Mountain Scholar :: Browsing by Author "Chicco, Adam, committee member"

Browsing by Author "Chicco, Adam, committee member"

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Embargo
A microphysiological system for studying barrier health of live tissues in real time
(Colorado State University. Libraries, 2024) Way, Ryan, author; Chen, Thomas W., advisor; Wilson, Jesse, committee member; Chicco, Adam, committee member
Epithelial cells create barriers that protect many different components in the body from their external environment. The gut in particular carries bacteria and other infectious agents. A healthy gut epithelial barrier prevents unwanted substances from accessing the underlying lamina propria while maintaining the ability to digest and absorb nutrients. Increased gut barrier permeability, better known as leaky gut, has been linked to several chronic inflammatory diseases. Yet understanding the cause of leaky gut and developing effective interventions are still elusive due to the lack of tools to maintain tissue's physiological environment while elucidating cellular functions under various stimuli ex vivo. This thesis presents a microphysiological system capable of recording real-time barrier permeability of mouse gut tissues in a realistic physiological environment over extended durations. Key components of the microphysiological system include a microfluidic chamber designed to hold the live tissue explant and create a sufficient microphysiological environment to maintain tissue viability; proper media composition that preserves a microbiome and creates necessary oxygen gradients across the barrier; integrated sensor electrodes and supporting electronics for acquiring and calculating transepithelial electrical resistance (TEER); and a scalable system architecture to allow multiple chambers running in parallel for increased throughput. The experimental results demonstrate that the system can maintain tissue viability for up to 72 hours. The results also show that the custom-built and integrated TEER sensors are sufficiently sensitive to distinguish differing levels of barrier permeability when treated with collagenase and low pH media compared to control. Permeability variations in tissue explants from different positions in the intestinal tract were also investigated using TEER revealing their disparities in permeability. Finally, the results also quantitatively determine the effect of the muscle layer on total epithelial resistance.
Open Access
Anatomic plasticity and functional impacts of neural – immune and neural – epithelial signaling in the intestine
(Colorado State University. Libraries, 2021) Schwerdtfeger, Luke A., author; Tobet, Stuart A., advisor; Chicco, Adam, committee member; Myers, Brent, committee member; Ryan, Elizabeth, committee member
The intestinal wall is a multicompartmental barrier tissue composed of over 25 distinct cell types with integrated and complex signaling both within and between compartments. The gut wall is also a large endocrine organ comprised of cells capable of producing dozens of peptides used for hormonal and other signaling functions. However, the mechanistic roles that neural secretions play in regulating the gut epithelial barrier in health and disease are not well known. Additionally, frequently used models available for studying intestinal function outside of the body lack the complexity to investigate neural – epithelial and neural – immune signaling interactions. Using a bifurcated approach to method development, we created two culture systems for maintaining the full thickness of the intestinal wall ex vivo. One method allows for culture of mouse or human organotypic intestinal slices that maintain the gut wall for 6 or 4 days, respectively. This system does not however, maintain a true luminal – epithelial barrier as seen in the in vivo gut. The second method, a microfluidic organotypic device (MOD) enables maintenance of explanted mouse or pig intestinal tissue for up to 3 days ex vivo, with an intestinal barrier intact. These two methods allow for investigating and cross-validating of numerous biological questions now previously possible using traditional culture models. Neuronal fiber proximity to gut epithelia has been shown, with goblet, tuft and enteroendocrine cells being closely opposed by fibers. Goblet cells secrete mucopolysaccharides, a first line of defense separating luminal microbiota from host tissue. I have recently shown that vasoactive intestinal peptide (VIP) can regulate goblet cell production in organotypic slices of mouse ileum. This peptide is also in close proximity to Paneth cells in the base of the crypt, and enteric mast cells. There were sex differences in baseline mast cell neuronal proximity, quantities, and cell size in mouse ileum. Further, mast cells showed a sex difference in responses to lipopolysaccharide challenge. Further investigation of neurosecretory factor regulation of immune and epithelial function is needed, both in goblet cells and other secretory epithelia like anti-microbial producing Paneth cells, and in immune components like mast cells. Graphical illustration of the dissertation project is included below.
