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Marine ecosystems are facing various threats, from population declines to diseases that impact their overall health. My dissertation investigates the complex interplay between microbiomes, pathogens, and environmental conditions in three distinct marine organisms: Olympia oysters (Ostrea lurida) in the Puget Sound, Pacific oysters (Crassostrea gigas) in San Diego Bay exposed to OsHV-1 SDB µvar, and white abalone (Haliotis sorenseni) afflicted by Abalone Withering Syndrome. The goal of combining and comparing these systems is to elucidate the crucial role of microbiota in understanding ecosystem and host health, including microbes’ response to environmental variables and their interaction with pathogens. The first chapter focuses on the Olympia oyster, a native species in the Puget Sound that has experienced a substantial population crash. To assess the impact of eelgrass habitat and geographical location on oyster microbiomes, Olympia oysters from a single parental family were deployed at multiple sites, both within and outside eelgrass (Zostera marina) beds. Using 16S rRNA gene amplicon sequencing, I demonstrate that gut-associated bacteria differ significantly from the surrounding environment. Regional differences in gut microbiota are associated with the oyster survival rates at different sites after two months of field exposure. However, eelgrass habitat does not influence microbiome diversity significantly. This research highlights the importance of understanding the specific bacterial dynamics associated with oyster physiology and survival rates in the Puget Sound. In the second chapter, I explore the OsHV-1 SDB µvar, a virus threatening oyster aquaculture globally, with a focus on its microvariant in San Diego Bay. The study investigates the influence of temperature on OsHV-1 SDB µvar infectivity. All microvariants of this virus exhibit limited replication and are unable to induce oyster mortality at lower water temperatures. Through experimental infections of hatchery-raised oysters at temperatures ranging from 15 to 24°C, I found that no oysters died at 15°C but most exposed oysters died above 18°C. The infection took hold faster at 21 and 24°C compared to 18°C. As oysters are often immunocompromised by this viral infection, I also chose to focus on the potential contribution of secondary bacterial infections to the disease. The microbiome of healthy, sick and dead oysters was compared using 16S rRNA gene amplicon sequencing to determine how the microbiome is disrupted by infection and which bacteria may be responsible for further progression of the disease. There is a clear shift in microbiome composition and decreases in evenness following infection with OsHV-1 SDB µvar. The third chapter centers on Abalone Withering Syndrome, characterized by the intracellular parasite Candidatus Xenohaliotis californiensus (CaXc) which disrupts gut morphology leading to starvation and possible death. Investigating the microbiome in endangered white abalone exposed to CaXc over an 11-month period reveals dynamic variations in the fecal microbiome and its distinctiveness from the internal tissue microbiomes. CaXc exposure notably impacts the anterior region of the digestive tract more than the distal tissues and feces, sometimes representing up to 99% relative abundance in the post esophagus samples. This comprehensive analysis incorporates qPCR to quantify pathogen loads over time and feces and in internal tissues. The pathogen is detected after 5 months of exposure and is most abundant in the post-esophagus tissue. The samples with the highest relative abundance of the pathogen were also shotgun sequenced to generate whole genome assemblies of bacteria. This led to the novel assembly of a 90% complete genome for CaXc, which is deposited in a public database. To pair these data with a more holistic understanding of the impact of this pathogen, RNA sequencing data was analyzed for differential gene expression patterns between exposed and unexposed abalone. While functional annotation and prediction was poor on the de novo assembled transcriptome, clear differences exist in gene-level response to CaXc between post esophagus and digestive gland tissue.

All opioids - whether they are addictive opioid analgesics or endogenous opioids like endorphins produced by our body - activate one of three receptors in our brain. However, different opioids cause different effects, including effects on pain and addiction. How opioids generate this variety in signaling has been a long-standing question in the field. My thesis tests the exciting new idea that specific opioids traffic receptors to different compartments of the cell, from where they can signal in a compartment-specific manner. In this dissertation I review the selective mechanisms that regulate G protein-coupled receptor (GPCR) trafficking, which highlight the complex framework underlying spatial regulation of receptor function. Lipid membrane dynamics, post-translational modifications, cell-specific protein interactors, and agonist-selectivity are all key determinants for selective GPCR trafficking. Specifically, I focus on how opioid peptides may cause distinct functional outcomes through activation of the same receptor. To address this question, I use high-resolution imaging assays to examine how different dynorphin peptides regulated kappa opioid receptor (KOR) trafficking and post-endocytic sorting. Interestingly, we observe that highly related dynorphin peptides caused distinct opioid receptor trafficking fates (recycling vs. degradation) as well as compartment-specific signaling from late endosomes and lysosomes, depending on the peptide ligand that activated the receptor. Specifically, we show that KOR activation by dynorphin A leads to receptor degradation and activation from late endosomal compartments, while KOR activation by the highly related dynorphin B leads to receptor recycling from Rab5 and Rab11 compartments, without endosomal signaling. Further, I investigate the dynamic relationship between receptor signaling and receptor trafficking. Initially, we observed that agonist washout caused a decrease in mu opioid receptor (MOR) recycling. This finding led us to hypothesize that signaling downstream of receptor activation regulated receptor trafficking back to the cell surface, which regulates resensitization. To test this, I used total internal reflection fluorescence (TIRF) microscopy to detect real-time exocytic fusion events in living cells. I tested various components of known signaling pathways downstream of MOR activation using pharmacological inhibitors and phosphodeficient and phosphomimetic receptor mutants to quantitate differences in MOR recycling. Our results indicate that MOR recycling is regulated via an agonist-dependent Gβγ signaling pathway, resulting in the phosphorylation of the receptor’s C-terminal tail to increase receptor recycling. I follow up on this work by further testing the role of receptor phosphorylation in regulating MOR trafficking and signaling. By understanding the signaling pathways that regulate receptor trafficking and resensitization, this work opens new avenues for potential druggable targets. Collectively, this work increases our understanding of opioid physiology. Further, because the opioid receptor is a prototype for GPCRs - the largest and highly conserved family of signaling receptors in humans - the principles could be relevant across many physiologically relevant members of this family.

Portable communication systems require, in part, high-efficiency radio frequency power amplifiers (RF PA) if battery lifetime is to be conserved. Conventional amplifier classifications and definitions are presented in a unified and concise format. The Bipolar Junction Transistor (BJT) and Metal-Semiconductor Field Effect Transistor (MESFET) are evaluated as active devices in high-efficiency RF PA designs. Two amplifier classes (class CE and class F) meet the system requirements of an 850 MHz operating frequency, a power output of 3 W, a battery supply voltage of 9 Vdc, and a sinusoidal-type signal to be amplified. Both classes are evaluated through recent research literature and simulated using the PSpice® computer simulation program. Class CE and class F are found to provide efficiencies exceeding 80 percent under the given system constraints.