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Optimization of muscle progenitor cell isolation techniques for production of cultivated meatSteele, Alexandra P January 2023 (has links)
Traditional meat production has major sustainability and ethical concerns. Cultivated meat helps to address these concerns by reducing the need for mass animal farming. Muscle progenitor cells (MPCs) harvested from skeletal muscle are a promising cell source for cultivated meat. While various protocols have been developed for MPC isolation, which protocol is best suited for the cultivated meat industry requires further investigation. Therefore, the purpose of this thesis was to optimize the MPC isolation technique to produce a pure myogenic cell population and provide the cultivated meat industry with standardized procedures for production. For these proof-of-concept experiments, skeletal muscles harvested from the hindlimb muscles of mice were used. Cells were isolated from the harvested muscle then subjected to one of three protocols for MPC enrichment: pre-plating, ice-cold treatment (ICT), or fluorescence activated cell sorting (FACS). The pre-plating and ICT protocols resulted in impure cell populations with few MPCs after one week in culture. Therefore, FACS using two cell-surface markers, NCAM and CD34, was employed as a more specific method for MPC sorting. CD34+NCAM1- cells grew quickly, however, unwanted cell types remained following FACS. In contrast, CD34+NCAM1+ cells had a consistent small, rounded shape and slow proliferation rate. These cells remained viable in culture for several months and had high Pax7 expression, indicating they were a pure population of myogenic cells. CD34+NCAM1+ cells maintained their capacity to differentiate after culturing for an extended period, demonstrating their potential use for cultivated meat production. The results of this study provide a better understanding of the differences between previously published MPC isolation techniques. Future studies will investigate the potential for CD34+NCAM1+ cells to be grown on a larger scale. These experiments provide insight into MPC populations that may exist in livestock species and will help to streamline the early stages of cultivated meat production. / Thesis / Master of Science (MSc) / Traditional meat production is associated with numerous challenges including animal welfare concerns, human health concerns, and harmful environmental consequences. The global population is predicted to reach 9.7 billion by 2050, emphasizing the importance of alternative food sources to meet the increased food demand. Cultivated meat is a promising new protein source, with the intended purpose of providing a sustainable food source with reduced ethical concerns compared to conventional meat. While there are several challenges to overcome throughout the production process, a major consideration in the early stages of cultivated meat production is cell sourcing. Muscle cells harvested from a tissue biopsy are one proposed starting cell source which has the potential to make up most of the tissue in cultivated meat products. This thesis aimed to improve upon previously published protocols used for muscle cell isolation and provide an optimized cell population for use in cultivated meat production. The cell sorting protocol described in this thesis provides a highly efficient technique for muscle cell purification and long-term growth. The resulting cell population has many characteristics that are pertinent to cultivated meat and may advance the early stages of production.
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The effects of hyaluronic acid and exercise on equine skeletal muscleGregg, Savannah Renee 18 August 2023 (has links)
Unaccustomed, strenuous exercise can cause skeletal muscle damage that subsequently induces an acute inflammatory response in the tissue which is marked by an infiltration of leukocytes into the damaged muscle. To try and suppress the initial pro-inflammatory response in skeletal muscle of horses performing a single exercise stress test, a commercial sodium hyaluronate (HA) treatment was administered and tested for anti-inflammatory properties. Unfit, adult Thoroughbreds were intravenously injected three times with HA or received no injection at all (CON) over a 3-week period before performing a single submaximal exercise test. Gluteal muscle biopsies were collected before and 1 h after the completion of exercise for RNA-Seq and staining. The results indicated that HA treatment in horses down regulated genes associated with lymphocyte activation and cytokine production (Il17RA, OSCAR, LYL1, TLR1, TLR2, TLR8, TLR10) but did not irreversibly down regulate these genes with the addition of exercise. Exercise as a stressor did cause an acute inflammatory response in muscle which was seen through global expression of macrophage and neutrophil surface markers (NCF2, ELANE, CD168I). These results determine that HA treatment does act as an anti-inflammatory in equine skeletal muscle but does not possess prolonged effects with the initiation of inflammation. / Master of Science / Horses subjected to an unaccustomed increase in exercise intensity can experience damage and subsequent acute inflammation within the skeletal muscle tissue that may hinder the performance of the horse by causing muscle swelling and soreness. Hyaluronic acid treatment may suppress this exercise-induced inflammatory response by acting as an anti-inflammatory in the muscle. Adult Thoroughbred horses were injected intravenously with a commercial sodium hyaluronate treatment in the weeks prior to performing an exercise stress test. Muscle biopsy samples were obtained before and after the exercise stress test was performed. The results indicate that horses receiving the hyaluronic acid treatment had decreased expression of inflammatory genes within skeletal muscle, but no genes remained suppressed after the induction of inflammation through exercise. These results demonstrate that hyaluronic acid treatment does act as an anti-inflammatory in skeletal muscle tissue but does not have long-term suppressive effects when inflammation does occur.