Open Access
Cardiovascular disease risk in middle-aged ultra-endurance athletes
(Colorado State University. Libraries, 2018) Bachman, Nate P., author; Dinenno, Frank, advisor; Richards, Jennifer, advisor; Chicco, Adam, committee member; Orton, Christopher, committee member
Background: It is widely accepted that aerobic exercise has the ability to reduce cardiovascular disease (CVD) risk. However, recent studies suggest that volumes of exercise that greatly exceed physical activity guidelines may be damaging to the heart. Currently, it is unclear if individuals who train for ultra-endurance races are at an elevated risk of developing CVD compared to those that perform lower amounts of physical activity. Purpose: To use traditional and novel measures of CVD risk to determine if individuals that train for ultra-endurance races have a greater CVD risk compared to participants that engage in recreational physical activity. Methods: We studied two groups of healthy, middle-aged adults (40-65 y); Control (CON, n=18) subjects included individuals who were meeting current physical activity guidelines and the athletes (ATH, n=25) had been training for ultra-endurance events for 10 years. We used cardiac computed tomography (CT) to calculate coronary artery calcium scores (CACS) and magnetic resonance imaging (MRI) to assess for myocardial fibrosis (MF). Vascular function was evaluated using carotid-femoral pulse wave velocity (cfPWA) and flow-mediated dilation (FMD). 10-Year coronary heart disease (CHD) risk was also determined using a risk score calculator. Results: CACS > 0 was observed in 2 CON and 8 ATH; however, the presence of CAC was not significantly different between groups (P>0.05). Additionally, no participants in CON or ATH had MF. CON had higher cfPWV compared to ATH (6.9±0.2 vs 6.2±0.2 m/s, P<0.05), while no differences in FMD were observed (CON; 5.6±1.2 vs ATH; 3.6±0.8 %, P>0.05). Furthermore, there were no group differences in CHD risk (CON; 1.6±0.3 vs ATH; 2.4±0.6 %, P>0.05). Conclusion: ATH training for ultra-endurance races are not at a greater risk of experiencing a cardiac event than individuals that meeting current physical activity guidelines.
Open Access
Characterization of the unique biomechanical behavior of right ventricle using experimental and constitutive modeling approaches
(Colorado State University. Libraries, 2022) Liu, Wenqiang, author; Wang, Zhijie, advisor; Puttlitz, Christian, committee member; Bark, David, committee member; Chicco, Adam, committee member
Ventricle dysfunction leads to high morbidity and mortality in heart failure patients. It is known that right and left ventricles (RV&LV) are distinct in their embryologic origins, anatomies and functions, as well as the pathophysiology of ventricular failure. However, how exactly the RV is distinct from the LV in their biomechanical properties remains incompletely understood. Furthermore, the prevalence of RV failure is significantly increased in the later stages of diseases such as pulmonary hypertension (PH) and heart failure with preserved ejection fraction, and the clinical management and treatment of RV failure are persistently challenging. This calls for a further understanding of the mechanisms of RV failure including the biomechanical mechanism. In addition, ventricular tissues are viscoelastic, which means both energy storage (originated from elasticity) and energy loss (originated from viscosity) are present during the deformation. However, the investigation of ventricular tissue viscoelasticity is much less than that of the elasticity, and it is largely unknown how the RV viscoelastic behavior changes during RV failure progression and impacts on the physiological function of the chamber. To fill these knowledge gaps, the overall goal of my study was to investigate the unique biomechanical properties of the RV in its physiological and pathological functions using experimental and constitutive modeling approaches. The Specific Aims are: 1) Develop the experimental protocols and characterize ventricular tissue passive static and dynamic mechanical properties in both large and small animals; 2) Adapted and performed constitutive modeling of ventricular tissue static and dynamic mechanical behaviors; 3) Quantify the changes in RV biomechanics during the maladaptive remodeling induced by pulmonary hypertension. At the completion of my study, I established the ex vivo testing protocols and provided fundamental data regarding static and dynamic mechanical differences between the healthy left and right chambers to delineate the unique biomechanical properties of the RV. I also adapted the constitutive models to capture static and dynamic mechanical behaviors of the RV. Finally, I quantified the biomechanical changes of the RV during the RV failure development and offered new insights in the contributions of the RV tissue biomechanics to the organ function. The findings were obtained from both large and small animals' species, which are translational to human diseases and a strong addition to the current literature of RV failure. More importantly, the investigation on the viscoelastic (dynamic) mechanical properties of the RV and the changes of viscoelasticity in RV failure progression is highly novel. The constitutive modeling of the RV biaxial viscoelastic behavior is pioneering and unique in the computational study of the RV. In summary, this study will deepen the understanding of the biomechanical mechanisms of RV failure and assist with the development of new computational tools for diagnosis and treatment strategies.
