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- Creators: Arizona State University
- Creators: Stabenfeldt, Sarah
Description
The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is essential for the innate immune response to danger signals. Importantly, the NLRP3 inflammasome responds to structurally and functionally dissimilar stimuli. It is currently unknown how the NLRP3 inflammasome responds to such diverse triggers. This dissertation investigates the role of ion flux in regulating the NLRP3 inflammasome. Project 1 explores the relationship between potassium efflux and Syk tyrosine kinase. The results reveal that Syk activity is upstream of mitochondrial oxidative signaling and is crucial for inflammasome assembly, pro-inflammatory cytokine processing, and caspase-1-dependent pyroptotic cell death. Dynamic potassium imaging and molecular analysis revealed that Syk is downstream of, and regulated by, potassium efflux. Project 1 reveals the first identified intermediate regulator of inflammasome activity regulated by potassium efflux. Project 2 focuses on P2X7 purinergic receptor-dependent ion flux in regulating the inflammasome. Dynamic potassium imaging revealed an ATP dose-dependent efflux of potassium driven by P2X7. Surprisingly, ATP induced mitochondrial potassium mobilization, suggesting a mitochondrial detection of purinergic ion flux. ATP-induced potassium and calcium flux was found to regulate mitochondrial oxidative signaling upstream of inflammasome assembly. First-ever multiplexed imaging of potassium and calcium dynamics revealed that potassium efflux is necessary for calcium influx. These results suggest that ATP-induced potassium efflux regulates the inflammasome by calcium influx-dependent mitochondrial oxidative signaling. Project 2 defines a coordinated cation flux dependent on the efflux of potassium and upstream of mitochondrial oxidative signaling in inflammasome regulation. Lastly, this dissertation contributes two methods that will be useful for investigating inflammasome biology: an optimized pipeline for single cell transcriptional analysis, and a mouse macrophage cell line expressing a genetically encoded intracellular ATP sensor. This dissertation contributes to understanding the fundamental role of ion flux in regulation of the NLRP3 inflammasome and identifies potassium flux and Syk as potential targets to modulate inflammation.
ContributorsYaron, Jordan Robin (Author) / Meldrum, Deirdre R (Thesis advisor) / Blattman, Joseph N (Committee member) / Glenn, Honor L (Committee member) / Arizona State University (Publisher)
Created2015
Description
Studies have demonstrated that anthocyanins can function as antioxidants, reduce inflammation, and improve dyslipidemia. Tart cherries are anthocyanin-rich, making them particularly attractive as a functional food to improve cardiovascular disease (CVD) risk. There have been few published studies to date examining the impact of tart cherries on biomarkers of dyslipidemia and inflammation, particularly in overweight and obese individuals at high risk for these conditions. This study evaluated the effect of consuming 100% tart cherry juice daily on blood lipids including total cholesterol, low-density lipoprotein cholesterol (LDL-C), calculated very low density lipoprotein cholesterol (VLDL-C), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), and the CVD risk ratios, as well as the inflammatory biomarkers interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein (CRP), monocyte chemotactic protein-1 (MCP-1), and erythrocyte sedimentation rate (ESR) following a 4-week period. Based on the high anthocyanin content of tart cherries, it was hypothesized that the lipid and inflammatory profiles would be significantly improved following the intervention. A total of 26 men and women completed this 4-week randomized, single-blind, placebo-controlled, crossover study. Participants were randomized to drink either 8 ounces of placebo beverage or tart cherry juice daily for 4 weeks. Following a 4-week washout period, the alternate beverage was consumed. Ultimately, this investigation demonstrated no statistically significant alterations in any of the lipid or inflammatory biomarkers when analyzed across time and between interventions (p > 0.05). As expected, glucose and insulin parameters remained stable over the duration of the study, as well as self-reported physical activity level, total calorie consumption, and macronutrient intake. However, trans-fat was reported to be significantly higher during the cherry arm of the study as compared to the placebo arm (p < 0.05), potentially confounding other results. Although the results of this study were equivocal, it is feasible that a higher dose, longer treatment duration, or more susceptible target population may be required to elicit significant effects. Consequently, further investigation is necessary to clarify this research.
