Both resistance and endurance exercise elicit an increase in muscle protein synthesis during recovery from exercise. Ingestion of amino acids augments the exercise-induced stimulation of muscle protein synthesis following resistance exercise. Our work showed that 8 wk of unilateral resistance training induced muscle hypertrophy only in the exercised limb. Importantly, using this unilateral model we showed that muscle hypertrophy was confined to the exercised leg and occurred without measurable changes in circulating anabolic hormones. We then went on to use the unilateral leg resistance exercise model to study how animal-derived (milk) and plant-derived (soy) proteins impacted acute post-exercise protein turnover. We observed that ingestion of soy or milk protein resulted in a positive net protein balance following resistance exercise. Moreover, milk promoted a greater net protein balance and muscle protein synthesis than soy protein. In the final study, a key finding was that acute endurance and resistance exercise differentially stimulated myofibrillar and mitochondrial protein synthesis and also differentially affected cellular signaling proteins involved in the regulation of the protein synthetic response. Specifically, the acute, untrained state response showed that resistance exercise stimulated myofibrillar and mitochondrial protein synthesis while endurance exercise stimulated mitochondrial protein synthesis. Following resistance training only myofibrillar protein synthesis increased after exercise, while mitochondrial protein synthesis was unchanged. Endurance exercise training did not affect the acute protein synthetic response and so following training mitochondrial protein synthesis was stimulated as it was acutely, prior to training. In conclusion, the studies within this thesis provided novel insights on the impact of intact dietary proteins and differing modes of exercise on the control skeletal muscle protein metabolism. / Thesis / Doctor of Philosophy (PhD)

AMPK is a master regulator of cellular energy homeostasis. The gain-of-function AMPKg3 R225W mutation in human skeletal muscle increases resistance to fatigue during exercise, mitochondrial content, and glycogen storage. We demonstrate that primary myotubes exhibit increased OCR, decreased ECAR, increased FAO, and increased activities of several mitochondrial complexes. To examine whether functional effects are attributable to mitochondrial content, we inhibited AMPK; differences between R225W and control were diminished. Glycogen phosphorylase inhibition demonstrated normal respiration independent of glycogen. We examined markers of quality/quantity control of mitochondria. In R225W muscle, fusion markers increased, biogenesis markers remained unchanged, mTOR pathway was inhibited, and there was greater capacity for autophagic flux and mitophagy. We thus determine that bioenergetic effects of R225W are in part due to active AMPK, but also due to capacity for more robust mitochondria. Overall, R225W provides a model for evaluating effects of AMPK, and new avenues toward treatment of metabolic disease.

In vitro and animal studies indicate that the response to heat stress is associated with beneficial adaptations that promote cell health and survival. Few studies to date have examined this finding in human subjects, and it is unclear how the adaptation compares in magnitude to exercise training. The purpose of this study was to investigate the skeletal muscle adaptations (namely mitochondrial biogenesis and capillarization) of 6 weeks of deep-muscle heat treatment relative to exercise training. We hypothesized that heat treatment (HT), applied through pulsed shortwave diathermy (2 hr, 3 days/week) over a 6-week intervention period would lead to increased mitochondrial content and capillarity within skeletal muscle, though to a lesser extent than single-leg knee extension exercise training (EX; 40 min, 3 days/week). We randomized 28 sedentary but otherwise healthy, young adults (ages 18–36; n = 13 female, n = 15 male) to receive either HT, EX, or sham heating sessions (CON; 2 hr, 3 days/week) over 6 weeks. Diathermy increased muscle temperature by 3.2 ± 0.33 C (P < 0.0001) within 20 minutes. Muscle biopsies were taken from the vastus lateralis at baseline, after 3 weeks of intervention and again after 6 weeks of intervention. Following 3 and 6 weeks of heat treatment, we did not observe significant changes in mitochondrial biogenesis or capillarization. However, exercise training was sufficient to elicit an increase in individual capillary-to-fiber ratio (P = 0.0003), capillary density (P = 0.0428), and the Capillary to Fiber Perimeter Exchange Index (P = 0.0089). Significant increases in the expression of mitochondrial protein Complexes I (P = 0.0073) and IV (P = 0.0015), were observed in the exercise group, but not the heat or control groups. These results indicate that 6 weeks of localized HT, when applied to young healthy individuals, is insufficient to induce mitochondrial biogenesis or capillarization in skeletal muscle. Additionally, our findings provide support for the extensive body of literature that connect exercise training to beneficial skeletal muscle adaptations.

