Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMPK, calcium, and circulating FFAs. Chronic treatment with either AICAR, a chemical activator of AMPK, or increasing circulating FFAs with a high fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high fat diet (HF), AICAR injections (AICAR), or a high fat diet and AICAR injections (HF+AICAR) for six weeks. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on LCAD, cytochrome c, and PGC-α protein, as well as on citrate synthase and β-HAD activity. In contrast, no additive effect was noted in the soleus and in the red quadriceps only β-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high fat diet-induced post transcriptional increase in PGC-α protein and AMPK mediated increase in PGC-α protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

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No description available.

The estuarine sheepshead minnow (Cyprinodon variegatus) is the most eurythermic fish species, with a thermal tolerance window between 0.6°C and 45.1°C. However, little is known about the physiological mechanisms that allow this species to survive this temperature range. In order to understand how sheepshead minnow physiology is affected by temperature acclimation and acute changes in temperature, I conducted research on this species using a multi-level approach. I began at the organismal level, and examined the effects of these temperature changes on the sheepshead minnow's metabolic rate and swimming performance. The next chapter investigated the effects of changing temperatures on cardiac function (i.e., tissue/organ specific effects). In the final chapter, I conducted research at the sub-cellular level, and determined how mitochondrial bioenergetics / function is impacted by changing temperatures. This research shows that while sheepshead minnows are able to sustain heart function and mitochondrial respiration over a broad range of temperatures; they also display a plastic temperature response which is associated with the downregulation of standard metabolic rate and cardiac remodeling to maintain force generation. Collectively, these physiological responses may contribute to the sheepshead minnow's ability to maintain physiological and organismal function across a large temperature range.

Mitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), which are essential for mitochondrial function. Mutations in the mitochondrial fission and fusion machinery lead to neurodegeneration. Thus, whether defective mitochondrial dynamics participates in ALS and HD requires further investigation. ALS is a fatal neurodegenerative disease characterized by upper and lower motor neuron loss. Mutations in Cu/Zn superoxide dismutase (SOD1) cause the most common familiar form of ALS by mechanisms not fully understood. Here, a new motor neuron-astrocyte coculture system was created and live-cell imaging was used to evaluate mitochondrial dynamics. Excessive mitochondrial fission was observed in mutant SOD1G93A motor neurons, correlating with impaired axonal transport and neuronal cell death. Inhibition of mitochondrial fission restored mitochondrial dynamics and protected neurons against SOD1G93A -induced mitochondrial fragmentation and neuronal cell death, implicating defects in mitochondrial dynamics in ALS pathogenesis. iv HD is an inherited neurodegenerative disorder caused by glutamine (Q) expansion in the polyQ region of the huntingtin (HTT) protein. In the current work, mutant HTT caused mitochondrial fragmentation in a polyQ-dependent manner in both primary cortical neurons and fibroblasts from human patients. An abnormal interaction between DRP1 and HTT was observed in mutant HTT mice and inhibition of mitochondrial fission or promotion of mitochondrial fusion restored mitochondrial dynamics and protected neurons against mutant HTT-induced cell death. Thus, mutant HTT may increase mitochondrial fission by elevating DRP1 GTPase activity, suggesting that mitochondrial dynamics plays a causal role in HD. In summary, rebalanced mitochondrial fission and fusion rescues neuronal cell death in ALS and HD, suggesting that mitochondrial dynamics could be the molecular mechanism underlying these diseases. Furthermore, DRP1 might be a therapeutic target to delay or prevent neurodegeneration.

To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.

The mechanisms by which mitochondria convert nutrients into cellular energy have been described in intricate detail, and yet, the regulation and compartmentalization of such metabolic pathways are poorly understood. As a result, the underlying causes of mitochondrial dysfunction and bioenergetic deficiency in diseases such as amyotrophic lateral sclerosis (ALS) remain elusive. To address this longstanding gap in the field, we first sought to understand how the metabolism of glucose and glucose-derived pyruvate are regulated in the cell. Previous research has suggested that this metabolic regulation is mediated by specialized lipid raft domains of the endoplasmic reticulum (ER) in close contact with mitochondria, referred to as mitochondria-associated ER membranes (MAM). Using density gradient ultracentrifugation and immunoblotting techniques, we found that MAM domains play a role in the compartmentalization of glycolysis by recruiting and promoting the interaction of specific glycolytic enzymes. We then performed a series of bioenergetic, proteomic, and lipidomic analyses to determine how the establishment of ER-mitochondria contact sites at MAM affects the biology of mitochondria attached at these domains. We observed a novel distinction between mitochondria in contact with ER-MAM domains (MER) and those that are free from the ER (FM), with MER displaying a higher capacity for pyruvate-driven respiration and FM being specialized for fatty acid-driven energy production. Finally, using cell and mouse models of ALS with mutations in superoxide dismutase 1 (SOD1), we found that the glycolytic deficiency in ALS is a direct consequence of the progressive disruption of MAM structure and function, which thereby hinders the use of glucose-derived pyruvate as a mitochondrial fuel. This triggers a shift in mitochondrial substrate from pyruvate to fatty acids that, when sustained over time, contributes to the death of motor neurons and the progression of this fatal disease. Overall, this work aims to advance our understanding of metabolic compartmentalization, mitochondrial substrate specificity, and the relevance of both to ALS etiology.

No description available.

Swedish populations of Wels catfish (Silurus glanis) experienced severe declines during the 19th and 20th centuries. The main causes for the decline were loss of suitable spawning habitat and fragmentation of populations. Currently, three native and two reintroduced populations remain in Sweden. Thanks to national protection and progress in restoring habitats, population sizes have increased over the past three decades. Previous studies that used microsatellite loci have found that genetic diversity and effective population sizes in Sweden are low, while population differentiation is high. A study that used whole genome sequencing (WGS) confirmed these results for native Swedish populations (those found in the Båven, Emån, and Möckeln water systems). The current project uses the same WGS methods and expands on the previous study by considering samples from non-Swedish populations (river Garonne, France; river Ebro, Spain; hatchery, Czech Republic), as well as improving read-depth coverage and sampling from the introduced Swedish population in the Helge å water system. Both a genome-wide SNP-set and full mitochondrial sequences were used to assess genetic diversity within each population, and differentiation among them. Genetic diversity in Swedish populations is lower than in non-Swedish populations. Native Swedish populations are strongly differentiated from one another. The introduced Helge å population is strongly differentiated from Emån and Möckeln, but less so from Båven. Despite Helge å individuals having heritage that can be predominantly traced to Båven, there are clear signs of admixture with the other two native populations. Swedish populations are all strongly differentiated from the non-Swedish populations. Altogether, evidence of admixture and slightly greater genetic diversity than native Swedish populations in Helge å can at the surface be seen as promising signs. However, it remains uncertain whether these improvements are durable over multiple generations. Considering the poor genetic status of Sweden taken as a whole, and the questionable nature of the improvements seen in Helge å, the long-term viability of Swedish catfish populations remains uncertain.

No description available.