Adult skeletal muscle is highly plastic and responds readily to environmental stimuli. One of the most commonly utilized methods to study skeletal muscle adaptations is immunofluorescence microscopy. By analyzing images of adult muscle cells, also known as myofibers, one can quantify changes in skeletal muscle structure and function (e.g. hypertrophy and fiber type). Skeletal muscle samples are typically cut in transverse or cross sections, and antibodies against sarcolemmal or basal lamina proteins are used to label the myofiber boundaries. The quantification of hundreds to thousands of myofibers per sample is accomplished either manually or semi-automatically using generalized pathology software, and such approaches become exceedingly tedious. In the first study, I developed MyoVision, a robust, fully automated software that is dedicated to skeletal muscle immunohistological image analysis. The software has been made freely available to muscle biologists to alleviate the burden of routine image analyses. To date, more than 60 technicians, students, postdoctoral fellows, faculty members, and others have requested this software. Using MyoVision, I was able to accurately quantify the effects of β-catenin knockout on myofiber hypertrophy. In the second study, I tested the hypothesis that myofiber hypertrophy requires β-catenin to activate c-myc transcription and promote ribosome biogenesis. Recent evidence in both mice and human suggests a close association between ribosome biogenesis and skeletal muscle hypertrophy. Using an inducible mouse model of skeletal myofiber-specific genetic knockout, I obtained evidence that β-catenin is important for myofiber hypertrophy, although its role in ribosome biogenesis appears to be dispensable for mechanical overload induced muscle growth. Instead, β-catenin may be necessary for promoting the translation of growth related genes through activation of ribosomal protein S6. Unexpectedly, we detected a novel, enhancing effect of myofiber β-catenin knockout on the resident muscle stem cells, or satellite cells. In the absence of myofiber β-catenin, satellite cells activate and proliferate earlier in response to mechanical overload. Consistent with the role of satellite cells in muscle repair, the enhanced recruitment of satellite cells led to a significantly improved regeneration response after chemical injury. The novelty of these findings resides in the fact that the genetic perturbation was extrinsic to the satellite cells, and this is even more surprising because the current literature focuses heavily on intrinsic mechanisms within satellite cells. As such, this model of myofiber β-catenin knockout may significantly contribute to better understanding of the mechanisms of satellite cell priming, with implications for regenerative medicine.

Epilepsy is a neurological disorder that is characterized by aberrant electrical activity in the brain resulting in at least two unprovoked seizures over a period longer than 24 hours. Approximately 60% of individuals with epilepsy are diagnosed with temporal lobe epilepsy (TLE) and about one third of those individuals do not respond well to anti-seizure medications. This places those individuals at high risk for sudden unexpected death in epilepsy (SUDEP). SUDEP is defined as when an individual with epilepsy, who is otherwise healthy, dies suddenly and unexpectedly for unknown reasons. SUDEP is one of the leading causes of death in individuals with acquired epilepsies (i.e. not due to genetic mutations), such as TLE. Previous studies utilizing genetic models of epilepsy have suggested that circuitry within the vagal complex of the brainstem may play a role in SUDEP risk. Gamma-aminobutyric acid (GABA) neurons of the nucleus tractus solitarius (NTS) within the vagal complex receive, filter, and modulate cardiorespiratory information from the vagus nerve. GABAergic NTS neurons then project to cardiac vagal motor neurons, eventually effecting parasympathetic output to the periphery. In this study, a mouse model of TLE was used to assess the effect of epileptogenesis on GABAergic NTS neuron function and determine if functional alterations in these neurons impact SUDEP risk. It was discovered that mice with TLE (i.e. TLE mice) have significantly increased mortality rates compared to control animals, suggesting that SUDEP occurs in this model. Using whole cell electrophysiology synaptic and intrinsic properties of GABAergic NTS neurons were investigated in TLE and control mice. Results suggest that during epileptogenesis, GABAergic NTS neurons become hyperexcitable, potentially due to a reduction in A-type potassium channel current and increased excitatory synaptic input. Increases in hyperexcitability have been shown to be associated with an increased risk of spreading depolarization and action potential inactivation leading to neuronal quiescence. This may lead to a decreased inhibition of parasympathetic tone, causing cardiorespiratory collapse and SUDEP in TLE.

