Autophagy delivers cytoplasmic material to the lysosome for degradation, and has been implicated in many cellular processes, including stress, infection, survival, and death. Although the regulation and role that autophagy plays in stress, infection, and survival is apparent, its involvement during cell death remains relatively unclear. In this thesis I summarize what is known about the roles autophagy can play in cell death, and the differences between the utilization of autophagy during nutrient deprivation and cell death. Utilizing Drosophila melanogaster as a model system, the roles autophagy plays in both of these contexts can be studied. The goal of this thesis is to provide a better understanding of the regulatory mechanisms that distinguish between autophagy as a survival mechanism and autophagy as a cell death mechanism. From my studies I was able to determine that microRNAs can regulate autophagy in vivo, and that the microRNA miR-14 controls autophagy specifically during the destruction of the larval salivary glands of Drosophila melanogaster. I found that miR-14 regulates autophagy through modulation of IP3 and calcium signaling, and this miR-14 control of IP3 and calcium signaling does not influence the induction of autophagy during nutrient deprivation. Therefore, this knowledge demonstrates how autophagy can be regulated to distinguish its use during cell survival and death providing insight into how autophagy can used to treat diseases.

Autophagy is a conserved process that cells use to degrade their own cytoplasmic components by delivery to lysosomes. Autophagy ensures intracellular quality control and is associated with diseases such as cancer and immune disorders. The process of autophagy is controlled by core autophagy (Atg) genes that are conserved from yeast to mammal. Most Atg proteins and their regulators were identified through pioneering studies of the single cell yeast Saccharomyces cerevisiae, and little is known about factors that systematically coordinate autophagy within the tissues of multicellular animals. The goal of this thesis is to identify new autophagy regulators and provide a better understanding of the regulatory mechanisms within multicellular animals. My research determined Macroglobulin complement-related (Mcr), a Drosophila complement orthologue, can activate autophagy during developmental cell death. Unlike most known autophagy regulators, Mcr functions in a cell non-autonomous manner to trigger autophagy in neighboring cells. To my knowledge, this is the first identified autophagy factor that cell non-autonomously activates autophagy. Additionally, I found that Mcr, a secreted protein, instructs the autophagy machinery through the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Lastly, Mcr is dispensable for both nutrient deprivation-induced autophagy in the fat body and developmentally programmed autophagy in the dying midgut of Drosophila. Therefore, this study unveils a mechanism in a multicellular organism by which autophagy is systematically controlled in distinct cell contexts.

Stretch activation (FSA) is the delayed increase in fiber specific tension (force per cross-sectional area) following a rapid stretch and can improve muscle performance during repetitive cyclical contractions. Historically considered minimal in skeletal muscle, our recent work showed the ratio ofstretch- to calcium-activated specific tension (FSA/F0) increased from 10 to 40% with greater inorganic phosphate (Pi) levels in soleus muscle fibers (Straight et al., 2019). Given Pi increases with muscle fatigue, we hypothesize that FSA helps maintain force generation during fatigue. To test this, FSA, induced by a stretch of 0.5% fiber length, was examined during Active (pCa 4.5 (pCa = -log([Ca2+]), pH 7.0, Pi 5 mM), High Ca2+ Fatigue (pCa 4.5, pH 6.2, Pi 30 mM) and Low Ca2+ Fatigue (pCa 5.1, pH 6.2, Pi 30 mM) in fibers expressing myosin heavy chain (MHC) I, IIA, IIX and IIB isoforms from soleus and extensor digitorum longus muscles of C57BL/6NJ mice. F0 of all MHC isoforms decreased from Active to High Ca2+ Fatigue to Low Ca2+ Fatigue, as expected. In MHC IIX and IIB fibers, FSA occurred under all conditions and FSA/F0 increased from Active (17-20%) to High Ca2+ Fatigue (32-35%) to Low Ca2+ Fatigue (42-44%). In MHC IIA fibers, FSA/F0 increased similarly to MHC IIX and IIB fibers from Active (14%) to High Ca2+ Fatigue (32%) but stayed elevated under Low Ca2+ Fatigue (35%). For MHC I fibers, no discernable FSA was apparent in either High – or Low Ca2+ Fatigue, leaving an FSA/F0 value in Active only ( 4%). These results show that FSA is a significant modulator of specific tension production under fatiguing conditions in fast-contracting muscle fibers. This mechanism could play an important physiological role during cyclical contractions, when the antagonistic muscle rapidly stretches the agonist muscle, by reducing the effect of fatigue on specific tension production.

