Browsing by Author "Hamilton, Karyn, committee member"
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Item Open Access A comprehensive approach to modeling musculoskeletal aging and injury: an emphasis on Nrf2-related pathogenesis(Colorado State University. Libraries, 2021) Andrie, Kendra M., author; Santangelo, Kelly S., advisor; Hamilton, Karyn, committee member; Goodrich, Laurie, committee member; Podell, Brendan, committee member; Muñoz Gutiérrez, Juan, committee member; Miller, Benjamin, committee memberOsteoarthritis (OA) is a degenerative joint disease that affects over 730 million people globally, over 30 million Americans, and is the leading cause of disability in adults. The underlying pathogenesis is multifactorial and largely undetermined, with a variety of cellular pathways and risk factors contributing to disease onset and progression. The crux of this work is that downregulation in nuclear factor erythroid-2 related factor-2 (Nrf2)-signaling in musculoskeletal tissue serves as a central driver for persistent low-grade inflammation, dysregulation of redox homeostasis, mitochondrial dysfunction, and protein dyshomeostasis, all of which contribute to OA progression. To explore the role of this pathway in OA, we utilized the Hartley OA-prone guinea pig model, which develops naturally occurring idiopathic disease with pathology that mimics human disease. My global hypothesis is supported by preliminary data that demonstrates that aging Hartley guinea pig knee joint tissues have decreased expression of Nrf2 mRNA and protein, which coincides with disease onset and remains decreased throughout OA progression. We investigated the utility of a novel nutraceutical and Nrf2-activator in delaying both the onset and progression of idiopathic OA in this model. The ultimate goal of this work is to (1) identify key molecular pathways involved in the etiopathogenesis of OA, with a particular focus on the contribution of the Nrf2 pathway; (2) investigate the utility of a novel nutraceutical and Nrf2-activator in delaying the onset and/or progression of OA in the Hartley guinea pig, and (3) examine the effects of Nrf2-activation on long bone strength. The inclusion of a musculoskeletal condition beyond OA was also pursued; as such, the clinical and histologic manifestations of a novel rectus femoris myotendinous junction injury model was characterized in rats. Ultimately, this work seeks to advance the understanding of musculoskeletal aging and injury through the analysis of key structural and functional outcome measures to further develop appropriate therapeutic targets for disease prevention and treatment.Item Open Access Caffeine augments the lactate and interleukin-6 response to moderate-intensity exercise in males but not females(Colorado State University. Libraries, 2022) Abbotts, Kieran Shay Struebin, author; Bell, Christopher, advisor; Hamilton, Karyn, committee member; Melby, Christopher, committee memberThe release of interleukin (IL)-6 from contracting skeletal muscle is thought to contribute to some of the health benefits bestowed by exercise. This IL-6 response appears proportional to exercise volume. Unfortunately, high volumes of exercise are not feasible for all people. Caffeine augments the magnitude of increase in circulating concentration of IL-6 in response to high-intensity and long-duration exercise, in males. Caffeine is also known to increase circulating concentrations of lactate during exercise. One of the mechanisms thought to contribute to IL-6 release from exercising skeletal muscle is lactate production. We hypothesized that caffeine, ingested prior to moderate-intensity exercise, would lead to greater circulating concentrations of lactate and IL-6 in a study population comprising both males and females. 15 healthy adults (9 males and 6 females, aged 26±7 years, (mean ± SD)) completed 30-minutes of moderate-intensity cycle ergometer exercise, equivalent to the ventilatory threshold, after ingesting either caffeine (6 mg/kg) or placebo. Arterialized-venous blood was collected throughout each of the exercise sessions. Compared with placebo, caffeine increased end-exercise circulating concentrations of lactate (5.72±3.95 vs. 7.14±4.66 mmol/L, P<0.001) but not end-exercise IL-6 (1.84±0.97 vs. 2.37±1.04 pg/mL, P=0.139). However, when females were excluded from the analysis, caffeine augmented (P=0.04) the magnitude of increase of end-exercise IL-6 concentration (1.80±0.86 vs. 2.57±1.21 pg/mL); this effect was further exaggerated after 30-minutes of inactive recovery (3.81±2.32 vs. 5.06±3.22 pg/mL). Noteworthy, caffeine evoked greater end-exercise lactate concentrations in data sets containing only males (P=0.02) and only females (P=0.002) but did not influence the IL-6 response in females (P=0.94). Our preliminary data imply that in males unable/unwilling to perform high-intensity and/or long-duration exercise, caffeine may potentially enhance the IL-6 mediated health benefits of relatively short, moderate-intensity exercise.Item Open Access Comparison of an antioxidant source and antioxidant plus BCAA on athletic performance and post exercise recovery of horses(Colorado State University. Libraries, 2022) Kent, Emily, author; Hess, Tanja, advisor; Coleman, Stephen, committee member; Hamilton, Karyn, committee