Schizophrenia is a chronic mental disorder affecting millions worldwide. It has no known cure. Current pharmaceutical treatments have shown efficacy in only one of the three symptom clusters of schizophrenia, providing little or no benefit in the other two. Furthermore, the current standard-of-care drugs, known as atypical antipsychotics, carry risks of severe side effects affecting multiple body systems. Most patients opt to discontinue drug therapy within two years of initiation due to lack of efficacy and/or preponderance of adverse effects. Previous findings have shown that chronic usage of atypical antipsychotics causes a 5-HT2A-dependent upregulation of histone deacetylase 2 (HDAC2), which in turn leads to downregulation of metabotropic glutamate receptor 2 (mGluR2), a G protein-coupled receptor with an important role in synaptic plasticity. The present study aims to characterize the extent to which this downregulation leads to specific functional outcomes, and in doing so, may help identify new targets for more effective treatment of schizophrenia.

The group II metabotropic glutamate receptors are known for their involvement in various psychiatric disorders. The mGluR2 in particular is linked with etiology of schizophrenia especially in the context of crosstalk with 5-HT2A. Thus, the mGluR2 has attracted attentions for its potential therapeutic applications. Despite numerous physiological evidences on the actions of mGluR2, its mechanism is still unclear to this day. It is partially due to the lack of understanding in characteristics of mGluR2 homodimer which is its functionally active form. Therefore, the characterization of dimeric interaction serves as a foundation to advanced understanding of the role of mGluR2. On that note, the role of the conserved cysteine residue (C121) in the ligand binding domain of mGluR2 has been evaluated in this study as they are known to play a critical part in homodimer formation. Collectively, C121 has been shown to affect the dimerization, subcellular localization, and pharmacokinetics of mGluR2. Lastly, the effect of mGluR2 on mouse behavior was examined in a partial effort to elucidate its role in crosstalk with 5-HT2A.

The field of cancer research has grown immensely in recent decades and has led to a better understanding of the causes of the disease, as well as greatly improved treatment for various types of cancers, especially breast cancer. One of the most effective treatments involves the chemotherapeutic drug doxorubicin (DOX). DOX is an effective tool against all types of breast cancer, especially against triple negative breast cancer. However, DOX causes adverse side effects that include damage to the heart and skeletal muscle, particularly above specific cumulative doses. Recent evidence suggests that embryonic stem cell-derived (ES) exosomes, nanoscale extracellular vesicles that carry proteins, messenger RNA, and microRNAs, may be able to mitigate some of the cardio- and cytotoxic effects of DOX without reducing its efficacy. The present study examined the effects of combined treatment with DOX (1 μM) and ES exosomes (10 μg/mL) on three cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-468. The DOX/ES exosomes treatment increased cell death and increased apoptosis specifically compared to control, as measured via dye exclusion assay and flow cytometry. The treatment also decreased cell growth compared to control, as measured via MTS cell proliferation assay. In addition, DOX/ES exosomes treatment also increased expression of pro-apoptotic Bax while decreasing the expression of anti-apoptotic Bcl-2, as measured via Western blot. Finally, the DOX/ES exosomes treatment decreased expression of miR-200c, a microRNA associated with preventing epithelial-mesenchymal transition, a process that is integral to metastasis. Although increased cell death and apoptosis and decreased cell proliferation implies that the DOX/exosomes treatment is effective against cancer, the decrease in miR-200c expression may suggest the opposite and will be investigated further in future studies. Even so, the results of this study suggest that exosomes may be an important component to reduce the harmful effects of cancer treatment in the future.