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Cellular and Molecular Changes Following Skeletal Muscle Damage: A Role for NF-kB and Muscle Resident PericytesHyldahl, Robert H 01 September 2011 (has links)
Skeletal muscle is dynamic and actively regenerates following damage or altered functional demand. Regeneration is essential for the maintenance of muscle mass and, when dysregulated as a result of disease or aging, can lead to losses in functional capacity and increased mortality. Limited data exist on the molecular mechanisms that govern skeletal muscle regeneration in humans. Therefore, the overall objective of this dissertation was to characterize early molecular alterations in human skeletal muscle to strenuous exercise known to induce a muscle regenerative response. Thirty-five subjects completed 100 eccentric (muscle lengthening) contractions (EC) of the knee extensors with one leg and muscle biopsies were taken from both legs 3 h post-EC. The sample from the non-EC leg served as the control. A well-powered transcriptomic screen and network analysis using Ingenuity Pathway software was first conducted on mRNA from the biopsy samples. Network analysis identified the transcription factor NF-kappaB (NF-kB) as a key molecular element affected by EC. Conformational qRT-PCR confirmed alterations in genes associated with NF-kappaB. A transcription factor ELISA, using nuclear extracts from EC and control muscle samples showed a 1.6 fold increase in NF-kB DNA binding activity following EC. Immunohistochemical experiments then localized the majority of NF-kB positive nuclei to cells in the interstitium, which stained positive for markers of pericyte cells and not satellite cells. To ascertain the mechanistic significance of NF-kB activation following muscle damage, in vitro analyses were carried out using a novel primary pericyte/myoblast co-culture model. Primary pericyte/myoblast co-culture experiments demonstrated that pericytes, transfected with a DNA vector designed to drive NF-kB activation, enhanced proliferation and inhibited myogenic differentiation of co-cultured skeletal muscle myoblasts. Furthermore, reduced NF-kB activation led to enhanced myogenic potential of primary pericytes. Taken together, the data in this dissertation suggest that NF-kB dependent signaling in pericytes regulates myogenic differentiation in a cell- and non-cell autonomous manner and may affect the early regenerative response following muscle damage by inhibiting differentition and promoting proliferation of muscle satellite cells.