Embargo
Comparative analysis of the role of redox active molecules on bioenergetically active membranes
(Colorado State University. Libraries, 2024) Dolan, Connor Cathal, author; Crans, Debbie, advisor; Kennan, Alan, committee member; Chicco, Adam, committee member
Transition metals play crucial roles in various biological processes, with vanadium and manganese being prominent examples due to their redox activity and impact on oxidative stress, mitochondrial function, and disease progression. This manuscript focuses on the role of transition metals, particularly vanadium, in biological functioning, with an emphasis on oxidative stress and mitochondria. Chapter 1 of this thesis discusses the respective role that vanadium plays on oxidative stress and how that influences biological systems. Due to its variety of speciation states and its ability to redox cycle as well as its structural and electronic properties, vanadium can affect biological systems in a variety of ways. These include the generation of reactive oxygen species, lipid peroxidation, protein inhibition, changes in membrane fluidity and potential. DNA damage and cell death. The effects that vanadium has is highly dependent on the speciation and state that they exist in. this can impact the system that is being affected and the outcome. Species such as decavanadate have a unique and profound biological effect. Changing of the species, oxidation state and complexation can alter the biological consequences associated with vanadium. Chapter 2 of this thesis explores the differences and similarities between vanadium and manganese on cardiac mitochondrial dysfunction and oxidative stress. Using varying vanadium and manganese compounds, we investigated the effects they had on isolated cardiac mitochondria using high resolution respirometry and UV-Vis spectroscopy. We found similarities between metal salts on inhibition of respiration as well as significant differences on the metals iii effect on mitochondrial swelling. We further investigated the role of transport proteins on vanadium induced swelling and found that the mitochondrial calcium uniporter played an important role in vanadium induced mitochondrial swelling. We further investigated the differences in species and oxidation state on function. We tested the difference between VV and VIV on mitochondrial swelling and found that VIV led to significantly greater swelling. We also found that there the VO(OH)3 - monomer and dimer were present in both VIV compounds and the Mn2+ ion was present in both manganese compounds. This speciation similarity between compounds may account for some of the similar effects seen within the same metal compounds as well as the differences seen when comparing manganese and vanadium together.
Open Access
Design and implementation of the SBX1: a smart environment chamber for biological research and discovery
(Colorado State University. Libraries, 2021) Ball, Daniel S., author; Chen, Thomas, advisor; Wilson, Jesse, committee member; Chicco, Adam, committee member
Modern biomedical laboratories make significant use of environmentally controlled chambers for incubation and examination of live cell samples. They require precise control over temperature, humidity, and gas concentration to mimic natural conditions for cell survival and growth. Many incubators and live cell imaging systems exist as commercial products; however, they are prohibitively expensive, costing tens or hundreds of thousands of dollars depending on capabilities of the system. This thesis presents the electrical, optical, mechanical, and software design of the SBX1Smart Environment Chamber. This device aims to fulfill the needs of most users at a lower cost than current commercial offerings, providing an opportunity for less funded labs to pursue biomedical research and development. The chamber provides temperature, humidity, and gas concentration controls, an internal microscope with an automated stage, and an integrated ARM microcomputer to with a graphical user interface for control and monitoring of the system. A patent has been filed for the SBX1; application no. US 2020/0324289 A1.
Open Access
Gene regulation by let-7 microRNAs during human and sheep placental development
(Colorado State University. Libraries, 2020) Ali, Asghar, author; Winger, Quinton A., advisor; Bouma, Gerrit J., advisor; Chicco, Adam, committee member; Garrity, Deborah, committee member
Intrauterine growth restriction (IUGR) is a major cause of perinatal morbidity and mortality and affects more than 30 million infants every year across the world. Its occurrence is 5–15% of all pregnancies in the United States and 10-55% in developing countries. Most common etiology of IUGR is impaired placental development. Structural and functional abnormalities in placenta can also lead to preeclampsia (PE), still birth and spontaneous abortion. Conditions like IUGR and PE are usually not detected until later stages of gestation. Hence, there is a need to better understand the placental development and function to improve diagnosis and treatment of placenta-associated disorders. In this study, we investigated genetic pathways regulated by let-7 miRNAs and their potential role in pathogenesis of malformed placenta. Let-7 miRNAs are markers of cell differentiation and reduce the expression of several genes by translational repression. Biogenesis of let-7 miRNAs is suppressed by LIN28 which is an RNA binding oncofetal protein with two paralogs, LIN28A and LIN28B. LIN28 is high and let-7 miRNAs are low in proliferating stem cells whereas LIN28 is low and let-7 miRNAs are high in differentiated cells. LIN28-let-7 axis determines fate of cells by regulating the expression of genes associated with cell proliferation and differentiation. In human placenta, LIN28 is mainly found in trophoblast cells and the fetal portion of placenta comprises mainly of trophoblast cells. We found that in term human placentas from IUGR pregnancies, LIN28 is low and let-7 miRNAs are high compared to placentas from control placentas. We further saw a reduction in the expression of AT-Rich Interaction Domain 3A (ARID3A) and AT-Rich Interaction Domain 3B (ARID3B). ARID3A and ARID3B promote cell proliferation by transcriptional regulation of stemness genes. In immortalized first trimester human trophoblast (ACH-3P) cells, ARID3A and ARID3B complex with lysine demethylase 4C (KDM4C) to make the ARID3B-complex. This complex binds the promoter regions of proliferation-associated genes such as high mobility group AT-hook 1 (HMGA1), transcriptional regulator Myc-like (c-MYC), vascular endothelial growth factor A (VEGF-A) and Wnt family member 1 (WNT1). These genes are also targeted by let-7 miRNAs. LIN28 knockout ACH-3P cells have significantly increased let-7 miRNAs and significantly reduced HMGA1, c-MYC, VEGF-A and WNT1. We also saw significant reduction in ARID3A and ARID3B in LIN28 knockout ACH-3P cells. ACH-3P cells with ARID3B knockout also showed significant reduction in HMGA1, c-MYC, VEGF-A and WNT1. Both LIN28 knockout and ARID3B knockout in ACH-3P cells resulted in reduced cell proliferation compared to control. These results suggest that proliferation-associated genes in trophoblast cells are regulated through LIN28-let-7-ARID3B pathway. Trophectoderm (TE) specific knockdown of LIN28 in sheep led to reduced conceptus elongation at day 16 of gestation. Furthermore, LIN28 knockdown day 16 TE had significantly increased let-7 miRNAs and significantly reduced expression of proliferation factors including insulin like growth factor 2 mRNA binding proteins (IGF2BPs), HMGA1, ARID3B and c-MYC. From these findings, we interpret that LIN28-let-7-ARID3B pathways regulates proliferation of trophoblast cells and is potentially associated with etiology of IUGR.