In Aedes aegypti, development and reproduction are regulated by juvenile hormone III (JH). This master regulatory hormone is synthesized by the corpora allata (CA), a pair of endocrine glands with neural connections to the brain. JH titers are largely determined by the rate of biosynthetic activity of the CA and are regulated by inhibitory and stimulatory factors. Like JH, the ecdysteroid 20-hydroxyecdysone (20E) is a key hormonal regulator and has been proposed as an allatoregulator in other insects. However, its part in the regulation of JH biosynthesis of mosquitoes was unknown. The specific aims of this dissertation were to (1) evaluate if 20E plays a role in the activation of the late pupal CA and (2) evaluate if 20E plays a role in the reactivation of JH synthesis in blood-fed females. To this end, we evaluated if 20E could prematurely activate JH biosynthesis in the CA of an early pupa (24h prior to eclosion or -24h). Remarkably, in vitro stimulation with 20E at -24h initiated JH synthesis at a time when transcript levels for most JH biosynthetic enzymes are low. Moreover, the application of 20E correlated with an increase in the enzymatic activity of juvenile hormone acid methyltransferase (JHAMT), a critical enzyme of the biosynthetic pathway. Additionally, separation of the CA from the brain increased JH synthesis. Together, these results indicate that 20E acts as a developmental mediator of CA maturation which overrides an inhibitory effect of the brain. In our previous aim we demonstrated that 20E mediates activation of the pupal CA which ensures the development of ovarian follicles of the newly emerged female. For mosquitoes, a blood-meal is required to complete vitellogenesis and results in suppression of CA activity. However, the CA must be reactivated to initiate the second gonotrophic cycle. Our findings show that in vitro stimulation with 20E at 24h post blood feeding reactivates the gland. Again, stimulation with the ecdysteroid resulted in increased activity of another key enzyme, farnesal dehydrogenase (FALDH). These results suggest a stimulatory role of 20E on the biosynthetic activity of the CA in the blood fed female.

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad. In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that b-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVb subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined. Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study.

The miR-17- 92 cluster is necessary for cell proliferation and development of the cardiovascular system. Deletion of this cluster leads to death in neonatal mice. The role of this cluster still needs to be defined following ischemia and reperfusion. Methods and Results: Adult male mice were injected with Tamoxifen- was to induce inducible cardiac-specific miR-17- 92-deficient (miR-17- 92-def: MCM:TG:miR-17- 92 flox/flox ) and wild type (WT: MCM:NTG:miR-17-92 flox/flox ) mice were subjected to 30 minutes of myocardial ischemia via left anterior descending coronary artery ligation followed by reperfusion for 24 hours. Post I/R survival (48%) and ejection fraction were reduced, while myocardial infarct size enlarged in miR-17- 92-deficient mice as compared to WT mice (survival: 71%). Necrosis (trypan blue staining) and apoptosis (TUNEL assay) both were higher in adult cardiomyocytes isolated from miR-17- 92-deficient mice as compared to WT mice subjected to simulated ischemia/reoxygenation with a concomitant reduction of mitochondrial membrane potential (JC1 staining). The electron transport chain was compromised through dysregulation of glutamate+malate as complex I substrate and malate dehydrogenase in the hearts of miR-17- 92-deficient mice compared to WT. After 4 hours of reperfusion, PTEN expression, a downstream target of miR-20A, increased, while phosphorylation of AKT reduced in the hearts of miR-17- 92-deficient mice in comparison to WT. The induced knockdown of cardiac miR-17- 92 increases myocardial I/R injury by ceasing suppression of PTEN, leading to decreased concentrations of AKT and mitochondrial dysfunction. These results suggest that innovative therapeutic strategies can focus on genetic upregulation of miR-17- 92 in patients with coronary artery disease.