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Muscle Weakness in Persons with Multiple SclerosisChung, Linda Haiwon 01 September 2010 (has links)
Skeletal muscle weakness is a problem for people living with Multiple Sclerosis (MS). Alterations in the central nervous system may be the primary source of muscle weakness because of the pathophysiology of MS. However, changes in peripheral mediators of force production may also contribute to muscle weakness in persons with MS. The main objective of the dissertation was to systematically identify key neural (motor unit discharge rates, spasticity) and muscular (muscle size, contractile function) mechanisms of force production that may explain lower isometric strength and dynamic power in persons with MS compared with age-matched controls. The knee extensor muscles of the weaker leg were studied, because this muscle group is commonly affected by MS. We showed that persons with MS had lower peak isometric torque and dynamic power compared with controls. Persons with MS had lower motor unit discharge rates, smaller muscle size, and lower specific power compared with controls. There was no difference in passive torque (spasticity), specific strength, or maximal rate of force development between groups. Because differences in isometric strength between persons with MS and controls were abolished when torque was normalized to muscle size, smaller muscle size may explain a large portion of lower isometric strength in persons with MS. Differences in dynamic power were reduced when peak power was normalized to muscle size, but remained lower in persons with MS compared with controls, suggesting that changes in neural factors (e.g., lower motor unit discharge rates) may explain lower dynamic power in persons with MS. These results suggest that different mechanisms may contribute to muscle weakness in MS, depending on the mode of contraction. Lower motor unit discharge rates and smaller muscle size were identified as key mechanisms of muscle weakness in persons with MS. Each of these mechanisms has been shown to improve with resistance training in controls. Thus, this dissertation provides an evidence-based rationale for resistance training interventions in persons with MS, to improve isometric strength and power production by increasing motor unit discharge rates and muscle size.
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Interaction of Loading and Feeding on Skeletal Muscle Anabolic Signaling and Protein Turnover in HumansGlover, Elisa I. January 2009 (has links)
<p> Resistance exercise and amino acids independently and synergistically stimulate muscle protein synthesis. Unloading of skeletal muscle depresses fasted state muscle protein synthesis, but the effect on the fed state response is unknown. Elucidation of the signaling pathways underlying the regulation of these processes in humans is in its infancy. Therefore, the purpose of this thesis was to determine how resistance exercise, feeding, and unloading interact to affect muscle protein turnover and its markers. In study 1 young men (N=9) underwent an acute bout of unilateral leg resistance exercise with or without feeding, with biopsies 6 h post exercise. Exercise dephosphorylated eiF2Bε and together with feeding potentiated the increase in phosphorylation of p70s6k and rps6. In study 2, 12 young people received primed constant infusions of 13C6-Phe in the fasted state and at one of two i.v. AA infusion rates (low, 42.5 mg/kg/h AA; high: 261 mg/kg/h AA) after 14 d of knee-brace mediated immobilization. Immobilization decreased fasted and fed state myofibrillar protein synthesis at both doses without obviously affecting translational signaling proteins. In study 3, two markers of muscle protein breakdown and oxidative damage were measured in 21 subjects (men, N=13, women, N=8) after 2 d and 14 d of knee-brace mediated immobilization. Protein ubiquitination was elevated after 2 d of immobilization but there was no sustained elevation in ubiquination at 14 d or increases in the 14kDa actin fragment or protein carbonyls and 4-hydroxy-2-nonenal. These studies support the concept that the responses of human muscle to changes in loading are primarily at the level of protein synthesis, and the p70 pathway appears to play a role in mediating the hypertrophic response. The currently known static markers of translational signaling and protein breakdown, however, are not very informative when attempting to account for an underlying molecular mechanism for disuse atrophy. </p> / Thesis / Doctor of Philosophy (PhD)
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Angiotensin II regulation of skeletal muscle regeneration, growth and satellite cell functionJohnston, Adam 12 1900 (has links)
