Increases in seawater temperatures have imposed physiological constraints which are partially thought to contribute to recently observed shifts in biogeographic distribution among closely related intertidal ectotherms. For instance, Mytilus galloprovincialis an introduced warm-adapted species from the Mediterranean, has displaced the native cold-adapted congener, M. trossulus, over large latitudinal expanses off the California coast. Several comparative physiological studies have revealed interspecific differences in thermal tolerance, including variation in aerobic metabolism and gape behavior, which suggest the invasive congener is better adapted to acclimate to increasing seawater conditions as predicted due to climate change. However, current analyses seek to discover the cellular process which contribute to thermal plasticity at the level of the whole organism in response to temperature stress. Since proteins represent the primary molecular machinery capable of responding to thermal stress, we quantified the proteomic response of the adductor muscles (AM) of M. galloprovincialis and M. trossulus to acute heat stress. After acclimation to 13°C, we exposed mussels to 24°C, 28°C and 32 °C (at a heating rate of 6C/h), kept mussels at the temperature for 1 h and then added a 24-h recovery period. Posterior adductor muscle samples were then excised and utilized for proteomic analysis. We were able to detect 273 protein spots within M. galloprovincialis and 286 protein spots within M. trossulus. Roughly 33% of these protein spots exhibited significant changes in abundance in response to heat stress within M. trossulus as compared to only 19% in M. galloprovincialis. In both data sets, most proteins changing abundance are part of the cytoskeleton or proteins controlling actin thin filament dynamics and stress fiber formation. Specifically, M. galloprovincialis increased the abundance of proteins involved in thin filament stabilization and cytoskeletal maintenance. In contrast, M. trossulus increased proteins involved in thin filament destabilization and filament turnover. In addition, only M. trossulus increased proteins involved in the cellular stress response at the highest temperature, suggesting its AM proteome is more thermolabile. In return, our results suggest that cytoskeletal architecture is more thermally stable in M. galloprovincialis. The differences in the proteomic responses suggest that M. galloprovincialis is capable of protecting itself from heat stress through valve closure at a higher temperature due to the increase in actin stabilizing proteins.

<p>Insects are continually exposed to potentially toxic endogenous compounds and xenobiotics that require rapid elimination from the body. Xenobiotic resistance in insects has evolved predominantly by increasing the activity of detoxification enzymes and/or by increasing toxin excretion via the Malpighian (renal) tubules. The tubules have long been known to transport organic anions at high rates. This thesis examines the mechanisms of excretion and detoxification of the organic anion methotrexate (MTX) by isolated tissues of the fruit fly <em>Drosophila melanogaster</em>. A radioisotope tracer technique and the Ramsay assay were used to measure MTX secretion. Quantitative PCR (qPCR) was used to evaluate the expression of the genes for putative organic anion transporters. My results show that MTX transport across the Malpighian tubule epithelium is active, saturable, Na⁺-independent and inhibited by a wide range of organic anions including MK-571, probenecid and Texas Red. Pharmacological studies and qPCR analyses suggest multiple transporters are involved in the movement of MTX across the Malpighian tubules. Moreover, chronic exposure of larvae to dietary MTX or salicylate dramatically increases the transepithelial transport of MTX by isolated Malpighian tubules, suggesting that excretion of MTX is upregulated by exposure to these organic anions in the diet. In addition, treatments known to increase expression of specific detoxification enzymes, such as the P450 monoxygenases (P450s) and the glutathione-S-transferases (GSTs), also led to an increase in expression levels of multidrug efflux transporter (MET), multidrug resistance like protein 1 (dMRP) as well as to increased secretion of MTX by the tubules. This latter finding suggests a coordinated response to toxin exposure, so that when detoxification pathways are increased, there is a corresponding increase in the capacity for elimination of the products of P450 and GST enzymes. Finally, the last section of this thesis has shown that RNAi knockdown of a single organic anion transporter gene in the principal cells of <em>D. melanogaster</em> Malpighian tubules is associated with reductions in the expression of multiple, functionally-related genes. Importantly, these results indicate that dMRP andMET are not the dominantMTX transporters in the tubules when flies are reared onMTX-enriched diets. However, reductions in the expression of organic anion transporting polypeptide (OATP) are associated with reduced secretion of the organic anionsMTX, fluorescein and Texas Red. Taken together, these results suggest that OATP and at least one additional transporter, as yet unidentified, are required forMTX secretion. In conclusion, the results of my research contribute to our understanding of the mechanisms of organic anion detoxification and excretion in flies exposed to dietary toxins.</p> / Doctor of Science (PhD)