member; Bruemmer, Jason, committee memberAntioxidant supplementation has been shown to decrease post-exercise oxidative stress but can lead to decreased post-exercise muscle protein synthesis. The objective of this study was to compare the effects of the supplementation with a control feed with low antioxidant content (CON) to a high antioxidant feed (AO), versus a high antioxidant and branched-chain amino acid feed (BCAO) on post-exercise protein synthesis and oxidative stress. Our hypothesis is that supplementing AO with BCAA will reduce oxidative stress without hindering muscle protein synthesis. Eighteen mixed breed conditioned polo horses were assigned to one of the three treatments. All horses consumed the CON diet for 30 days and were then assessed using a lactate threshold test (LT). One hour later and following subsequential LT, horses were assigned to the experimental groups and given their treatments. Follow-up LTs were conducted on days 15 and 30 of supplementation. Blood was collected before, two and four hours after LT, and oxidative stress was assessed by determining glutathione peroxidase, superoxide dismutase and malondialdehyde concentrations by ELISA. Muscle biopsies were taken before and 4 hours after LT and analyzed for the expression of protein synthesis by RT-PCR. Results were analyzed in a mixed model by ANOVA and compared by LSM. A reduction of oxidative stress was found over time (P<0.050) with no treatment effect (P>0.050) when using the measured oxidative stress parameters mentioned above. An upregulation in the production of mRNA transcripts related to muscle protein synthesis after exercise was found for muscle primers CD36, CPT1, PDK4, MyF5, and Myogenin (P<0.050). There was a treatment by exercise effect for MyoD (P=0.0041), where AO was upregulated the most after exercise compared to BCAO and CON. MRF4 had a time by treatment effect (p=0.045) where AO was upregulated from day 0 to day 15 and 30 compared to BCAO and CON. This study demonstrated post-exercise muscle synthesis with no advantage of AO plus BCAA compared to AO.Item Open Access Evaluation of acupuncture as an alternative therapy in a rodent model of spontaneous osteoarthritis(Colorado State University. Libraries, 2020) Personett, Alexa R., author; Santangelo, Kelly, advisor; Kendall, Lon, committee member; Hamilton, Karyn, committee memberOsteoarthritis (OA) is a degenerative joint disease affecting nearly 250 million people globally. With clinical signs of severe and persistent joint pain, OA is a leading cause of physical disability throughout the world. When faced with the frustration of chronic discomfort and restricted mobility due to OA, many individuals have turned to acupuncture as an alternative therapy. Acupuncture is a traditional Chinese practice of medicine for pain alleviation that involves insertion of thin needles into the skin and underlying tissue. The needles may be manipulated via manual or electrical stimulation, referred to as manual acupuncture and electroacupuncture, respectively. However, the efficacy of acupuncture in managing OA pain is uncertain, as much of the evidence is of questionable quality. The overall goal of this project was to evaluate acupuncture in a rodent model of human OA such that unbiased conclusions regarding its effectiveness for symptom modification could be drawn. Unfortunately, the majority of laboratory models of OA are artificially induced via chemicals or surgery and may not adequately represent the spontaneous disease process that occurs in humans. In contrast, the Dunkin Hartley guinea pig is a natural disease model, with primary OA pathology that mirrors human disease. As the major symptoms of OA are painful and decreased mobility, we were interested in evaluating the effect of acupuncture on a variety of mobility parameters using treadmill-based gait analysis and open-field enclosure monitoring. Additionally, as OA is an inflammatory disorder, we were interested in evaluating the effect of acupuncture on systemic inflammation, as well as any potential effects on normal physiology. This is commonly done in veterinary species with minimally invasive blood tests, such as complete blood counts and serum biochemistries. Despite their widespread use in biomedical research, there are few published studies investigating normal reference ranges for these diagnostics in the Dunkin Hartley guinea pig. Therefore, the goal of the first study was to develop hematology and serum biochemistry reference intervals for this strain. Data from complete blood counts and serum biochemistries were compiled from control Dunkin Hartley guinea pigs from previous studies to establish reference ranges for this model. Data were stratified by sex to determine specific reference intervals for males and females, and significant differences in parameters were investigated based on age and sex. The results of this study provide a foundation for interpreting these common diagnostic and laboratory blood tests in the Dunkin Hartley strain. The second and third studies evaluated electroacupuncture and manual acupuncture for the treatment of OA utilizing the Dunkin Hartley guinea pig model. Results of these studies demonstrated that electroacupuncture improved mobility, but not structural changes, in the knee joint. Conversely, manual