Open Access
Hemocompatibility of hyaluronan enhanced linear low-density polyethylene for heart valve leaflet applications
(Colorado State University. Libraries, 2018) Simon-Walker, Rachael, author; Popat, Ketul C., advisor; Reynolds, Melissa, committee member; Orton, Christopher, committee member; Chicco, Adam, committee member
Heart valve disease is a major concern in both developed countries with advanced ageing populations and undeveloped countries which experience a high incidence of rheumatism leading to valvular disease. To reduce mortality and improve quality of life, heart valve implantations have been widely used to assist in improving function of the native cardiovascular system. While mechanical heart valves and tissue-based heart valves have been successfully used to improve quality of life compared to untreated valvular disease, draw-backs are inherent. Mechanical heart valves are prone to thrombosis and require life-long supplemental anti-coagulation therapy. Tissue-based valves are more hemocompatible, but lack the durability required for long-term implantation. To address these issues, polymeric heart valves have been highly sought after due to polymers' abilities to enhance durability and be manufactured to be similar to the native heart valve leaflet. In addition, their surfaces can be modified to increase hemocompatibility. In this work we explore the hemocompatibility and immune response to a novel polymer for use in heart valve leaflet applications; hyaluronan enhanced linear low-density polyethylene. It is proposed that the combination of linear low-density polyethylene with hyaluronan will create a highly durable material that will reduce thrombosis and inflammation due to the anionic and hydrophilic nature of the glycosaminoglycan.
Open Access
Host directed therapy targeting M. tuberculosis infected macrophages
(Colorado State University. Libraries, 2016) Lakey, Natalie, author; Basaraba, Randall, advisor; Podell, Brendan, committee member; Perera, Rushika, committee member; Chicco, Adam, committee member
With the rise of drug resistant strains of Mycobacterium tuberculosis (Mtb) and lags in antimicrobial drug development, it is imperative to explore alternative methods of treatment through host-directed adjunct therapies. Hallmarks of Mtb infection are altered host cell glucose metabolism and non-diabetic hyperglycemia, which increase disease severity and bacterial burden. This can be targeted using a combination of metformin and 2-deoxyglucose (2DG) to lower systemic blood glucose and increase metabolic stress in infected macrophages to induce apoptotic cell death, enabling Mtb clearance and antigen presentation to activate cell-mediated immune responses. We hypothesized that bacterial survival is aided by glycolysis-dependent macrophages, which can be targeted using a combination of metformin and 2DG to strengthen host immune responses. Using an in vitro model of guinea pig bone marrow derived macrophages under normal and high glucose conditions, we determined that both basal respiration and glycolytic activity increased after infection. When singly treated, metformin inhibited basal respiration while increasing glycolysis while 2DG inhibited both processes. In combination metformin and 2DG treatment inhibited basal respiration more effectively than metformin treatment alone and inhibited glycolysis as effectively as 2DG by itself. Efficacy of metformin-2DG treatment is dependent on high cellular glycolytic activity, a characteristic of granulomatous cells. Metformin-2DG treatment decreased cell survival 48 hours post-treatment by increasing apoptotic cell death and decreased Mtb survival more effectively than metformin or 2DG alone. We conclude that apoptotic induction of macrophages by metformin-2DG treatment may be a viable adjunct treatment to antimicrobial drugs to reduce bacterial burden and increase an effective host adaptive immune response.