<p>Aortic bodies (ABs) are putative peripheral arterial chemoreceptors located near the aortic arch. They are hypothesized to contribute to O₂ homeostasis by sensing arterial O₂ content and initiating cardiovascular reflexes during hypoxia; however, information on their cellular physiology is lacking. The primary goal of this thesis was to elucidate chemosensory mechanisms among mammalian (rat) AB cells, located specifically at the bifurcation of the left vagus nerve and recurrent laryngeal nerve (RLN), where they are found in association with a group of local neurons (>30). In vagus nerve-RLN whole-mounts, AB chemoreceptor (type I) cells were immunoreactive against the vesicular acetylcholine (ACh) transporter, and were surrounded by nerve terminals immunopositive for purinergic P2X2 and P2X3 receptor subunits, suggesting that ACh and ATP may act as neurotransmitters as in the related carotid body. In a novel dissociated AB culture model, subsets of type I cells exhibited elevated intracellular Ca²⁺ responses to hypoxia, isohydric hypercapnia, isocapnic acidosis, and acidic hypercapnia, demonstrating their direct chemosensitivity for the first time. Interestingly, surviving local neurons also responded to these chemostimuli, suggesting that they are sensory. Patch clamp electrophysiological and Ca²⁺ imaging studies revealed functional heteromeric P2X2/3 and nicotinic ACh receptors on local neurons, consistent with ACh and/or ATP mediating chemotransmission between receptor cells and local neurons. These neurons were also found to be interconnected by electrical synapses. Finally, the short-term survival of red blood cells (RBCs) in AB cultures, along with the finding that blood-borne factors (e.g. ATP released from RBCs) may have access to AB nerve terminals <em>in situ</em>, implicates RBCs as O₂-sensors in AB function. Altogether, these results suggest an important role for purinergic P2X2/3 receptors on local neurons/nerve terminals and ATP release from type I cells and RBCs, in the unique ability of ABs to sense and process information about blood O₂ content.</p> / Doctor of Philosophy (PhD)

<p>Prediabetes can lead to Type II Diabetes Mellitus, yet Prediabetes is a disease in its own right with its own physiological complications. Despite the pervasiveness of Prediabetes in our society and the negative impact on current and future health the extent of myopathy, short of muscle insulin resistance, and the mechanisms behind development of muscle insulin resistance remains unclear. Animal models of diet-induced obesity (DIO) have been employed to assess development of muscle insulin resistance and changes to muscle health. However there is a lack of clarity as to the molecular mechanisms leading to muscle insulin resistance. The goal of the studies presented here was to elucidate changes to muscle health and potential mechanisms contributing to muscle insulin resistance in response to DIO. Since the ability to perform exercise is to date one of the best therapies for Prediabetes and exercise contributes to a healthy muscle mass, the ability of muscle to undergo proper regeneration was also assessed following DIO. The results presented in this work demonstrate that skeletal muscle tissue adapts to increased dietary lipid by an early increase in functional lipid oxidation, mitigating IMCL deposition, despite glucose intolerance. Unfortunately this adaptive response is reversed with prolonged dietary fat intake and the development of muscle insulin resistance. Of note was the stronger link between IMCLs and muscle insulin resistance, compared to inflammation. DIO also led to decrements in satellite cell functionality which, along with physiological changes to HGF content and signaling, likely resulted in the observed impairment in regenerative ability. The results reported here improve our understanding of changes to muscle health and the mechanisms behind development of muscle insulin resistance with DIO. These findings have implications for therapies and treatments for Prediabetes.</p> / Doctor of Philosophy (Medical Science)

Rationale: Tissue Factor (TF) is a transmembrane glycoprotein that canonically functions as the initiator of the coagulation cascade. Increased levels of TF have been associated with inflammatory airway diseases. Since lipopolysaccharide (LPS) is known to elicit and inflammatory response in airway epithelium, we hypothesized that airway epithelial cells release TF when exposed to LPS. Since TF aids in local wound healing, we also hypothesized that inhibition of TF would decrease NHBE growth. The specific aim of this work was to evaluate the effects of LPS exposure on TF production and release from airway epithelia and determine the signaling pathways involved. A secondary aim was to evaluate the effects of TF inhibition on NHBE growth. Methods: Normal human bronchial epithelial cells were grown in submerged cell culture and exposed to LPS as well as several intracellular signaling pathway agonist and inhibitors. Measurements: Tissue Factor mRNA and protein were measured in culture media and cell lysate by reverse-transcriptase polymerize chain reaction and enzyme-linked immunosorbent assay, respectively. Signaling pathways were evaluated using selective agonists and inhibitors. Main results: TF protein levels increased nearly two-fold in cell media after exposure to LPS (p < 0.01). This did not occur in the presence of an MEK/ERK inhibitor (PD98059) or a SMAD inhibitor (SB431542). TF protein levels also increased nearly ten-fold in the presence of TGF-beta (p < 0.05). mRNA of TF and TGF-beta was not altered by LPS or TGF-beta exposure. NHBE grown in the presence of Tissue Factor Pathway Inhibitor grew significantly slower than those grown in standard media (P < 0.05). Conclusions: NHBE release TF when exposed to LPS. This phenomenon is post-translational and may be mediated by an autocrine mechanism involving MEK/ERK signaling that increases TGF-beta which then leads to the release of TF. Our data suggest that this airway epithelium release of TF serves as a local repair function.