acupuncture did not improve mobility parameters, but trended toward a decrease in knee joint histology scores compared to control groups. The results of these studies provide evidence that short-term treatment with electroacupuncture, but not manual acupuncture, is effective for symptom modification in this animal model of OA. Long-term studies are still needed to determine mechanisms for disease modification with these modalities.Item Open Access Exercise training improves exercise capacity despite persistent muscle mitochondrial dysfunction in the taz shRNA mouse model of human Barth Syndrome(Colorado State University. Libraries, 2013) Claiborne, Michael Scott, author; Chicco, Adam J., advisor; Hamilton, Karyn, committee member; Miller, Benjamin, committee member; Tamkun, Michael, committee memberBarth Syndrome is a mitochondrial disease associated with exercise intolerance and cardioskeletal myopathy resulting from mutations in the tafazzin (taz) gene. The present study characterized skeletal muscle mitochondrial function and exercise capacity of a taz shRNA mouse model of Barth Syndrome (90% taz-deficient), and examined the effect of exercise training on these parameters. Mitochondrial respiratory function was assessed, in mitochondria freshly isolated from hindlimb muscles, using an Oroboros O2K respirometer with pyruvate + malate as substrates, oligomycin as an ATP synthase inhibitor, and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) to establish maximal activity. A pre-training GXT revealed profound exercise intolerance, which corresponded to reduced respiratory capacity, citrate synthase (CS) and ETC complex 1 protein content of muscle mitochondria in the taz vs. age-matched wild-type (WT) mice. Based on the pre-training GXT, exercise training was conducted at 12-17 m/min, 0% grade for 60 min/d, 5d/wk, for 4 wks. Exercise training elicited a 99% increase in GXT run time in the taz mice (P < 0.01 vs. pre-training), but failed to increase times to those of sedentary WT mice. Training significantly decreased state 3 respiratory capacity of muscle mitochondria from exercised mice (wild type sedentary (WTS): 4992.59 ± 371.35, wild type exercised (WTX): 3779.60 ± 561.43, taz sedentary (TazS): 2978.50 ± 383.53, TazS: 1827.55 ± 525.17 (pmolO2/(s*mg), P = 0.02, Sed. vs. Ex.), and significantly decreased mitochondrial CS activity in taz mice (WTS: 4.48 ± 0.51, WTX: 3.87 ± 0.69, TazS: 3.21 ± 0.54, taz exercised (TazX): 1.63 ± 0.69 (relative absorbance/gram of protein) (RU/g), P = 0.01). Training also tended to reduce mitochondrial lactate dehydrogenase (LDH) and monocarboxylate transporter 1 (MCT1) activities, MnSOD content, and 4-hydroxnonenal-protein adducts (index of oxidative stress), but tended to increase mitochondrial UCP3 in exercised WT and taz mice. Interestingly, training significantly increased muscle levels of CS (WTS: 1.491 ± 0.112, WTX: 1.792 ± 0.143, TazS: 1.325 ± 0.108, TazX: 1.550 ± 0.143 (RU/g), P = 0.05 Sed. v. Ex.), suggesting increased muscle mitochondrial content with training. This study indicates that exercise training improves functional capacity of taz deficient mice and induces selective mitochondrial protein remodeling during mitochondrial biogenesis that perhaps mitigates oxidative stress while adapting to increased metabolic demand.Item Open Access Identifying novel molecular mechanisms of healthspan using multi-omics(Colorado State University. Libraries, 2023) Smith, Meghan Elizabeth, author; LaRocca, Tom, advisor; Hamilton, Karyn, committee member; Broussard, Josiane, committee member; Ehrhart, Nicole, committee memberAn important goal in research on aging is to extend healthspan, the period of life spent healthy and disease-free. Next-generation sequencing and other emerging bioinformatics technologies (e.g., RNA-seq/transcriptomics, epigenetic profiling, and proteomics) have made it possible to broadly profile potential molecular mediators of aging, and perhaps identify therapeutic targets. The studies in this dissertation focus on using transcriptomics and complementary "multi-omics" strategies to characterize novel cellular mechanisms of aging, and to determine their relevance to systemic/functional health in humans. With the guidance of my mentoring team, I completed three studies in which I identified novel mediators of healthspan-related exercise training responsiveness, age-related inflammation, and cognitive/motor function decline in middle-aged and older adults. One particularly novel focus among these studies was the role of non-coding repetitive RNAs (derived from transposable elements) in healthspan. Transposable elements have been linked to known mechanisms of aging, and this topic is reviewed at the start of this dissertation to provide perspective on their role in the context of research on aging biology. Collectively, my findings represent new ideas for targetable genes and proteins that may influence human healthspan.Item Open Access Impact of timing of milk intake on nitrogen balance in hypocaloric exercising older individuals(Colorado State University. Libraries, 2011) Heusinger, Daniel, author; Miller, Benjamin, advisor; Melby, Christopher, advisor; Hamilton, Karyn, committee member; Melanson, Ed, committee member; Hickey, Matthew, committee memberWe have previously shown that in older