ContributorsColes, Katie (Author) / Martin, Keith R. (Thesis advisor) / Traustadottir, Tinna (Committee member) / Vega-Lopez, Sonia (Committee member) / Arizona State University (Publisher)
Created2012
Description
Approximately 2.8 million Americans seek medical care for traumatic brain injury (TBI) each year. Of this population, the majority are sufferers of diffuse TBI, or concussion. It is unknown how many more individuals decline to seek medical care following mild TBI. This likely sizeable population of un- or self-treated individuals combined with a lack of definitive biomarkers or objective post-injury diagnostics creates a unique need for practical therapies among diffuse TBI sufferers. Practical therapies stand to decrease the burden of TBI among those who would otherwise not seek treatment or do not meet clinical diagnostic criteria upon examination. For this unique treatment niche, practical therapies for TBI are defined as having one or more of the following qualities: common availability, easy administration, excellent safety profile, and cost-effectiveness. This dissertation identifies and critically examines the efficacy of four classes of practical treatments in improving rodent outcome from experimental diffuse traumatic brain injury.
Over-the-counter (OTC) analgesics, omega-3 fatty acids, specialized pro-resolving mediators (SPMs), and remote ischemic conditioning (RIC) were administered before or following midline fluid percussion injury. Behavioral, histological, and molecular analyses were used to assess treatment effects on functional outcome and secondary injury progression. Acute administration of common OTC analgesics had little effect on post-injury outcome in mice. Dietary supplementation with omega-3 fatty acid docosahexaenoic acid (DHA) prior to or following diffuse TBI significantly reduced injury-induced sensory sensitivity and markers of neuroinflammation with no effect on spatial learning. Intraperitoneal administration of omega-3 fatty acid-derived SPM resolvin E1 significantly increased post-injury sleep and suppressed microglial activation. Aspirin-triggered (AT) resolvin D1 administration improved both motor and cognitive outcome following diffuse TBI. RIC treatment in mice demonstrated little effect on functional outcome from diffuse TBI. Untargeted proteomic analysis of plasma samples from RIC-treated mice was used to identify candidate molecular correlates of RIC. Identification of these candidates represents a vital first step in elucidating the neuroprotective mechanisms underlying RIC. The overall findings suggest that omega-3 fatty acid supplementation, SPM administration, and RIC may serve as effective practical therapies to reduce the somatic, cognitive, and neurological burden of diffuse TBI felt by millions of Americans.
Over-the-counter (OTC) analgesics, omega-3 fatty acids, specialized pro-resolving mediators (SPMs), and remote ischemic conditioning (RIC) were administered before or following midline fluid percussion injury. Behavioral, histological, and molecular analyses were used to assess treatment effects on functional outcome and secondary injury progression. Acute administration of common OTC analgesics had little effect on post-injury outcome in mice. Dietary supplementation with omega-3 fatty acid docosahexaenoic acid (DHA) prior to or following diffuse TBI significantly reduced injury-induced sensory sensitivity and markers of neuroinflammation with no effect on spatial learning. Intraperitoneal administration of omega-3 fatty acid-derived SPM resolvin E1 significantly increased post-injury sleep and suppressed microglial activation. Aspirin-triggered (AT) resolvin D1 administration improved both motor and cognitive outcome following diffuse TBI. RIC treatment in mice demonstrated little effect on functional outcome from diffuse TBI. Untargeted proteomic analysis of plasma samples from RIC-treated mice was used to identify candidate molecular correlates of RIC. Identification of these candidates represents a vital first step in elucidating the neuroprotective mechanisms underlying RIC. The overall findings suggest that omega-3 fatty acid supplementation, SPM administration, and RIC may serve as effective practical therapies to reduce the somatic, cognitive, and neurological burden of diffuse TBI felt by millions of Americans.