<p> Local renin-angiotensin systems (RASs) have been described in many mammalian tissues. However, the role of angiotensin II (Ang II) in skeletal muscle is poorly understood with initial reports suggesting it may function to regulate overload-induced hypertrophy. Therefore, the purpose of this thesis was to 1) investigate the potential that adult skeletal muscle and muscle stem cells possess a local RAS. 2) Describe its role in regulating skeletal muscle regeneration and growth following injury and 3) demonstrate its capacity to regulate muscle stem cell activity and myogenesis. We report that cultured primary and C2C12 myoblasts and myotubes possess a local Ang II signalling system evidenced by the differential expression of angiotensinogen, angiotensin converting enzyme (ACE), and both angiotensin type 1 and 2 (AT1, AT2) receptors. Interestingly, myoblasts demonstrated the capacity to produce Ang II in spite of lacking renin expression. Furthermore, angiotensin receptors demonstrated differential localization with AT1 associated with actin filaments in proliferating myoblasts, and localized to the nucleus in differentiated myotubes. We also report that a local angiotensin system is present in vivo and responsive to myotrauma as cardiotoxin injection (to induce muscle injury) resulted in the increased staining intensity of angiotensinogen and AT1 during myogenesis with a progressive downregulation throughout the regenerative timecourse. </p> <p> To investigate the effects of Ang II signalling blockade on muscle growth and regeneration we induced muscle injury in mice supplemented with captopril (ACE inhibitor) or mice devoid of the AT1 a receptor. Histological analysis
revealed that ACE inhibition resulted in a decreased muscle fibre growth,
increased proportion of small myofibres, an inability to accrete myonuclei and a robust hyperplasia of muscle fibres. Similarly, AT1 a receptor ablation resulted in decreased muscle fibre growth following injury suggesting that these effects are
receptor specific. </p> <p> To investigate the mechanisms underlying these effects we assessed the role of Ang II in regulating muscle satellite cell function. In vitro experiments revealed that Ang II had the ability to regulate the early response of satellite cells to muscle injury by acting as a potent transcriptional activator of quiescent myoblasts and directing their subsequent migration. Furthermore, these migratory effects were mediated through an Ang 11-induced increase in matrix metalloproteinase 2 (MMP2) content and reorganization of the actin cytoskeleton. Interestingly, Ang II may also participate in the fusion of myoblasts as captopril treatment suppressed the expression of markers of differentiation (myogenin) and maintained the expression of markers of proliferation (Pax7, Myf5). In agreement with this, IHC analysis revealed that ACE inhibition also induced a strong trend for a decrease in the proportion of myogenin positive cells following injury. Collectively, these results implicate the activation of local Ang II signalling system as a pleiotropic regulator of skeletal muscle growth. </p> / Thesis / Doctor of Philosophy (PhD)
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The Metabolic and Structural Response of Human Skeletal Muscle to Acute Exercise and Nutritional ManipulationCermak, Naomi M. 09 1900 (has links)
<p> The work in this thesis describes the metabolic and structural response of human skeletal muscle to acute exercise and nutritional manipulation. Over a series of three studies, healthy young men performed acute bouts of either endurance or resistance exercise, and a range of invasive and non-invasive techniques were applied to examine the muscle adaptive response during exercise and recovery. Study 1 investigated the hypothesis that co-ingestion of protein with carbohydrate during exercise would improve oxidative energy metabolism and attenuate ultrastructural disruption during prolonged 90 min of cycling at ~70% VO2peak. While protein ingestion increased blood amino acids, there was no difference between treatments in glycogen degradation or the content of TCA cycle intermediates during exercise, or the blood concentration of plasma creatine kinase (CK) after 24 h of recovery. Given the limitations associated with traditional indirect markers of muscle injury, study 2 examined the potential for a non-invasive imaging technique, diffusion tensor magnetic resonance imaging (DT-MRI), to detect exercise-induced changes in skeletal muscle structure. Subjects performed 300 eccentric actions of the leg extensors, a protocol previously shown to induce histological evidence of muscle disruption. DT-MRI revealed changes consistent with muscle disorganization 24 h post-exercise