adults, consumption of protein immediately after aerobic exercise, rather than earlier in the day, enhances nitrogen balance when energy balance is maintained. Since some older individuals consume lower calorie diets, it is important to know if these benefits also occur during hypocaloric feeding. The purpose of the study was to investigate if consumption of protein immediately after aerobic exercise rather than earlier in the day can improve nitrogen balance in older individuals consuming a hypocaloric diet. In a randomized crossover design, healthy sedentary male (n=2; age=67.0±1.0 years; BMI=27.4±0.3 kg/m²) and female (n=8; age=63.0±1.8 years; BMI=22.3±0.6 kg/m²) subjects completed two separate 3-day exercise and nutrition interventions. Exercise (60 minutes of stationary cycling at 55% of VO2max) was performed daily. Diets were hypocaloric (-15% daily intake), with a protein+carbohydrate (PRO+CHO) or carbohydrate only (CHO) drink consumed in the morning and the opposite drink consumed after exercise. Both diets (15% protein, 30% fat, and 55% carbohydrate) were isonitrogenous and isocaloric with only the timing of the drinks differing. A 24-hour stay in a metabolic chamber confirmed negative energy balance, while 24-hour urine collections determined nitrogen balance. The 3-day mean nitrogen balance was not significantly greater in the PRO+CHO trial (0.097±0.526g N) trial than the CHO trial (-0.070 ±0.520g N) (p=0.280). Thus, older individuals in negative energy balance do not maintain a significantly more positive nitrogen balance by consuming protein after aerobic exercise as opposed to earlier in the day. These results differ from our previous work and indicate that energy balance is an important determinant of the anabolic effect of protein feeding.Item Open Access Impact of timing of protein intake on nitrogen balance in exercising older individuals on a hypercaloric diet(Colorado State University. Libraries, 2011) Minor, Brian, author; Miller, Benjamin, advisor; Hamilton, Karyn, committee member; Melanson, Ed, committee member; Hickey, Matthew, committee member; Melby, Christopher, committee memberWe have previously shown that in older adults, consumption of protein in the form of chocolate milk immediately after exercise enhances nitrogen balance (NBAL) when energy balance is maintained. Since it is known that hypercaloric diets increase nitrogen (N) retention, it is important to know if the timing of protein intake after aerobic exercise provides further increases in N retention compared to the consumption of carbohydrate only post exercise. PURPOSE: To investigate if consumption of protein and carbohydrate (PRO + CHO) immediately after exercise as opposed to earlier in the day can improve NBAL in older individuals consuming a hypercaloric diet. METHODS: In a randomized cross-over design, subjects completed two separate 3-day exercise and nutrition interventions. Exercise (60 minutes of stationary cycling at 55% of VO2max) was performed daily at 4:30 PM. Diets were hypercaloric (calculated at +15% daily intake), with a PRO+CHO or carbohydrate only (CHO) drink consumed at 10 am and the opposite drink consumed after exercise (5:30 PM). Both diets (1.2 g protein/kg bodyweight, 30% fat, and balance as carbohydrate) were isonitrogenous and isocaloric with only the timing of the drinks differing. A 24 hour stay in a metabolic chamber confirmed positive energy balance while 24-hour urine collections determined NBAL. RESULTS: The 3-day mean NBAL was not significantly different (p=.0881) (n=6) between the CHO trial (.970 ± .517 g N) and the PRO + CHO trial (1.659 ± .430 g N) although a trend toward increased NBAL with PRO+CHO was apparent. The mean energy balance was not significantly different (p=.2906) between the CHO trial (+13.09 ± 1.94%) and the PRO + CHO trial (+ 14.28 ± 1.75%). Further analyses comparing the positive energy balance cohort to previously completed negative, and even energy balance cohorts distinguished the role of energy balance and timing of nutrition effects. CONCLUSION: Older individuals in positive energy balance do not maintain a significantly more positive NBAL balance by consuming protein after aerobic exercise as opposed to earlier in the day although energy balance does change the effect of protein timing on NBAL.Item Open Access Investigation of molecular effects of the soy-derived phytoestrogen genistein on cardiomyocytes by proteomic analysis(Colorado State University. Libraries, 2011) Sun, Zeyu, author; Reardon, Kenneth, advisor; Hamilton, Karyn, committee member; Orton, Christopher, committee member; Reisfeld, Bradley, committee memberThe soy-derived phytoestrogen genistein (GEN) has received attention for its potential to benefit the cardiovascular system by providing protection to cardiomyocytes against pathophysiological stresses. Although GEN is a well-known estrogen receptor (ER) agonist and a non-specific tyrosine kinase inhibitor, current understanding of the complex cellular and molecular effects of GEN in cardiomyocytes is still incomplete. The overall goal of this dissertation is to use high throughput proteomics methodologies to better understand the molecular action of GEN in cardiomyocytes and to identify proteins and pathways that respond to GEN treatment. The first study of this project