Alcoholic Fatty Liver Disease (AFLD) is caused by excessive alcohol consumption and is a leading cause of liver related mortalities, with currently no treatments available. The goal of this project was to establish the effect of alcohol consumption on adipose tissue-derived secreted factors, adiponectin and C1q TNF Related Proteins 1-3 (CTRP1-3). We propose that excessive alcohol consumption will reduce circulating levels of adiponectin and CTRPs 1-3. Mice were fed a Lieber-Decarli control or alcohol diet for 10-days with a gavage (NIAAA model) or 6-weeks with no gavage (chronic model). Serum and adipose tissue were collected and CTRPs 1-3 and adiponectin levels were examined by immunoblot analysis. Our results indicate that long-term alcohol consumption effects adipokine secretion in a sex specific manner. Further research will be needed to explore the physiological relevance of these findings, to determine if these changes are beneficial to combat the negative effects of excessive alcohol consumption.

Insulin plays a vital role in whole-body metabolism and provides a major anabolic stimulus for cellular signaling pathways, including those involved in the metabolism of glucose and protein. Consequently, insulin dysregulation (ID) is known to alter molecular signal transduction in insulin-sensitive tissues such as skeletal muscle, thereby disrupting glucose metabolism and compromising protein synthetic capacity. Our first objective was to induce ID in healthy horses by administering dexamethasone (DEX), a potent glucocorticoid, for 21 days. We evaluated the effects on insulin-stimulated muscle protein signaling components involved in the mammalian target of rapamycin (mTOR) pathway. DEX-induced ID reduced insulin-stimulated activation of downstream (rpS6, 4EBP-1) mTOR signaling and increased atrogin-1 abundance, a marker for protein breakdown (P < 0.05). Additionally, 21 days of DEX elevated plasma amino acids levels in insulin-stimulated conditions, indicative of reduced uptake or increase release into circulation (P < 0.05). The second objective was to evaluate the short-term effects of DEX treatment in healthy horses. Plasma insulin, glucose and amino acid dynamics and activation of mTOR signaling pathways following an oral sugar test (OST) or intake of a high protein meal were evaluated before and after 7 days of DEX treatment, and after 7 days of no treatment. Seven days of DEX treatment increased basal levels of glucose, insulin and several amino acids (P < 0.05). Additionally horses treated with DEX had an exacerbated insulin response to the OST and consumption of the high protein meal in comparison to control horses (P < 0.05). The majority of blood metabolites returned to basal levels after 7 days of recovery from DEX treatment, indicating these effects were transient. Short-term DEX treatment decreased overall activation of mTOR and FoxO3 but increased total FoxO3 and IRS-1 abundance (P < 0.05). Postprandial activation of rpS6 was greater in horses treated with DEX for 7 days but was lower in those horses after 7 days of recovery from treatment (P < 0.05). Postprandial activation of ULK and AMPK tended to be greater in DEX treated horses (P < 0.1). Akt phosphorylation and mysotatin abundance were lower after the OST in DEX treated horses (P < 0.05). The final objective was to evaluate whether similar changes in postprandial metabolic responses would be seen in horses with naturally occurring ID. Plasma insulin, glucose and amino acid responses following ingestion of a high protein meal were determined in mature horses with equine metabolic syndrome (EMS). Horses with EMS had higher basal plasma insulin concentrations but lower levels of aspartate, glutamate, asparagine and plasma urea nitrogen in comparison to healthy controls (P < 0.05). Consumption of a high protein meal resulted in a 9-fold greater insulin response and higher postprandial levels of various amino acids (P < 0.05). Together this research indicates that ID affects whole body protein metabolism by altering cellular signaling pathways in healthy and diseased horses.

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.