Sympathetic stimulation occurs in the heart after injuries such as ischemia/reperfusion (I/R) and myocardial infarction and affects myocardial remodeling. Prolonged sympathetic stimulation can result in myocardial dysfunction through its effects on cardiac myocyte apoptosis and myocardial fibrosis. Ubiquitin (UB) is well known for its role of tagging old or damaged proteins for degradation via the UB-proteosome pathway. The role of exogenous UB however, is not fully understood. Previously, our lab showed that β-adrenergic receptor (β-AR) stimulation increased levels of extracellular UB in the conditioned media of adult rat ventricular myocytes and that UB inhibits β-AR-stimulated apoptosis. This study investigates the role of extracellular UB after myocardial I/R injury in terms of infarct size, function, inflammation and proteomic changes in vivo as well as the effects of extracellular UB on cardiac fibroblast function in vitro. First, we validated a method of consistently measuring real-time myocardial ischemia and reperfusion in vivo. Second, cardiac function was studied 3 days post I/R injury in the presence or absence of UB infusion. Echocardiographic analysis determined UB infusion increased cardiac function after I/R injury in terms of ejection fraction and fractional shortening. UB decreased infarct size and infiltration of inflammatory cells including neutrophils and macrophages as well as reduced activity of neutrophils. UB increased protein levels of matrix metalloproteinase (MMP)-2 and transforming growth factor-β1 and increased activity of MMP-9. Third, in adult rat primary cardiac fibroblasts, we demonstrate that extracellular UB interacts with CXCR-4. UB treatment decreased serum-mediated increases in fibroblast proliferation and enhanced the contraction of fibroblast-populated collagen gels. Thus, extracellular UB likely interacts with CXCR-4 to influence fibroblast function and proliferation. Additionally, UB influences cardiac remodeling in terms of heart function, infarct size, inflammatory response and proteomic profile.

Background: Northern elephant seals transition from terrestrial nursing pups to pelagic foraging juveniles in a short period of just 8-12 weeks. During the post-weaning period, pups rely solely on the energy reserves gained during nursing for their caloric demands and water supply. The prolonged absence of food after weaning is the first of many fasts for which the seals have evolved adaptations such as decreased urine production and increased blubber reserves. The stressors experienced from learning to dive for the first time are also stressors that they will experience frequently as an adult and for which they have evolved adaptations. The purpose of this study was to understand the tissue-specific molecular fasting- and diving- induced adaptive responses of pups during this critical transition. Methods: To investigate these adaptive responses to fasting and diving, we collected skeletal muscle and (inner and outer) adipose tissue from early-fasting (< 1 week post-weaning) and late-fasting (8 weeks post-weaning) pups. We analyzed the samples with mass-spectrometry-based proteomics using two-dimensional gel electrophoresis. Proteomics is an invaluable tool for analyzing marine mammal physiology, as it provides a large, unbiased data set of proteins that offer a comprehensive set of mechanisms involved with the cellular processes being studied. Proteomics has only been used as analytical tool for marine mammal biology in two other studies, and it can be used as a tool leading to the discovery of novel, unanticipated results. Results and Discussion: Because muscles are utilized during locomotion, we expected the proteome of skeletal muscle to highlight important physiological changes as the pups learn to dive. Inner adipose is more metabolically active than outer adipose, so we anticipated it would show important changes in metabolism throughout their fast. Outer adipose was useful to detect changes in the proteome due to thermoregulation, as it experiences the most drastic change in temperature and pressure while the pups learn to dive. In all tissues, we found significant shifts in energy metabolism proteins that show a decrease in lipid metabolism and urine production, and an increase in alternative metabolic pathways, such as the pentose phosphate pathway, which produces precursors for nucleic acid synthesis. We also found increases in cytoskeletal proteins, skeletal muscle proteins, and oxygen-binding proteins that facilitate the development of diving ability in late-fasting pups. Lastly, changes in the abundance of oxidative stress related proteins showed increased use of antioxidant proteins to control the production of reactive oxygen species in late-fasting pups. This study provides insight into cellular and physiological responses in marine mammals during ontogeny and their adaptive capacity during a key transition from a terrestrial to aquatic lifestyle.