focused on the concentration-dependent proteome changes in cultured HL-1 cardiomyocytes due to GEN treatments. Proteins from HL-1 cardiomyocytes treated with 1 μM and 50 μM GEN were prefractionated into hydrophilic and hydrophobic protein fractions and were analyzed by two-dimensional electrophoresis followed by protein identification using tandem mass spectrometry (MS). In total, 25 and 62 differential expressed proteins were identified in response to 1 μM and 50 μM of GEN treatment, respectively. These results suggest that 1 μM GEN enhanced the expression of heat shock proteins and anti-apoptotic proteins, while 50 μM GEN down-regulated glycolytic and antioxidant enzymes, potentially making cardiomyocytes more susceptible to energy depletion and apoptosis. The second study, employing a two-dimensional liquid chromatography and tandem MS shotgun proteomics workflow, was carried out to dissect the cellular functions changed in cardiomyocytes by ER-dependent or ER-independent actions of GEN. In this study, primary cardiomyocytes isolated from male adult SD rats were treated with 10 μM GEN without or with 10 μM ER antagonist ICI 182,780 (ERA) before proteomics comparison. A total of 14 and 15 proteins were found differentially expressed in response to the GEN, and the GEN+ERA treatment, respectively. Cellular functions such as glucose and fatty acid metabolism and cardioprotection were found to be modulated by GEN in an ER-dependent fashion, while proteins involved with steroidogenesis and estrogen signaling were identified as novel effectors of GEN via ER-independent actions. In this study, a consensus-iterative searching strategy was also developed to enhance the sensitivity of the shotgun proteomic approach. In the last study, an attempt to explore the response to a GEN stimulus in the signaling pathways, we developed a phosphopeptide enrichment method to assist the detection of protein phosphorylation in a complex peptide mixture. The quantitative performance of a sequential immobilized metal affinity chromatography (SIMAC) protocol was evaluated. We further conducted a preliminary application of this protocol in a large-scale, quantitative, label-free phosphoproteomics study to explore the alterations of protein phosphorylation patterns due to ER-independent GEN action in the SD rat cardiomyocytes. This project demonstrates the usefulness of proteomics methodologies to screen novel molecular targets influenced by GEN in cardiomyocytes. This is also the first investigation of the complex cellular impact of this soy-derived phytoestrogen in cardiomyocytes via a systems biology perspective.Item Open Access Investigations of telomeres and telomerase following ionizing radiation exposure(Colorado State University. Libraries, 2014) Sishc, Brock James, author; Bailey, Susan, advisor; Bedford, Joel, committee member; Hamilton, Karyn, committee member; Liber, Howard, committee member; Miller, Benjamin, committee memberTelomeres are critical structures located at the termini of eukaryotic chromosomes that regulate the replicative lifespan of human cells. Telomeres shorten with cell division, a process that eventually leads to telomere based growth arrest and cellular senescence. Telomere length is maintained through the activity of the reverse transcriptase, telomerase that functions in embryonic and adult stem cells to elongate telomeres and prolong replicative lifespan. Telomerase is repressed in the vast majority of human somatic cells, and its reactivation is a critical early step in carcinogenesis. Thus, telomerase and telomere maintenance are critical factors in the processes of carcinogenesis, tumor maintenance, and tumor recurrence following interventional therapy. Ionizing radiation (IR) has long been acknowledged as both a potent carcinogen and an effective agent in the treatment of cancer. To investigate the role telomeres and telomerase play in the cellular response to IR exposure, we tracked telomerase activity and telomere length in a panel of cancer and immortalized non-cancer cell lines following both acute and low dose rate (LDR) exposures to γ-rays. We observed elevations of telomerase activity in cancer, but not non-cancer, cell lines following acute exposures to IR. Further, telomere length was significantly reduced in both cancer and non-cancer cells post-acute IR exposure. Taken together, these studies suggest telomerase activity is playing a role in accelerated tumor repopulation following radiation therapy and that the associated telomere loss may be contributing to genomic instability. As IR induced enrichment of cancer stem cells (CSC) in established cancer cell lines was recently suggested to play a role in accelerated tumor repopulation following radiation therapy, we investigated a potential role for telomerase in the IR induced enrichment of CSCs. Consistent with previous reports, we detected a significant enrichment of putative breast CSCs in MCF-7 mammary carcinoma cells at 5 days post exposure, and demonstrate significant enrichment of putative CSCs in the non-tumorigenic MCF-10a, WTK1, and LCL15044 cell lines. Further small molecule inhibition of telomerase activity was able to effectively block CSC enrichment in both MCF-7 and MCF-10a cells. Together, these results suggest that telomerase inhibition is a significant