ContributorsHarrison, Jordan L (Author) / Lifshitz, Jonathan (Thesis advisor) / Neisewander, Janet (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Willyerd, Frederick A (Committee member) / Pirrotte, Patrick (Committee member) / Arizona State University (Publisher)
Created2017
Description
The prevalence of obesity and obesity-related disorders have increased world-wide. In the last decade, the intestinal microbiome has become a major indicator of metabolic and gastrointestinal health. Previous research has shown that high-fat diet (HFD) consumption can alter the microbial composition of the gut by increasing the abundance of gram-positive bacteria associated with the onset of obesity and type 2 diabetes. Although, the most common form of obesity and metabolic syndrome intervention is exercise and diet, these recommendations may not improve severe cases of obesity. Thus, an important relevance of my project was to investigate whether the intake of an organometallic complex (OMC) would prevent the onset of metabolic and gastrointestinal complications associated with high-fat diet intake. I hypothesized that the consumption of a HFD for 6 weeks would promote the development of metabolic and gastrointestinal disease risk factors. Next, it was hypothesized that OMC treatment would decrease metabolic risk factors by improving insulin sensitivity and decreasing weight gain. Finally, I hypothesized that HFD-intake would increase the abundance of gram-positive bacteria associated with gastrointestinal disease. My preliminary data investigated the effects of a 6-week HFD on the development of hepatic steatosis, intestinal permeability and inflammation in male Sprague Dawley rats. I found that a 6-week HFD increases hepatic triglyceride concentrations, plasma endotoxins and promotes the production of pro-inflammatory cytokines in the cecum wall. I then investigated whether OMC treatment could prevent metabolic risk factors in male Sprague-Dawley rats fed a HFD for 10 weeks and found that OMC can mitigate risk factors such hyperglycemia, liver disease, impaired endothelial function, and inflammation. Lastly, I investigated the effects of a 10-week HFD on the gastrointestinal system and found an increase in liver triglycerides and free glycerol and alterations of the distal gut microbiome. My results support the hypothesis that a HFD can promote metabolic risk factors, alter the gut microbiome and increase systemic inflammation and that OMC treatment may help mitigate some of these effects. Together, these studies are among the first to demonstrate the effects of a soil-derived compound on metabolic complications. Additionally, these conclusions also provide an essential basis for future gastrointestinal and microbiome studies of OMC treatment.
ContributorsCrawford, Meli'sa Shaunte (Author) / Sweazea, Karen L (Thesis advisor) / Deviche, Pierre (Thesis advisor) / Al-Nakkash, Layla (Committee member) / Whisner, Corrie (Committee member) / Hyatt, Jon-Philippe (Committee member) / Arizona State University (Publisher)
Created2019
Description
One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs that could be used to treat diseases in the CNS. Previous studies have shown that activation of the adenosine receptor signaling pathway through the use of agonists has been demonstrated to increase BBB permeability. For example, regadenoson is an adenosine A2A receptor agonist that has been shown to disrupt the BBB and allow for increased drug uptake in the CNS. The goal of this study was to verify this property of regadenoson. We hypothesized that co-administration of regadenoson with a non-brain penetrant macromolecule would facilitate its entry into the central nervous system. To test this hypothesis, healthy mice were administered regadenoson or saline concomitantly with a fluorescent dextran solution. The brain tissue was either homogenized to measure quantity of fluorescent molecule, or cryosectioned for imaging with confocal fluorescence microscopy. These experiments did not identify any significant difference in the amount of fluorescence detected in the brain after regadenoson treatment. These results contradict those of previous studies and highlight potential differences in injection methodology, time windows, and properties of brain impermeant molecules.
