor play a central role in haemostasis and in cardiovascular diseases, being involved in both excess clotting that causes heart attacks and strokes, and excess bleeding. Herein, we outline a method for leveraging paper microfluidics with the aim of developing a WHO ASSURED criteria compliant point-of-care device for the diagnosis of von Willebrand Disease, as well as work to increase the understanding of mechanical force generation by platelets through Traction Force Microscopy.Item Open Access Vascular endothelium glycocalyx-mimetic surfaces designed for blood contacting devices(Colorado State University. Libraries, 2021) Vlcek, Jessi, author; Kipper, Matthew, advisor; Reynolds, Melissa, advisor; Popat, Ketul, committee member; Olver, Christine, committee memberEach year millions of blood-contacting devices are used in clinical scenarios, with contact durations designed to range anywhere from hours to years. Current blood-contacting devices can perform their intended purposes well but require the assistance of systemic drugs to inhibit failure via unfavorable interactions between the material's surface and the surrounding biology. The drugs used to inhibit failure of these devices are associated with side effects that can cause increased morbidity of patients because of their systemic administration. Thus, there is a need to design materials that can inhibit thrombus, inflammation, and infection locally at the surface of a device for the device's lifetime. In this work bio-inspired surfaces were engineered to reduce unfavorable blood-material reactions. The success of the designed surfaces was tested by evaluating their cell-material interactions, whole blood interactions, enzymatic stability, and mechanical durability. The inspiration behind these surfaces is the vascular endothelial glycocalyx, which is the luminal side of blood vessels and inhibits blood coagulation during hemostasis. The vascular endothelial glycocalyx is a meshwork of glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins which are predominantly negatively charged that acts as mediator between the blood and the underlying tissue. The surfaces proposed in this work are made to mimic the glycocalyx in its topography and chemistry by adsorbing polyelectrolyte multilayers (PEMs) onto a substrate, and subsequently adsorbing PG mimics on top of the PEMs. There are two different PG mimics used for this project which are: polyelectrolyte complex nanoparticles (PCNs) and proteoglycan mimetic graft copolymers (GC); both of which will present either heparin (HEP) or chondroitin sulfate (CS). Some of the surfaces will also be made to release nitric oxide (NO) from the surfaces through a modified version of chitosan (CHIT). Additionally, further modifications were made to the PEM surfaces to make them more mechanically durable by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) crosslinking of the CHIT and hyaluronan (HA) layers, and the addition of an initial polydopamine (PDA) layer. In the first chapter of this work outlines the current approaches to blood-contacting materials and their limitations, along with the biological components and processes that they will need to interact. The second chapter evaluates PCN and PEM surfaces that do and do not release NO via their cell-material interactions with key cell types in the processes of thrombosis, inflammation, and infection. Chapter three examines the interactions between two different PG-mimics (PC or GCs) and enzymes when suspended in solution or adsorbed onto PEM surfaces. The fourth chapter includes an evaluation of the mechanical durability of PEM and mechanically improved PEM surfaces, and whole blood evaluations of PG, GC, and modified and unmodified PEM surfaces. Taken together, this work produces multiple bioinspired surfaces that have varying degrees of success in their blood compatibility and longevity.