player in the IR induced enrichment of putative CSCs in both cancer and non-cancer mammary epithelial cells. Further, this process seems to be driven by non-canonical roles of telomerase.Item Open Access New evidence for age differences, within-person declines and plasticity in the aging white matter: new MRI techniques and analytical approaches(Colorado State University. Libraries, 2023) Mendez Colmenares, Andrea, author; Thomas, Michael L., advisor; Burzynska, Agnieszka Z., advisor; Davalos, Deana, committee member; Fisher, Gwen, committee member; Hamilton, Karyn, committee memberWhite matter deterioration leads to cognitive impairments in healthy aging, Alzheimer's disease, and related dementias. Therefore, it is critical to identify interventions that can slow the white matter deterioration. Animal studies have suggested that the white matter plays an active role in brain plasticity and learning. However, evidence for experience-induced plasticity in adult human white matter remains scarce and inconsistent, especially in older age. To accurately predict the effects of interventions on the white matter, we first need to understand the direction and magnitude of naturally occurring within-person changes across adulthood. To date, white matter in aging, Alzheimer's disease, and related dementias have been studied almost solely using diffusion MRI, which provides limited information about the white matter microstructure. Because there is little evidence of white matter plasticity in adult humans, white matter has rarely been considered as a target for interventions against dementia. This dissertation comprises three scientific articles investigating the mechanisms of white matter decline and plasticity. The first article presents a study using a novel technique (T1w/T2w imaging) to examine the effects of aerobic exercise on aging white matter in a randomized controlled trial. The second article is a meta-analysis and systematic review of within-person changes in white matter. The third article shows the first application of a multimodal fusion analysis to study healthy aging white matter. Through these innovative approaches, this dissertation provides new insights into the mechanisms of white matter decline and plasticity, paving the way for the development of effective interventions to promote healthy brain aging.Item Open Access Novel transcriptomic mechanisms of brain aging(Colorado State University. Libraries, 2023) Cavalier, Alyssa Nicole, author; LaRocca, Thomas, advisor; Lark, Daniel, committee member; Hamilton, Karyn, committee member; Weir, Tiffany, committee memberAs the world ages, the incidence of age-related diseases like dementia is expected to increase. Brain aging is characterized by declines in cognitive function that may develop into mild cognitive impairment, which increases the risk for dementia. In fact, age is the primary risk factor for late-onset Alzheimer's disease, which is the most common age-related dementia. The adverse cellular and molecular processes that underlie cognitive decline with aging in the brain are known collectively as the "hallmarks of brain aging." Advances in next-generation sequencing (e.g., transcriptomics/RNA-seq) have made it possible to investigate age- and disease-related changes in the brain at the broad gene expression level, and to identify potential therapeutic targets. With the support of my committee and mentoring team, I completed three studies using transcriptomics that characterize novel mechanisms that underlie brain aging. My findings include: (1) doxorubicin chemotherapy accelerates brain aging at the gene expression level, (2) apigenin nutraceutical supplementation targets age-related inflammation in the brain and rescues cognitive impairment in old mice, and (3) epigenetic dysregulation of transposable elements (remnants of viral infection in the genome) with aging contributes to age-related inflammation in Alzheimer's disease. Together, my work provides insight into transcripts and cellular/molecular pathways that are modifiable and may be therapeutic targets to delay or prevent consequences of brain aging.Item Open Access Oncofetal proteins regulate proliferation and differentiation in placental cells(Colorado State University. Libraries, 2018) West, Rachel Claire, author; Winger, Quinton, advisor; Bouma, Gerrit, advisor; Anthony, Russ, committee member; Hamilton, Karyn, committee memberThe chromatin associated transcription factor HMGA2 is a downstream target of let-7 miRNAs and binds to chromatin to regulate gene expression, inducing rapid cell proliferation during embryogenesis. Inhibition of let-7 miRNAs by RNA binding proteins LIN28A and LIN28B is necessary during early embryogenesis to ensure stable expression of HMGA2 and proper cell proliferation. In addition to LIN28, HMGA2 is regulated by a BRCA1/ZNF350/CtIP repressor complex. In normal tissues, the BRCA1/ZNF350/CtIP complex binds to the HMGA2 promoter to prevent transcription. However, in many cancers the oncomiR miR-182 targets BRCA1, preventing BRCA1 translation and allowing for increased HMGA2. Little is known about the regulation of HMGA2 during early placental development therefore we hypothesized that both LIN28 and BRCA1 can regulate HMGA2 in placental cells. Using siRNA and CRISPR gene editing