Open Access
Mechanisms of exercise hyperemia during elevated oxygen delivery in humans
(Colorado State University. Libraries, 2020) Anna, Jacob L., author; Dinenno, Frank, advisor; Richards, Jennifer, advisor; Lark, Daniel, committee member; Chicco, Adam, committee member
The coupling between skeletal muscle oxygen delivery (O2D) and metabolic demand is largely attributed to the integration of feedback, and feedforward vascular mechanisms. It has been demonstrated that blood flow responses remain intact despite pharmacological elevations in resting blood flow, suggesting the existence of a vasodilator capable of augmenting hyperemia independent of tissue oxygen demand. We hypothesized that the change in forearm blood flow (FBF) from rest to steady-state exercise is preserved independent of baseline O2D, and that reciprocal reductions in oxygen extraction coincide with elevated O2D (Protocol 1). Additionally, pharmacological blockade of Kir channels and ATPase will reduce the change in FBF. In 10 young healthy adults, we quantified forearm blood flow (FBF; Doppler ultrasound), venous oxygen saturation (SvO2), oxygen extraction (O2 extraction; deep venous blood samples), and forearm oxygen consumption (mVO2) at rest and throughout 5 minutes of mild-intensity (10% maximal voluntary contraction; MVC) rhythmic handgrip exercise under control (CON) conditions and following intra-arterial infusion of the vasodilator sodium nitroprusside (SNP) to elevate local FBF and O2D. In Protocol 1, we elevated resting FBF and O2D to levels that matched (MAT) and exceeded (EXC) steady-state FBF (FBF: MAT; 166 ± 25 ml/min, P=NS, EXC; 219 ± 27 ml/min, P<0.05) during control (CON) exercise trials (FBF: CON; 172 ± 24). Changes in blood flow remained intact (ΔFBF: CON; 135 ± 20 ml/min vs. MAT; 132 ± 19 ml/min vs. EXC; 167 ± 26 ml/min, P=NS across all conditions), despite elevations in resting FBF which were adequate to sustain steady-state contractile activity under CON conditions. Reciprocal reductions in O2 extraction were observed in the MAT (O2 extraction: CON; 63 ± 3% vs. MAT; 43 ± 5%, P<0.05) and EXC trials (O2 extraction: CON; 63 ± 3% vs. EXC; 35 ± 5%., P<0.05) compared to CON during exercise. Additionally, we measured venous K+ in a subset of participants (N=6) to evaluate changes in K+ efflux (venous [K+] x FBF/1000) as an index of K+ release during exercise, alluding to K+-mediated activation of Kir channels and the ATPase. Five participants completed Protocol 2 which included control and elevated FBF trials (Saline + Block and SNP + Block) with the addition of intra-arterial infusion of barium chloride (BaCl2) and ouabain to inhibit Kir channels and the ATPase, respectively. Blockade of these pathways reduced the change in FBF that persisted during the Protocol 1 MAT trial (ΔFBF: MAT; 148 ± 21 ml/min vs. SNP + Block; 96 ± 13 ml/min, P<0.05). From this data, we are able to determine that changes in blood flow during exercise persist despite elevations in resting O2D (via SNP) prior to the start of exercise and that trends in O2 extraction follows changes in total O2D. We believe that local skeletal muscle K+ release is capable of activating Kir channels and the ATPase in a feed-forward manner which initiates a hyperpolarizing signal, thus augmenting blood flow independent of tissue oxygen demand.
Open Access
Mechanisms of impaired red blood cell ATP release in older adults: implications for altered vascular control with age
(Colorado State University. Libraries, 2018) Racine, Matthew L., author; Dinenno, Frank A., advisor; Amberg, Gregory, committee member; Chicco, Adam, committee member; Gentile, Christopher, committee member
The following dissertation is comprised of a series of experiments with the overall aim of determining the mechanisms of impaired ATP release from red blood cells (RBCs) of healthy older adults in response to hemoglobin deoxygenation and identifying a potential role of this impairment in the declines in vascular control of peripheral blood flow with advancing age. Advancing age is the primary risk factor for cardiovascular disease (CVD), which is the leading cause of death in societies today and is strongly associated with arterial dysfunction. Furthermore, impairments in vascular control and the subsequent regulation of tissue blood flow and oxygen delivery contribute to vascular pathologies such as atherosclerosis and ischemic disease, as well as the age-associated declines in functional capacity, exercise tolerance, and overall quality of life. Thus, understanding the mechanisms of the age-related impairments in vascular control and identifying potential therapeutic targets holds significant potential for reducing the healthcare burden associated with a rapidly aging population. Accordingly, the ultimate goal of this dissertation is to determine if an in vivo pharmacological approach can be utilized to treat the age-related declines in RBC ATP release, thereby restoring circulating ATP responses and subsequent vascular control during the physiological stimuli of hypoxia and exercise in healthy older adults. The key novel findings of this dissertation are that (i) age-associated declines in RBC deformability are the primary mechanism of impaired deoxygenation-induced ATP release from RBCs of healthy older adults; (ii) primary (healthy) aging is not associated with a global decline in RBC function given that inhibition of cyclic AMP hydrolysis by phosphodiesterase 3 did not improve deoxygenation-induced ATP release from RBCs of older adults and that the cellular responses to Gi protein activation remained intact with age; and (iii) that systemic Rho-kinase inhibition via administration of fasudil improves the age-related impairments in vascular control and circulating ATP during systemic hypoxia and exercise, which may be related to enhanced RBC ATP release and NO bioavailability. These findings are the first to identify a role for Rho-kinase inhibition in improving these physiological responses in healthy older adults and are therefore clinically significant for aging population in which impaired vascular control contributes to elevations in cardiovascular disease risk and declines in exercise tolerance, functional independence and overall quality of life.