The transformation of genomic data into functionally relevant information about the composition of biological systems hinges critically on the field of computational genome biology, at the core of which lies comparative genomics. The aim of comparative genomics is to extract meaningful functional information from the differences and similarities observed across genomes of different organisms. We develop and test a novel framework for applying complex models of nucleotide evolution to solve phylogenetic and comparative genomic problems, and demonstrate that these techniques are crucial for accurate comparative evolutionary inferences. Additionally, we conduct an exploratory study using vertebrate mitochondrial genomes as a model to identify the reciprocal influences that genome structure, nucleotide evolution, and multi-level molecular function may have on one another. Collectively this work represents a significant and novel contribution to accurately modeling and characterizing patterns of nucleotide evolution, a contribution that enables the enhanced detection of patterns of genealogical relationships, selection, and function in comparative genomic datasets. Our work with entire mitochondrial genomes highlights a coordinated evolutionary shift that simultaneously altered genome architecture, replication, nucleotide evolution and molecular function (of proteins, RNAs, and the genome itself). Current research in computational biology, including the advances included in this dissertation, continue to close the gap that impedes the transformation of genomic data into powerful tools for the analysis and understanding of biological systems function.

Circulating cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress and disease which predicts mortality and is associated with various disease states. First, I present a systematic review of the literature that shows cf-mtDNA levels respond to common real-world stressors. The review discusses current knowledge on the mechanisms of cf-mtDNA release, its molecular forms of transport, potential physiological functions, cellular and neuroendocrine triggers, and factors that may contribute to cf-mtDNA removal from the circulation. Second, to evaluate the contribution of cf-mtDNA to health and disease states, standardized high-throughput procedures are needed to quantify cf-mtDNA in relevant biofluids. I describe the development of MitoQuicLy: Mitochondrial DNA Quantification in cell-free samples by Lysis. I demonstrate high agreement between MitoQuicLy and a commonly used column-based method, although MitoQuicLy is faster, cheaper, and requires a smaller input sample volume. I find that cf-mtDNA levels between concurrently collected plasma, serum, and saliva from the same individual differ on average by up to two orders of magnitude and are poorly correlated with one another, pointing to different cf-mtDNA biology or regulation between commonly used biofluids in clinical and research settings. Finally, I deploy MitoQuicLy in a highly phenotyped cohort of participants with mitochondrial disease and healthy controls to quantify the impact of a brief psychological stressor on cf-mtDNA levels in three biofluids and evaluate how cf-mtDNA release varies between individuals, biofluids, and mitochondrial disease status. These studies set the stage for a research agenda identifying novel links between psychological stress and physiological response, which may improve our understanding of how stressful experiences increase disease risk. In the future, cf-mtDNA release or its downstream effects may be targeted to modulate the impact of psychological stress on human health.

As sequencing technology has taken off since the late 1990's, a unique phenomenon has been observed repeatedly: genes with little to no conservation across species. For a while, the predominant theory that arose to explain these genes was duplication and subsequent evolution of conserved genes. While this theory can explain some, still many genes have now been proven to arrive de novo - from previously non-coding DNA. This work further investigates the origins of these de novo evolved genes and their practical application relevance in crop biotechnology. This work demonstrates the dynamic nature of plant mitochondrial genomes between even closely related species, and the integral role of mitochondrial genomes in the origin of de novo orphan genes in plants. To better understand the functional potential of plant orphan genes, the network of the orphan gene Qua Quine Starch (QQS) is further elucidated. This analysis demonstrates the broad functionality of an orphan gene as a "fine-tuning knob" in many plant pathways. Further, QQS' role in protein and starch allocation and plant defense is tied to the Nuclear Factor Y subunit C4 (NF-YC4) transcription factor - this knowledge leads us to manipulate expression of native NF-YC4 transcription factors in important crop species to successfully increase seed protein, increase broad disease resistance, and expedite maturation in soybean. A key goal in biotechnology is creating non-transgenic plants with advantageous traits. To accomplish this, the CRISPR-CAS9 system was used to target and delete repressive cis-regulatory elements in the GmNF-YC4-1 promoter sequence. This has allowed us to modify the activity of GmNF-YC4-1 and thus increase soybean seed protein, making it possible to get a non-transgenic plant by segregating out the CRISPR-CAS9 T-DNA and keeping the regulatory deletion. Overall, this work uncovers a novel mechanism of orphan gene evolution and uses the study of the orphan gene QQS to develop important crop biotechnology.

No description available.

No description available.

No description available.

No description available.