ContributorsWohlleb, Gregory Michael (Author) / Sirianni, Rachael (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Background: Despite the reported improvements in glucose regulation associated with flaxseeds (Linum usitatissimum) few clinical trials have been conducted in diabetic participants. Objective: To evaluate the efficacy of ground flaxseed consumption at attenuating hyperglycemia, dyslipidemia, inflammation, and oxidative stress as compared to a control in adults with non-insulin dependent type 2 diabetes (T2D). Design: In a randomized parallel arm controlled efficacy trial, participants were asked to consume either 28 g/d ground flaxseed or the fiber-matched control (9 g/d ground psyllium husk) for 8 weeks. The study included 17 adults (9 male, 8 females; 46±14 y; BMI: 31.4±5.7 kg/m2) with a diagnosis of T2D ≥ 6 months. Main outcomes measured included: glycemic control (HbA1c, fasting plasma glucose, fasting serum insulin, and HOMA-IR), lipid profile (total cholesterol, LDL-C, HDL-C, total triglycerides, and calculated VLDL-C), markers of inflammation and oxidative stress (TNF-alpha, TBARS, and NOx), and dietary intake (energy, total fat, total fiber, sodium). Absolute net change for measured variables (week 8 values minus baseline values) were compared using Mann-Whitney U non-parametric tests, significance was determined at p ≤ 0.05. Results: There were no significant changes between groups from baseline to week 8 in any outcome measure of nutrient intake, body composition, glucose control, or lipid concentrations. There was a modest decrease in TNF-alpha in the flaxseed group as compared to the control (p = 0.06) as well as a mild decrease in TBARS in the flaxseed as compared to the control group (p = 0.083), though neither were significant. Conclusions: The current study did not detect a measurable association between 28 g/d flaxseed consumption for 8 weeks in T2D participants and improvements in glycemic control or lipid profiles. There was a modest, albeit insignificant, decrease in markers of inflammation and oxidative stress in the flaxseed group as compared to the control, which warrants further study.
ContributorsRicklefs, Kristin (Author) / Sweazea, Karen L (Thesis advisor) / Johnston, Carol S (Committee member) / Gaesser, Glenn (Committee member) / Vega-Lopez, Sonia (Committee member) / Gonzales, Rayna (Committee member) / Arizona State University (Publisher)
Created2015
Description
The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could provide new insight into disease mechanisms. Although protocols to differentiate hiPSCs and hESCs to neurons have been established, standard practice relies on two dimensional (2D) cell culture systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment.
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
ContributorsPetty, Francis (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2016
Description
An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells.
ContributorsRaman, Sreedevi (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2017
Description
ABSTRACT
Objective: The purpose of this randomized, placebo-controlled trial was to investigate the effect a daily coconut oil supplement (2 grams) would have on a common serum marker of systemic inflammation (C-reactive protein) and an indicator of oxidative stress (TBARS) when compared to the control group receiving a placebo capsule (white flour) in healthy, sedentary adults between the ages of 18-40 in Phoenix, Arizona.
Design: This study was designed as secondary analyses of blood samples originally collected to study the effects of coconut oil supplementation on blood lipids and body composition. The original study consisted of 32 healthy, adult volunteers recruited from the Arizona State University campus in Phoenix, Arizona. Participants followed no food restrictions or special diets, exercised less than 150 minutes per week, had no diagnoses of chronic disease, were not taking statin medications, were non-smokers, and no female participants were pregnant. Participants were randomized into either the Coconut Oil group (CO) or the Placebo group (PL) at week 0, and baseline blood samples and anthropometric measurements were obtained. Each participant completed an 8-week protocol consisting of two supplement capsules daily (coconut oil or placebo). Final fasting blood samples and anthropometric measurements were taken at week 8. This study analyzed the blood samples for measurements of C-reactive protein (CRP) and thiobarbituric reactive substance (TBARS).