techniques, we found that knockdowns of both LIN28A and LIN28B increase HMGA2 levels in ACH-3P cells. These cells also demonstrated deficiencies in cell differentiation towards the syncytiotrophoblast, secreting higher amounts of hCG and displaying upregulated ERVW-1. Additionally, we found that a knockout of both LIN28A and LIN28B caused a significant increase of miR-182 and a decrease in BRCA1 which allows HMGA2 mRNA levels to increase and protein levels to remain the same. Using chromatin immunoprecipitation, we saw binding of the BRCA1 repressor complex to HMGA2. We also saw a decrease in binding to HMGA2's promoter in the LIN28A/B knockout cells. These findings suggest a novel role for BRCA1 during early human placental development. To test this hypothesis, we used CRISPR-Cas9 gene editing to knockout BRCA1 in the Swan71 cell line as the Swan71 cells had significantly higher BRCA1 levels compared to ACH-3P cells. HMGA2 mRNA and protein was significantly increased in the BRCA1 KO cells compared to control cells. Chromatin immunoprecipitation was used with an antibody for ZNF350 and PCR was run using primers for the promoter region for HMGA2. We saw a loss of BRCA1 repressor complex binding to HMGA2 in the knockout cells compared to our control cells, leading us to conclude that increased HMGA2 was due to decreased binding of the BRCA1 repressor complex. Additionally, we tested levels of apoptosis in our cells. After serum starving cells for 16 hours, we found that Caspase 3 and 7 levels were significantly higher in our BRCA1 KO cells compared to controls. This data suggests that BRCA1 is an important factor in the regulation of the oncofetal protein HMGA2 and promotes cell survival in human placental cells.Item Open Access Pharmacokinetic investigation of commercially available edible marijuana products in humans: potential influence of body composition and influence on glucose control(Colorado State University. Libraries, 2021) Ewell, Taylor, author; Bell, Christopher, advisor; Hickey, Matthew, committee member; Hamilton, Karyn, committee member; Weir, Tiffany, committee memberOur investigation of five commercially available edible marijuana products containing 10mg of delta-9-tetrahydrocannabinol (THC) aimed to describe the pharmacokinetics of these products, investigate the potential influence of body composition on THC bioavailability, and, based on epidemiological research completed in the last decade, determine if acute marijuana ingestion influences glucose tolerance when compared to a THC-free gummy. We studied seven regular marijuana users. We utilized a single-blind randomized controlled crossover study design in which participants self-administered edible marijuana or a THC-free gummy. Thirty minutes following marijuana ingestion a standard oral glucose tolerance test was initiated via consumption of a 75g glucose drink. There was, at minimum, a four-day washout period between trials. Average time to peak plasma THC concentration ranged from 35 to 90 minutes, and average peak THC concentrations ranged from 3.2 to 5.5 ng/ml. Significant differences between products were identified twenty- and thirty-minutes post-ingestion. Several measures of body composition had significant correlations with plasma THC, although none of these correlations persisted across all products. There were no differences in indices of glycemic control between marijuana products or the THC-free gummy. Following acute edible marijuana ingestion in habitual users, significant differences in THC pharmacokinetics existed between similar products, possibly due to body composition, although glucose control was not impacted. In summary, these data may inform recreational users to the proper dose for marijuana ingestion to achieve the desired outcome and to avoid overdose.Item Open Access Temporal examination of myoglobin and myosin heavy chain expression patterns in vitro(Colorado State University. Libraries, 2020) Larson, Ashley Mari, author; Kanatous, Shane, advisor; Hamilton, Karyn, committee member; Mykles, Donald, committee memberMyoglobin is a hemoprotein expressed in vertebrate muscle that is typically known to follow an established oxidative muscle fiber type, found in aerobic muscles. However, more recent evidence has demonstrated changes in myoglobin expression without a change in fiber type, indicating myoglobin expression could be regulated by different pathways and may not always be dependent on a prior expressed fiber type. Myoglobin structure is characterized by a globin backbone that supports a nonprotein heme prosthetic group containing iron, which is responsible for the reversible binding of several ligands such as oxygen, nitric oxide, and carbon monoxide. Naturally, it has been implicated in oxygen transport and storage, nitric oxide and reactive oxygen species scavenging, and cellular lipid transport. Mixed lipid supplementation alone and coupled with hypoxia elevates skeletal muscle myoglobin levels, but it is unknown how these culture treatments affect myoglobin expression relative to the fiber type. Given the uncertainty of when myoglobin is expressed independently from oxidative fiber types, we aimed to determine when differentiating C2C12 cells begin to express myoglobin compared to when they express oxidative isoforms of myosin heavy chain