Polarography was used to determine functional differences in isolated SS and IMF mitochondria between lean (37 ± 3 yrs; n = 10) and obese (35 ± 3 yrs; n = 11) subjects during either saline (control) or amino acid (AA) infusions. AA infusion increased ADP-stimulated respiration (i.e., coupled respiration), non-ADP stimulated respiration (i.e., uncoupled respiration), and ATP production rates in SS, but not IMF mitochondria in lean (n = 10; P < 0.05). Neither infusion increased any of the above parameters in muscle SS or IMF mitochondria of the obese subjects.
Using label free quantitative mass spectrometry, we determined differences in proteomes of SM SS and IMF mitochondria between lean (33 ± 3 yrs; n = 16) and obese (32 ± 3 yrs; n = 17) subjects. Differentially-expressed mitochondrial proteins in SS versus IMF mitochondria of obese subjects were associated with biological processes that regulate: electron transport chain (P<0.0001), citric acid cycle (P<0.0001), oxidative phosphorylation (P<0.001), branched-chain amino acid degradation, (P<0.0001), and fatty acid degradation (P<0.001). Overall, these findings show that obesity is associated with redistribution of key biological processes within the mitochondrial reticulum responsible for regulating energy metabolism in human skeletal muscle.
Assessment of DNA methylation was performed on human skeletal muscle and blood using reduced representation bisulfite sequencing (RRBS) for high-throughput identification and pyrosequencing for site-specific confirmation. Sorbin and SH3 homology domain 3 (SORBS3) was identified in skeletal muscle to be increased in methylation (+5.0 to +24.4 %) in the promoter and 5’untranslated region (UTR) in the obese participants (n= 10) compared to lean (n=12), and this finding corresponded with a decrease in gene expression (fold change: -1.9, P=0.0001). Furthermore, SORBS3 was demonstrated in a separate cohort of morbidly obese participants (n=7) undergoing weight-loss induced by surgery, to decrease in methylation (-5.6 to -24.2%) and increase in gene expression (fold change: +1.7; P=0.05) post-surgery. Moreover, SORBS3 promoter methylation was demonstrated in vitro to inhibit transcriptional activity (P=0.000003). The methylation and transcriptional changes for SORBS3 were significantly (P≤0.05) correlated with obesity measures and fasting insulin levels. SORBS3 was not identified in the blood methylation analysis of lean (n=10) and obese (n=10) participants suggesting that it is a muscle specific marker. However, solute carrier family 19 member 1 (SLC19A1) was identified in blood and skeletal muscle to have decreased 5’UTR methylation in obese participants, and this was significantly (P≤0.05) predicted by insulin sensitivity.
These findings suggest SLC19A1 as a potential blood-based biomarker for obese, insulin resistant states. The collective findings of SORBS3 DNA methylation and gene expression present an exciting novel target in skeletal muscle for further understanding obesity and its underlying insulin resistance. Moreover, the dynamic changes to SORBS3 in response to metabolic improvements and weight-loss induced by surgery.
Over 40% of adults in the United States are considered obese. Obesity is known to cause abnormal metabolic effects and lead to other negative health consequences. Interestingly, differences in metabolism and contractile performance between obese and healthy weight individuals are associated with differences in skeletal muscle fiber type composition between these groups. Each fiber type is characterized by unique metabolic and contractile properties, which are largely determined by the myosin heavy chain isoform (MHC) or isoform combination that the fiber expresses. In previous studies, SDS-PAGE single fiber analysis has been utilized as a method to determine MHC isoform distribution and single fiber type distribution in skeletal muscle. Herein, a methodological approach to analyze MHC isoform and fiber type distribution in skeletal muscle was fine-tuned for use in human and rodent studies. In the future, this revised methodology will be implemented to evaluate the effects of obesity and exercise on the phenotypic fiber type composition of skeletal muscle.
Staufen is a double-stranded RNA binding protein (dsRBP) with discovered homologs in a diverse range of animals, insects, and other multicellular organisms. Staufen acts on secondary structures in mRNA transcripts to modulate translation of many targets through several mechanisms of action. It has roles in microtubule-dependent subcellular localization of mRNA transcripts, translational activation, transcript stability, Staufen-mediated mRNA decay (SMD), is a known component of RNA granules, and has been implicated in several cellular processes, one being myogenesis. Mammals have two Staufen orthologs–Staufen1 and Staufen2. Staufen1 has four conserved dsRNA binding domains (dsRBDs), each with distinct functional characteristics. This study finds that cultured MuSCs show distinct patterns of Staufen1 transcriptional expression from quiescence throughout the myogenic differentiation program characterized by high expression in quiescent satellite cells, less expression in proliferating myoblasts, and fairly high, sustained expression throughout differentiation and myotube formation. The temporal expression pattern is compared with recently reported novel Staufen1 functions in myogenesis. This research highlights that Staufen1 is able to act on transcripts in several overlapping ways to assist in the regulation of myogenesis, and more extensive characterization of Staufen1 as well as high-confidence identification of Staufen binding sites (SBS), will be necessary to fit Staufen1 into a model of translational regulation in myogenesis.
Cellular hypertrophy is an anaerobically-based, adaptive process that mammalian skeletal muscle undergoes in response to damage resulting from unaccustomed force generation by the muscle. Hypertrophy allows for the muscle tissue to recover from the immediate injury and also to be rebuilt more capable of withstanding producing the same amount of force without injury, should it happen again. This means the end result of an adapted muscle is an overall more efficient tissue. The ability to regenerate after damage to the structure and function of the muscle tissue is a highly orchestrated event involving multiple steps and key events to occur. Most briefly, a mechanical load is attempted to be lifted but due to demanding a high amount of contractile force to lift, it causes microdamage to the structural and contractile elements of muscle fiber’s sarcomeres. In addition to an inflammatory response, satellite cells, as a part of a myogenic response, are activated to invade the fiber and then permanently reside inside to produce new proteins that will replace the damaged and necrotized proteins. This addition of cellular content, repeated over multiple times, results in the increased diameter of the fibers and manifests in the visual appearance of skeletal muscle hypertrophy. These steps have been listed off devoid of the contexts in which it takes for these to occur and will be addressed within this thesis.
for Unmanned Aerial Vehicles.
Towards enabling a UAV to autonomously sense and avoid moving obstacles, this thesis makes the following contributions. Initially, an image-based reactive motion planner is developed for a quadrotor to avoid a fast approaching obstacle. Furthermore, A Dubin’s curve based geometry method is developed as a global path planner for a fixed-wing UAV to avoid collisions with aircraft. The image-based method is unable to produce an optimal path and the geometry method uses a simplified UAV model. To compensate
these two disadvantages, a series of algorithms built upon the Closed-Loop Rapid Exploratory Random Tree are developed as global path planners to generate collision avoidance paths in real time. The algorithms are validated in Software-In-the-Loop (SITL) and Hardware-In-the-Loop (HIL) simulations using a fixed-wing UAV model and in real flight experiments using quadrotors. It is observed that the algorithm enables a UAV to avoid moving obstacles approaching to it with different directions and speeds.