Results: Eight weeks of 2 grams per day coconut oil supplementation, in comparison to placebo treatment, did not significantly reduce serum CRP ( -13% and +51% respectively, p=0.183) but did significantly increase TBARS ( +16% and -27% respectively, p=0.049).
Conclusions: Coconut oil supplementation (2 g/day) may impact lipid peroxidation as indicated by an increase in plasma TBARS concentration. Future trials are necessary to corroborate these results using other indices of fatty peroxide formation.
Objective: The purpose of this randomized, placebo-controlled trial was to investigate the effect a daily coconut oil supplement (2 grams) would have on a common serum marker of systemic inflammation (C-reactive protein) and an indicator of oxidative stress (TBARS) when compared to the control group receiving a placebo capsule (white flour) in healthy, sedentary adults between the ages of 18-40 in Phoenix, Arizona.
Design: This study was designed as secondary analyses of blood samples originally collected to study the effects of coconut oil supplementation on blood lipids and body composition. The original study consisted of 32 healthy, adult volunteers recruited from the Arizona State University campus in Phoenix, Arizona. Participants followed no food restrictions or special diets, exercised less than 150 minutes per week, had no diagnoses of chronic disease, were not taking statin medications, were non-smokers, and no female participants were pregnant. Participants were randomized into either the Coconut Oil group (CO) or the Placebo group (PL) at week 0, and baseline blood samples and anthropometric measurements were obtained. Each participant completed an 8-week protocol consisting of two supplement capsules daily (coconut oil or placebo). Final fasting blood samples and anthropometric measurements were taken at week 8. This study analyzed the blood samples for measurements of C-reactive protein (CRP) and thiobarbituric reactive substance (TBARS).
Results: Eight weeks of 2 grams per day coconut oil supplementation, in comparison to placebo treatment, did not significantly reduce serum CRP ( -13% and +51% respectively, p=0.183) but did significantly increase TBARS ( +16% and -27% respectively, p=0.049).
Conclusions: Coconut oil supplementation (2 g/day) may impact lipid peroxidation as indicated by an increase in plasma TBARS concentration. Future trials are necessary to corroborate these results using other indices of fatty peroxide formation.
ContributorsNorman, Lisa (Author) / Johnston, Carol (Thesis advisor) / Shepard, Christina (Committee member) / Ellis, Melissa (Committee member) / Arizona State University (Publisher)
Created2017
Description
Traumatic brain injury (TBI) is a leading cause of death in individuals under the age of 45, resulting in over 50,000 deaths each year. Over 80,000 TBI patients report long-term deficits consisting of motor or cognitive dysfunctions due to TBI pathophysiology. The biochemical secondary injury triggers a harmful inflammatory cascade, gliosis, and astrocyte activation surrounding the injury lesion, and no current treatments exist to alleviate these underlying pathologies. In order to mitigate the negative inflammatory effects of the secondary injury, we created a hydrogel comprised of hyaluronic acid (HA) and laminin, and we hypothesized that the anti-inflammatory properties of HA will decrease astrocyte activation and inflammation after TBI. C57/BL6 mice were subjected to mild-to-moderate CCI. Three days following injury, mice were treated with injection of vehicle or HA-Laminin hydrogel. Mice were sacrificed at three and seven days post injection and analyzed for astrocyte and inflammatory responses. In mice treated with vehicle injections, astrocyte activation was significantly increased at three days post-transplantation in the injured cortex and injury lesion. However, mice treated with the HA-Laminin hydrogel experienced significantly reduced acute astrocyte activation at the injury site three days post transplantation. Interestingly, there were no significant differences in astrocyte activation at seven days post treatment in either group. Although the microglial and macrophage response remains to be investigated, our data suggest that the HA-Laminin hydrogel demonstrates potential for TBI therapeutics targeting inflammation, including acute modulation of the astrocyte, microglia, and macrophage response to TBI.
ContributorsGoddery, Emma Nicole (Author) / Stabenfeldt, Sarah (Thesis director) / Addington, Caroline (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05