compared to baseline, including decreased fractional anisotropy (FA) and increased tensor eigenvalue λ3. The exercise protocol also induced changes in traditional direct and indirect markers of muscle injury, including Z-band streaming, increased blood CK and a decrease in force-generating capacity. Study 3 examined the potential for DT-MRI to detect structural changes in response to an acute bout of work, previously shown to induce muscle damage that more closely simulated normal endurance exercise. Subjects performed 45 min of downhill running (-10° grade) and DT-MRI revealed increased ADC and tensor eigenvalue λ3 24 h post-exercise compared to baseline, in addition to increased plasma CK and decreased force-generating capacity. The main finding from the thesis is the application of DT-MRI to non-invasively detect exercise-induced changes in skeletal muscle structure as verified using well understood direct and indirect measures of muscle damage.</p> / Thesis / Doctor of Philosophy (PhD)
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Analyses of the effects of 17β-estradiol on skeletal muscle and global gene expression following acute eccentric exerciseMacNeil, Lauren January 2010 (has links)
<p> Introduction: 17β-estradiol (E2) has proposed anti-oxidant and membrane stabilizing properties that may attenuate exercise-induced damage, inflammation and alter gene expression. The purpose of this thesis was to determine if acute E2 supplementation would affect the oxidative stress, membrane damage, inflammation and global mRNA expression induced by eccentric exercise. Methods: 18 healthy young males were randomly assigned to 8 days of placebo (CON) or E2 (EXP) supplementation. Blood and muscle samples were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after 150 single-leg eccentric contractions. Blood samples were analyzed for hormone concentration, creatine kinase (CK) activity and total antioxidant capacity (T AC). Inflammation was quantified by neutrophil and macrophage infiltration. Genes selected a priori for oxidative stress defense, membrane homeostasis and growth were analyzed with real-time RT-PCR. High density oligonucleotide based microarrays were screened for novel differences in mRNA expression. Results: A primary finding was that increased serum E2 did not affect anti-oxidant capacity, creatine kinase efflux or mRNA content of genes related to oxidative stress defence and membrane homeostasis. E2 did attenuate neutrophil infiltration into muscle but did not affect macrophage density. Microarray analysis revealed that exercise induced differential expression of 611 genes at 3H and confirmed that E2 did not affect mRNA content. Genes were manually clustered into biological categories and from this dataset the signaling pathways for RhoA and NF AT were identified as transcriptionally active. Both pathways regulate hypertrophic signaling through the AP-1 transcription factor complex. Conclusions and significance: A major contribution ofthis thesis is that E2 may affect exercise induced inflammation through mechanisms that that do not affect oxidative stress or membrane stability. Additionally, the transcriptional activation ofSTARS/RhoA/APl and NFAT/APl indicates that both are important for early repair and remodelling signaling after a single bout of unaccustomed eccentric exercise. </p> / Thesis / Doctor of Philosophy (PhD)
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Impact of Diabetes Mellitus and Associated Changes on Skeletal Muscle and its Stem Cell Population / Satellite Cells in Diabetes MellitusD'souza, Donna M. January 2016 (has links)
Diabetes Mellitus is chronic lifelong condition that continues to be a global health concern. Despite the development of insulin therapy in 1921, many diabetics are likely to endure a number of co-morbidities that impact their quality of life. Today, the search for additional diabetic therapies incorporates the investigation of various organ systems for their potential in attenuating disease development. Skeletal muscle is a striated tissue that is integral to metabolism, movement, and overall wellbeing, yet its significance to Diabetes Mellitus remains understudied, as compared to other metabolic tissues. Previous work has identified that diabetes promotes adverse changes to skeletal muscle physiology, function, and morphology, contributing to a complication referred to as diabetic myopathy. The capacity to adapt to changing internal and external cues, as achieved through skeletal muscle plasticity, permits the maintenance of skeletal muscle health; a term encompassing its metabolism, function, and/or structure. This malleability is primarily regulated by the function of muscle progenitor stem cells, referred to as satellite