Open Access
Mechanisms of vascular dysfunction in obesity and type 2 diabetes: role of the gut microbiota and endoplasmic reticulum stress
(Colorado State University. Libraries, 2018) Battson, Micah Lee, author; Gentile, Christopher, advisor; Cox-York, Kimberly, advisor; Weir, Tiffany, committee member; Pagliassotti, Michael, committee member; Chicco, Adam, committee member
Vascular dysfunction, characterized by arterial stiffness and endothelial dysfunction, is a key antecedent to overt cardiovascular disease in obesity and type 2 diabetes. Although the mechanisms underlying the development of vascular dysfunction in obese and type 2 diabetic individuals are not fully known, a growing body of evidence suggest that adverse cellular processes, including endoplasmic reticulum (ER) stress, inflammation and oxidative stress, are primarily responsible for the disruption of normal vascular function in these two metabolic diseases. Therefore, identifying effective strategies to mitigate one or more of these adverse processes may lead to novel therapies for the treatment of vascular dysfunction in obesity and/or type 2 diabetes. In addition, ascertaining the initial triggering factor(s) that promote these adverse processes will inform innovative ways to prevent or control the progression of vascular dysfunction. The goals of this dissertation research were to 1) examine the underlying causes of vascular dysfunction in obesity and type 2 diabetes and 2) identify potential strategies to mitigate vascular dysfunction in these metabolic diseases. To this end, we conducted three separate studies in murine models of obesity and/or type 2 diabetes aimed to modulate key factors that can affect vascular function. In all three studies, we measured aortic pulse wave velocity and endothelium-dependent dilation as clinically relevant indices of arterial stiffness and endothelial dysfunction, respectively. We also conducted various biochemical analyses to explore the potential mechanisms by which our experimental interventions altered vascular function. In our first study (Chapter 2), we examined the role of ER stress in diabetic vascular dysfunction. In type 2 diabetic (db/db) mice, we found that chronic administration of the ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), significantly reduced arterial stiffness and endothelial dysfunction. These vascular improvements were associated with reduced expression of ER stress-related genes within the aorta and surrounding perivascular adipose tissue (PVAT). Next (Chapter 3), we examined the role of the gut microbiota in the development of vascular dysfunction in obesity. We found that Western diet (WD)-induced obesity increased arterial stiffness, impaired endothelial function, and promoted endotoxemia-related inflammation. Antibiotic treatment to suppress the gut microbiota in WD-fed mice reduced arterial stiffness, improved endothelial function, and attenuated systemic and vascular inflammation. In our final study (Chapter 4), we examined whether gut dysbiosis represents a causal factor in the development of obesity-related vascular dysfunction. We found that transplant of gut microbiota from obese (ob/ob) to control mice promoted the development of arterial stiffness, and this was associated with reduced abundance of a symbiotic bacterium, Akkermansia muciniphila, decreased short-chain fatty acid levels, and increased gut permeability. In contrast, transplant of control microbiota to obese mice did not attenuate arterial stiffness. Collectively, these studies in mice provided evidence that 1) mitigation of ER stress improves vascular function in type 2 diabetes, 2) gut dysbiosis contributes to vascular dysfunction in WD-induced obesity, and 3) an obese-type microbiota can promote arterial stiffening independent of body weight. Future clinical trials and mechanistic studies are needed to translate our findings to humans and to further examine the molecular mechanisms linking gut dysbiosis to vascular dysfunction.
Open Access
Metabolic interventions in the treatment of Mycobacterium tuberculosis infection
(Colorado State University. Libraries, 2023) Ackart, David, author; Basaraba, Randall, advisor; Podell, Brendan, committee member; Chicco, Adam, committee member
Tuberculosis remains a global threat. For the first time in over a decade, there was an increase in deaths in 2021. Current antimicrobial treatments are lengthy and costly, which leads to non-compliance. Host-directed therapies have emerged as a possible adjuvant to antimicrobial treatment. It has become clear that immune cell function and metabolic pathways are intertwined and a target for therapy. Previously, metformin, a partial inhibitor of complex I, was associated with decreased disease burden in the guinea pig model. Based on these findings, we hypothesized that limiting glycolysis would reduce lesion burden and bacterial viability. To test our hypothesis, we infected guinea pigs for thirty days and then administered metformin or 2-deoxy-glucose alone, and in combination. After 90 days of infection, histological analysis revealed increased healing in the combination treatment. To determine the effect of treatment on cellular metabolism, we evaluated treatments in guinea pig bone marrow-derived macrophages. We found that the combination treatment reduced bacterial viability, prevented mitochondrial damage, and increased apoptosis, which helps support in vivo findings. These findings suggest that the metabolic switch of immune cells to glycolysis alone is insufficient to control Mycobacterium tuberculosis infection; it is essential, as is oxidative phosphorylation.