Periodontal disease affects 47% of Americans over 30. Characterized by microbial dysbiosis and unregulated inflammation, severe periodontitis causes degradation of bone and soft tissue around teeth. Current treatments have limited regenerative outcomes and frequent reinfection by harmful bacteria. Human mesenchymal stem cells (hMSCs) have been shown to promote wound healing and tissue regeneration. Many therapeutic benefits of hMSCs are due to their secretome products, like exosomes. Our long-term goal is to develop periodontal therapies with hMSC exosomes. The objectives of this study were to determine the effect of hMSC-derived exosomes on cellular activity of hMSCs and investigate whether hMSC exosome treatment reduces pro-inflammatory cytokine production in LPS-activated RAW264.7 cells. The specific aims of this study were: 1) Determine the characteristics of hMSC-derived exosomes, 2) Determine the biological effect of exosomes on cellular activity of hMSCs, 3) Determine whether exosomes treatment can inhibit cytokine production in activated RAW264.7 cells, and 4) Determine the role of exosomal miRNA in pro-inflammatory cytokine production of RAW264.7 cells. To investigate, exosomes were first harvested from hMSCs culture media through ultracentrifugation. Exosomes were then observed under a transmission electron microscope (TEM) and assessed for surface markers using Western Blot. A transwell migration assay was used to evaluate the chemotactic effect of exosomes. To study the effect of exosomes on stem cell proliferation, exosomes were administered to hMSCs. The immunogenicity of MSC exosome was also evaluated. After 72 hours, cells were lysed and DNA was measured. To study anti-inflammatory effects of exosomes, LPS stimulated RAW264.7 cells were treated with exosomes. Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) levels of supernatant were measured by ELISA. To study exosomal miRNA, exosomal miRNAs were overexpressed in RAW264.7 cells and these cells were stimulated with LPS. IL-6 and TNFα were measured by ELISA. TEM images showed that exosomes are nano-sized vesicles (~100 nm). Western blot images showed that CD63 and CD81 are enriched in exosomes compared to total cell lysates. Exosome treatment increased cell proliferation and migration in hMSCs. At the doses that are chemotactic and mitogenic, MSC exosomes had minimal effect on the inflammatory cytokine IL-6 production. Treatment with exosomes significantly decreased IL-6 and TNFα production in RAW264.7 cells activated by LPS. Transfecting RAW264.7 cells with exosomal miR-760 significantly decreased IL-6 production, but had minimal effect on TNFα. Our results indicate that exosomes have a pleiotropic activity, which includes stimulating stem cell migration and proliferation, and mitigating the inflammatory response. Therefore, hMSC exosome delivery is promising for the treatment of periodontal diseases.

It is well known that 24-hour day-night (light-dark) cycles can entrain the circadian rhythms of most species possessing circadian clocks. However, much less is understood about how other environmental cycles operate as entraining signals (zeitgebers) to synchronize (entrain) the internal clock with the outside world. Potential non-photic zeitgebers include daily cycles of temperature, food availability, and social signals. This project’s goal is to evaluate the efficacy of temperature cycles of varying amplitudes, ranging from 1°C to 10°C, as potential zeitgebers for two different circadian rhythms, eclosion and locomotor activity, in the flesh fly (Sarcophaga crassipalpis). Both rhythms were monitored in individual flies, using infrared motion detectors, under precisely controlled 24-hour temperature cycles (12 hours of high temperature [thermophase] alternating with 12 hours of low temperature [cryophase]) in constant darkness. Our results show clear entrainment of eclosion, a once-in-a-lifetime event, and locomotor activity, reflecting daily sleep-wake rhythms, in response to temperature cycles at amplitudes of 2.5, 5, and 10o C. At 1o C amplitudes, the evidence indicates a partial effect of temperature on the phasing of the two behaviors but not true entrainment (phase regulation), suggesting that 1o C amplitudes may be near the threshold for detection by the entrainment pathways communicating with the circadian clock. Interestingly, although both light and temperature cycles entrain the locomotor activity rhythm, the entrainment profiles are remarkably different, thereby suggesting that light and temperature cycles activate different behavioral programs. Finally, flies placed under different constant temperatures have endogenous circadian periods of locomotor activity that are remarkably similar, indicating a high level of temperature compensation. Our results provide conclusive evidence that temperature is a strong circadian zeitgeber in flesh flies, thereby expanding the known repertoire of environmental cues these organisms use to sync their internal clock with the world around them. These findings also set the stage for future experiments designed to explore the interactions between light and temperature entrainment mechanisms – these zeitgeber interactions almost certainly occur in nature but have received little or no attention.