cells. While past research has shown that satellite cells are hindered in various diabetic states, the precise mechanisms through which these observations occur remain to be elucidated. The data presented herein identify impaired satellite cell activation in two sub-types of diabetes (Pre-Diabetes and Type 1 Diabetes), and shows that such results are mediated by alterations to intrinsic signalling cascades. Additional insight into a potential unifying mechanism mediating this response led to the identification of Lipocalin-2 and its influence on satellite cell function and muscle plasticity. The results uncovered in these studies have enhanced our understanding of the response of satellite cells in diabetes, and have identified a prospective therapeutic target for the attenuation of diabetic myopathy. / Dissertation / Doctor of Philosophy (PhD)
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Temporal Diffusion MRI of Post-Exercise Human Calf MusclesRockel, Conrad P January 2017 (has links)
Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) method that resolves structures by three-dimensional measurement of water movement, and has shown to benefit studies of anisotropic tissues such as brain and skeletal muscle. While many studies have used DTI to non-invasively study static tissue architecture, little attempt has been made to use this technique to temporally characterize muscle during post-exercise recovery. Thus, the goal of this work was to use DTI to study the timecourse of changes in skeletal muscle following exercise. The first study was performed to test stability of DTI eigenvectors (ε1 ε2 ε3), and to determine how time-expensive parameters such as increased number of diffusion directions (NDD) or signal averages (NSA) improve vector stability. The ε2 vector was found to have more directional variability than ε1, and showed less improvement than ε1 with increased NDD or NSA. Furthermore, decreasing directional variability of ε1 was correlated with increasing NDD, not NSA (p<0.0008), while decreased variability of ε2 was correlated with increasing NSA, not NDD (p<0.0005). The variation in ε2 indicated that combining the corresponding minor eigenvalues into a measure of Radial Diffusivity is more robust than analyzing λ2 and λ3 eigenvalues separately. The second study tested the use of DTI to characterize temporal calf muscle changes following a mild in-bore dorsiflexion-eversion exercise. DTI volumes were acquired before and immediately after exercise. Anterior tibialis (ATIB), extensor digitorum longus (EDL), and peroneus longus (PER) showed significantly-elevated mean diffusivity (MD) post-exercise, while soleus (SOL) and lateral gastrocnemius (LG) did not (p<0.0001). The EDL showed greater initial MD increase and remained significantly elevated across more time points than ATIB or PER (p<0.05 to p=7.41x10−10). Significant signal increases were observed in post-exercise EDL b=0s/mm2 volumes (S0) relative to other muscles across the majority of timepoints (p<0.01 to p<0.001). The notable differences of EDL temporal MD and S0 relative to ATIB and PER may be related to the physiology of the increased Type-II fiber content in this muscle. The third and final study investigated the feasibility of a ’sliding window’ multiple-timescale temporal DTI approach, intended to acquire data with high temporal resolution and ongoing structural representation. Continuous diffusion data was acquired in the calf before and after four plantarflexion tasks, which varied by number of flexions (10 or 60), and weight load (10% or 40% of individual max). Apparent diffusion coefficient (ADC) and S0 were calculated from 3-direction subunits, while 15-direction subunits produced DTI measures such as mean diffusivity (MD). Four different post-exercise temporal patterns were observed for ADC, S0, and MD amongst the measured muscles: ’elevated-decline’, ’latent peak’, ’sub-to-peak’, and ’horizontal’. The 10-flex 10% condition elicited ’elevated-decline’ in active muscles, particularly SOLlat. Exercise of greater intensity produced ’latent peak’ and ’sub-to-peak’ patterns, with peak height related to greater workload. The 10-flex 40% trial produced a ’sub-to-peak’ pattern across all subjects only in the LG and MG, but ’latent-peak’ in these muscles 60-flex 40%. The specificity of temporal diffusion patterns according to muscle and task indicate that this technique could be beneficial to future studies of muscle function. These experiments have demonstrated the limits of DTI in the study of skeletal muscle, yet established a basis for future investigation of muscle dynamics using temporal diffusion methods. / Dissertation / Doctor of Philosophy (PhD)
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