Open Access
Metformin: a tool to better understand T cell mediated protection against Mycobacterium tuberculosis
(Colorado State University. Libraries, 2020) Haugen Frenkel, Jessica D., author; Basaraba, Randall J., advisor; Obregón-Henao, Andrés, committee member; Podell, Brendan K., committee member; Chicco, Adam, committee member; Avery, Anne, committee member
To view the abstract, please see the full text of the document.
Open Access
Multi-modal investigation of tendon healing: tendinopathic injury models to novel rehabilitative strategies
(Colorado State University. Libraries, 2022) Johnson, Sherry, author; Frisbie, David, advisor; King, Melissa, advisor; Selberg, Kurt, committee member; Chicco, Adam, committee member
To view the abstract, please see the full text of the document.
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 member
Mycobacterium 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.
Open Access
Protective effects of sulforaphane on nitric oxide induced mitochondrial dysfunction
(Colorado State University. Libraries, 2019) Acerbo, Evan R., author; Hanneman, William, advisor; Legare, Marie, committee member; Chicco, Adam, committee member
Sulforaphane (SFN), an isothiocyanate compound that is formed in the breakdown process of cruciferous vegetables, has demonstrated the ability to interfere with dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. The present study investigated whether SFN can protect cells exhibiting persistent mitochondrial fission induced by nitrosative stress (S-nitrosoglutathione; GSNO), and shed light on the mechanism by which this occurs. Results show that SFN (5 μM) prevents decreases in the rate of mitochondrial oxidative phosphorylation (ATP production) in SH-SY5Y neuroblastoma cells treated with 200-600 μM GSNO, which was associated with significant improvements in cell viability at all doses. Based upon the understood activation mechanism of Drp1, we further explored the possibility that SFN interferes with phosphorylation of Drp1 at serine residue 616 (pDrp1-Ser616). Indeed, SFN significantly reduced GSNO-mediated increases in pDrp1-Ser616, suggesting a possible mechanism of cytoprotection. However, due to the various reported targets of SFN, it remains unclear if SFN interferes directly with Drp1 phosphorylation or with other targets upstream of this event.
Open Access
Role of the endothelium in modulating sympathetic vasoconstriction in contracting skeletal muscle of young and older adults
(Colorado State University. Libraries, 2016) Hearon, Christopher M., author; Dinenno, Frank A., advisor; Amberg, Gregory, committee member; Chicco, Adam, committee member; Gentile, Christopher, committee member
Aerobic capacity is a powerful independent predictor of all-cause mortality in healthy and disease populations. Healthy (primary) ageing is associated with a decline in maximal aerobic capacity, exercise intolerance and elevated risk for ischemic cardiovascular disease. Specifically, ageing is characterized by impaired regulation of vascular tone during exercise, due in part to lower vasodilatory signaling and elevated sympathetic vasoconstrictor activity in the peripheral vasculature. Impaired regulation of peripheral vascular tone results in attenuated blood flow and oxygen delivery to contracting skeletal muscle during exercise and is a primary contributor to the age-associated decline in aerobic capacity. The overall aim of this dissertation is to determine the vascular signaling mechanisms responsible regulating sympathetic vasoconstrictor signaling during exercise in young healthy adults and translate these findings to improve vascular function during exercise in older adults. The regulation of blood flow and oxygen delivery during exercise depends on the proper integration of local vasodilatation and neural sympathetic vasoconstriction. In healthy humans, the integration of these competing signals results in attenuation of sympathetic vasoconstriction, or “sympatholysis”, to ensure adequate blood flow to contracting skeletal muscle. The signaling mechanisms responsible for sympatholysis in healthy humans are unknown. To date, the only exogenous vasodilator shown to mimic exercise in its ability to attenuate sympathetic vasoconstriction in humans is adenosine triphosphate (ATP). The first aim of this dissertation is to determine if smooth muscle cell hyperpolarization (via activation of inwardly-rectifying potassium (KIR) channels), the primary vasodilatory pathway of ATP, is responsible for ATP-mediated attenuation of sympathetic vasoconstriction. In contrast to smooth muscle specific signaling, vasodilatory stimuli such as ATP and exercise can act through endothelium-dependent pathways. The second aim of this dissertation tests the hypothesis that endothelium-dependent signaling is capable of attenuating sympathetic vasoconstriction during exercise in young healthy humans. With age, impaired endothelial function and elevated sympathetic vasoconstrictor activity results in impaired functional sympatholysis. The third aim is to determine if augmentation of endothelium-dependent signaling during exercise improves age-associated impairments in functional sympatholysis. The primary findings of this dissertation are that 1) similar to exercise, the ability of ATP to attenuate sympathetic vasoconstriction is independent of smooth muscle cell hyperpolarization via activation of KIR channels, 2) activation of endothelium-dependent signaling during exercise significantly enhances the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction, and 3) that augmentation of endothelium-dependent signaling during exercise significantly improves functional sympatholysis in older adults. These findings are the first to identify endothelium-dependent modulation of sympathetic vasoconstriction in humans, and identifies vascular signaling pathways capable of improving the regulation of vascular tone during exercise in older adults. These findings are clinically significant for patient populations and disease states characterized by impaired functional sympatholysis including ageing, hypertension, and heart failure.