The development of complex neural networks relies on a careful balance of environmental cues to guide and shape both ends of the eventual connection. However, the correct wiring of circuits whose components share molecular profiles depends on a more elaborate phenomenon, competition. Despite being highly studied, there is still a lack of understanding as to the mechanism that allows molecularly identical cells to form exclusive connections with their targets. To address this complex question, we turned to a simple circuit within the genetically tractable fly. Responsible for the escape reflex, the Giant Fiber System is comprised of bilaterally symmetrical axons that innervate the ipsilateral "jump" motorneuron, TTMn in a 1:1 ratio. However, if a TTMn is unilaterally ablated prior to circuit formation, this ratio is disrupted and the deprived axon forms its presynaptic terminal on the opposite side. Midline crossing by the deprived axon led to exploration of a known pathway in giant fiber development, midline repulsion via Slit and Roundabout. Axons in which Roundabout levels were reduced through a natural pathway antagonist, Commissurelesss, crossed the midline freely, confirming a native, if normally restricted ability to do so. However, unlike the overlapping giant fiber terminals seen following ablation, these axons retained their wild exclusivity, elaborating their terminals toward a single TTMn. This supported our initial aim of uncovering a competitive force in giant fiber target selection. In addition to repulsion, I also examined the attractive pathway of Netrin and Frazzled for a possible role in target identification. Varying the levels of Frazzled receptors led to increased midline crossing and overlapped terminals, suggesting a connection between this attractive receptor and the repulsion pathway first examined. Frazzled has been shown to induce commissureless expression independent of its ligand, making it an important linchpin in the regulation of giant fiber guidance and competition. In fact, when allowed to traverse the midline, giant fibers responded to a Netrin increase with overlapping synaptic terminals. In this dissertation, I present a model in which giant fibers possess competitive machinery, driven by Netrin and triggered by Frazzled, underneath the naturally restrictive midline repulsion.

Background: Vitamin D is crucial for skeletal muscle function. 25-hidroxyvitamin D (25(OH)D) has been correlated with skeletal muscle mass and intramyocellular lipid (IMCL) content. The purpose of this study was to understand how calcitriol, the active vitamin D metabolite, directly affects myocellular size and lipid partitioning. Methods: C2C12 myotubes were treated with calcitriol (100nM) or vehicle control for 24 or 96 h. Myotube diameter and protein synthesis rate were measured to determine effects of calcitriol on myocellular size. Intramyocellular triacylglycerol (IMTG), diacylglycerol (DAG), and ceramide content were measured by LC/MS. Expression of genes involved in lipid packaging and lipolysis were measured by RT-PCR. Insulin-stimulated phosphorylated Akt (Thr 308) was determined by western blot. Results: Calcitriol did not affect myocellular size or protein synthesis rate. Calcitriol increased total DAG and ceramides in a sub-species specific manner. Calcitriol increased IMTG area, but did not affect total IMTG content. Calcitriol reduced mRNA content of diglyceride acyltransferase and increased mRNA content of lipid packaging genes. Calcitriol did not negatively affect insulin-stimulated pAkt. Conclusions: These results suggest calcitriol directly alters lipid content and packaging in skeletal muscle cells. Altering the expression of lipid packaging genes and increasing IMCL subspecies content may be mechanisms by which vitamin D improves skeletal muscle function in vivo.