when subjected to factors known to increase myoglobin expression; hypoxia, lipid and/or caffeine treatments. We found that under control and hypoxic conditions, regardless of lipid supplementation, myoglobin expression occurred before oxidative fiber expression. Conversely, cells receiving caffeine stimulation expressed myoglobin following oxidative fiber type expression. Cells exposed to hypoxia and lipid supplementation displayed elevated functional myoglobin expression compared to caffeine stimulated cells, suggesting that this combination of treatments may be more effective at increasing myoglobin than stimulation alone. Overall, the work presented here has determined conditions under which expression of myoglobin precedes oxidative fiber type expression and within these treatments, conditions that also increase functional myoglobin concentration. These findings can act as a step in the process to assist in revealing more about how myoglobin can be expressed in skeletal muscle. Examination of alternate routes of myoglobin expression that are not reliant on prior expression of a particular fiber type could yield potential therapeutic benefits of expressing myoglobin in tissues to combat ischemic diseases seen in humans.Item Open Access The effect of lipid and oxygen concentration on myosin heavy chain isoform expression in c2c12 and cultured Weddell seal (Leptonychotes weddellii) skeletal muscle cells(Colorado State University. Libraries, 2014) Garcia, Teresa Maria, author; Kanatous, Shane, advisor; Hamilton, Karyn, committee member; Mykles, Don, committee memberThe Weddell seal (Leptonychotes weddellii), a diving mammal, has large stores of myoglobin as compared to terrestrial mammals. Understanding the regulation of myoglobin in this unique species has therapeutic potential in people at risk of muscle infarction; to pursue this end, we established a successful protocol for the isolation and culture of primary Weddell seal skeletal muscle cells utilizing lipid-supplemented culture media. Using these cells, a study was designed to determine if increasing lipid in the media in either normoxic or hypoxic conditions would affect the expression of myosin heavy chain (MHC), which might explain a previously observed increase in myoglobin expression with increased lipid. Gel electrophoresis consistently resolved two putative MHC isoforms from seal and c2c12 cells. LC-MS/MS determined that the resolved bands were heterogeneous, and that all bands contained minimal adult MHC (10% or less, as measured by NSAF). Despite the minimal expression of adult MHC isoforms, myoglobin was detected in each treatment group. This study suggests that electrophoresis methods alone may not be sufficient to detect changes in MHC isoform abundance in cell culture, and that, in cell culture, regulatory pathways may exist for myoglobin that are independent of adult fiber type.Item Open Access Vitamin C supplementation: a comparison of delivery methods and the ability to attenuate oxidative stress induced by ischemia-reperfusion(Colorado State University. Libraries, 2015) Davis, Janelle Lynn, author; Bell, Christopher, advisor; Hamilton, Karyn, committee member; Frye, Melinda, committee memberIntravenous delivery of vitamin C to adult humans decreases indices of oxidative stress and in some instances improves physiological function. Oral delivery of vitamin C is more practical than intravenous but typically results in lower circulating vitamin C concentrations. The hypotheses for this study were, oral consumption of vitamin C encapsulated in liposomes would: 1) result in higher circulating vitamin C concentrations than a traditional oral supplement, and 2) better attenuate oxidative stress induced by ischemia-reperfusion. Eleven overweight/obese adults [age: 52±7 years; body mass index: 34.1±1.0 kg/m²; mean±SE] were administered a 4 g supplement of placebo, or vitamin C via different delivery methods, on four separate occasions, in a random order. The four treatments were: placebo, oral vitamin C, liposomal vitamin C, and intravenous (IV) administration of vitamin C. Concentrations of ascorbic acid, thiobarbituric acid reactive substances (TBARS), and oxidized low-density lipoproteins (Ox-LDL) were measured in venous blood at baseline, and over four hours following supplement administration. At three hours a blood pressure cuff was placed around the upper arm and inflated to 200 mmHg for 20 minutes to evoke an ischemia-reperfusion injury. Plasma ascorbic acid concentrations were significantly greater after IV vitamin C compared with all other treatments at all time points (P<0.01). At two hours, all subsequent ascorbic acid concentrations were greater after liposomal vitamin C treatment compared with oral vitamin C and placebo treatments. Plasma ascorbic acid concentrations were greater after oral vitamin C compared with placebo (P<0.01). Neither vitamin C nor ischemia-reperfusion influenced Ox-LDL. In the placebo condition, ischemia-reperfusion increased plasma TBARS concentration; all of the vitamin C treatments prevented this increase. These data suggest that liposomal encapsulation of vitamin C increases bioavailability of oral vitamin C. Additionally, the antioxidant protection provided by liposomal vitamin C is not inferior to intravenously administered vitamin C.