Open Access
Sex differences in cortical-hypothalamic control of stress reactivity and cardiovascular susceptibility
(Colorado State University. Libraries, 2023) Schaeuble, Derek, author; Myers, Brent, advisor; Chicco, Adam, committee member; Gentile, Chris, committee member; Tobet, Stu, committee member
Major depressive disorder (MDD) is characterized by prolonged sadness and a loss of interest, and it impacts an estimated 21 million adults in the United States. The onset of MDD is multifactorial and rates of MDD have increased due to the psychosocial and economic factors associated COVID-19 pandemic. This poses a substantial threat to population health as MDD is projected to be the leading cause of disability by 2030. Even throughout the pandemic, cardiovascular disease (CVD) is still the highest mortality rate of any disease worldwide average of 17.9 million deaths per year. More importantly, MDD and CVD have devastating comorbidity that is poorly understood. MDD doubles the risk of developing cardiovascular disease and significantly increases the chance of morbidity following cardiovascular events. Thus, we need to address mental health disabilities and cardiovascular disease susceptibility. Interestingly, both diseases are exacerbated by chronic life stressors, which increase the prevalence of mood disorders and can alter sympathetic nervous activity increasing heart rate and blood pressure. Studying how stress affects the brain may yield important information on how to treat these two diseases. In this series of experiments, I examine how the ventral medial prefrontal cortex (vmPFC) alters stress responding through its downstream connections to provide a mechanism for MDD and cardiovascular disease comorbidity. I will provide a brief background of the structure and function of the vmPFC and describe how neurons from this region can alter stress responding through synapses in the hypothalamus. Chapter 2 is the first of a series of experiments where I show decreased activity of the vmPFC interacts with chronic stress to predispose male rats to cardiovascular disease susceptibility. Because mood disorders are more common in women and cardiovascular disease is more prevalent in post-menopausal women compared to men, chapter 3 examines whether activating vmPFC projection neurons is sufficient to influence behavior, stress responding, and cardiovascular activity in both sexes of rats. This work uncovered that output of vmPFC glutamate neurons has sexually divergent outcomes on neuroendocrine and autonomic cardiovascular responses to stress. Furthermore, it became evident that altered vmPFC activity predisposes males but not females to cardiovascular disease susceptibility. The vmPFC does not directly project to autonomic or neuroendocrine effector regions, so chapter 4 investigates whether the vmPFC is sufficient to control stress autonomic and neuroendocrine responding through downstream intermediary synapses. The intermediate region of interest is the posterior hypothalamus (PH) which can regulate endocrine and cardiovascular activity and receives dense innervation from the vmPFC. In chapter 5, I am exploring the necessity of this vmPFC-PH circuit to regulate cardiovascular activity and stress reactivity following chronic stress exposure. Altogether these data identify novel neurocircuitry linking stress exposure to cardiovascular disease risk.
Open Access
Study of real-time spatial and temporal behavior of bacterial biofilms using 2D impedance spectroscopy
(Colorado State University. Libraries, 2019) Begly, Caleb R., author; Chen, Thomas W., advisor; Wilson, Jesse, committee member; Chicco, Adam, committee member
The study of biofilms and their effect on disease treatment, prevention, and cures has been increasing in importance in recent years. Bacterial biofilms are colony formations developed by bacteria that allow them to anchor onto a surface and survive hostile environments. The formation of harmful bacteria biofilms on some surfaces can be troublesome, particularly in the case of medical implants. The continuing rise of antibiotic-resistant bacteria over the past decade had escalated the need to study and understand biofilms. This thesis presents the design of a multi-channel impedance spectroscopy instrument to allow 2D spatial and temporal evaluation of biofilm growth. The custom-designed circuits allow measurement updates once per second on the entire set of impedance sensors. The distance between the neighboring sensors is 220 micrometers, allowing realtime observation of biofilm growth. The initial results show that the proposed 2D impedance spectroscopy tool provides the needed accuracy to predict the existence of bacteria biofilm at a given sensor location. The initial results were validated using optical images with fluorescent staining.