The L-type calcium channel (LTCC) provides trigger Ca2+ for sarcoplasmic reticulum Ca2+-release and LTCC function is influenced by interacting proteins including the LTCC Distal Carboxyl-terminus (DCT) and calmodulin. DCT is proteolytically cleaved, and re-associates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (IBa,L) in reconstituted channel complexes, yet the contribution of DCT to ICa,L in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte ICa,L. We measured LTCC current and Ca2+ transients with DCT co-expressed in murine cardiomyocytes. We also heterologously co-expressed DCT and CaV1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, CaV1.2Δ1801, and a shorter deletion corresponding to well-studied construct, CaV1.2Δ1733. DCT inhibited IBa,L in cardiomyocytes, and in HEK 293 cells expressing CaV1.2Δ1801 and CaV1.2Δ1733. Ca2+-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of IBa,L combined with Ca2+-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca2+. We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca2+, and show that DCT reduced diastolic Ca2+ at low stimulation frequencies but spared high frequency Ca2+-entry. DCT reduction of diastolic Ca2+ and relief of block at high pacing frequencies, and under conditions of supraphysiological bath Ca2+ suggests that a physiological function of DCT is to increase the dynamic range of Ca2+ transients in response to elevated pacing frequencies. Our data motivates the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

The discovery that bile acids act as endogenous ligands of the membrane receptor TGR5 and the nuclear receptor FXR increased their significance as regulators of cholesterol, glucose and energy metabolism. Activation of TGR5, expressed on enteroendocrine L cells, by bile acids caused secretion of GLP-1, which stimulates insulin secretion from pancreatic β cells. Expression of TGR5 on pancreatic islet cells and the direct effect of bile acids on the endocrine functions of pancreas, however, are not fully understood. The aim of this study was to identify expression of TGR5 in pancreatic islet cells and determine the effect of bile acids on insulin secretion. Expression of TGR5 was identified by quantitative PCR and western blot in islets from human and mouse, and in α (αTC1-6) and β (MIN6) cells. Release of insulin, glucagon and GLP-1 were measured by ELISA. The signaling pathways coupled to TGR5 activation were identified by direct measurements such as stimulation of G proteins, adenylyl cyclase activity, PI hydrolysis and intracellular Ca2+ in response to bile acids; and confirmed by the use of selective inhibitors that block specific steps in the signaling pathway. Our studies identified expression of TGR5 receptors in β cells and demonstrated that activation of these receptors by both pharmacological ligands (oleanolic acid (OA) and INT-777) and physiological ligand (lithocholic acid, LCA) induced insulin secretion. TGR5 receptors are also expressed in α cells and, activation of TGR5 by OA, INT-777 and LCA at 5 mM glucose induced release of glucagon, which is processed from proglucagon by the selective expression of prohormone convertase 2 (PC2). However, under hyperglycemia, activation of TGR5 in α cells augmented the glucose-induced increase in GLP-1 secretion, which in turn, stimulated insulin secretion. Secretion of GLP-1 from α cells reflected TGR5-mediated increase in PC1 promoter activity and PC1 expression, which selectively converts proglucagon to GLP-1. The signaling pathway activated by TGR5 to mediate insulin and GLP-1 secretion involved Gs/cAMP/Epac/PLC-ε/Ca2+. These results provide insights into the mechanisms involved in the regulation of pancreatic α and β cell function by bile acids and may lead to new therapeutic avenues for the treatment of diabetes.

Mitochondrial diseases encompass a broad range of devastating disorders that typically affect tissues with high-energy requirements. These disorders have been difficult to diagnose and research because of the complexity of mitochondrial genetics, and the large variability seen among patient populations. We have devised and carried out a mechanistic study to generate a cell based model for Leigh’s disease caused by mitochondrial DNA mutation 8993 T>G. Leigh’s disease is a multi-organ system disorder that depends heavily on the mutation burden seen within various tissues. Using new reprogramming and sequencing technologies, we were able to show that Leigh’s disease patient fibroblasts reprogrammed to induced pluripotent stem cells maintain the same level of mutation burden seen in the original patient cell line. Mutation burden was maintained through several passages and spontaneous differentiation. This cell based model could be useful for future pathogenesis studies, or therapeutic drug screenings in a patient and tissue specific manner.