RNA interference or RNAi was first discovered as an experimental approach that induces potent sequence-specific gene silencing. Remarkably, subsequent studies on dissecting the molecular mechanism of the RNAi pathway reveal that RNAi is conserved in most eukaryotes. In addition, genes and mechanisms related to RNAi are employed to elicit the regulation of endogenous gene expression that controls a variety of important biological processes. To investigate the mechanism of RNAi in the nematode C. elegans, we performed genetic screens in search of RNAi deficient mutants (rde). Here I report the summary of the genetic screens in search of rde mutants as well as the identification of two novel genes required for the RNAi pathway, rde-3 and rde-8. In addition, we demonstrate that some of the rde genes, when mutated, render the animals developmentally defective, suggesting that these rde genes also function in developmental gene regulation. This work presents novel insights on the components of the RNAi pathway and the requirement of these components in the regulation of endogenous gene expression.

Feeding is one of the most important behaviors for an animal’s survival. At a gross level, it is known that the nervous system plays a major role in the expression of this complex behavior, yet a detailed understanding of the neural circuits directing feeding behavior remains unknown. Here we identify a command neuron in Drosophila melanogaster whose artificial activation, using dTrpA1, a heat-activated cation channel, induces the appearance of complete feeding behavior. We use behavioral, genetic, cellular and optical imaging techniques to show that the induced behavior is composed of multiple motor programs and can function to uptake exogenous, even noxious, material. Furthermore, we resolve the neuron’s location to the subesophageal ganglion, characterize its pre and post-synaptic sites, and determine its responsiveness to sucrose stimulation. Interestingly, the neuron’s dendritic field is proximal to sweet sensing axon terminals and its baseline activity corresponds to the fly’s satiation state, suggesting a potential point of integration between sensory, motor and motivational systems. The identification of a command neuron for feeding in a genetically tractable organism provides a useful model to develop a deeper understanding of the neural control of this ubiquitous and evolutionarily ancient behavior.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active players in the development and integrity of the nervous system. The formation of functional neuronal circuits requires the precise addition of new synapses. Mounting evidence implicates glial function in synapse remodeling and formation. However, the precise molecular mechanisms governing these functions are poorly understood. My thesis work begins to define the molecular mechanisms by which glia communicate with neurons at the Drosophila neuromuscular junction (NMJ). During development glia play a critical role in remodeling neuronal circuits in the CNS. In order to understand how glia remodel synapses, I manipulated a key component of the glial engulfment machinery, Draper. I found that during normal NMJ growth presynaptic boutons constantly shed membranes or debris. However, a loss of Draper resulted in an accumulation of debris and ghost boutons, which inhibited synaptic growth. I found that glia use the Draper pathway to engulf these excess membranes to sculpt synapses. Surprisingly, I found that muscle cells function as phagocytic cells as well by eliminating immature synaptic ghost boutons. This demonstrates that the combined efforts of glia and muscle are required for the addition of synapses and proper growth. My work establishes that glia play a crucial role in synapse development at the NMJ and suggests that there are other glial-derived molecules that regulate synapse function. I identified one glial derived molecule critical for the development of the NMJ, a TGF-β ligand called Maverick. Presynaptically, Maverick regulates the activation of BMP pathway confirmed by reducing the transcription of the known target gene Trio. Postsynaptically, it regulates the transcription of Glass bottom boat (Gbb) in the muscle suggesting that glia modulate the function of Gbb and consequently the activation of the BMP retrograde pathway at NMJ. Surprisingly, I also found that glial Maverick regulates the transcription of Shaker potassium channel, suggesting that glia potentially could regulate muscle excitability and consequently modulate synaptic transmission. Future work will elucidate such hypothesis. My work has demonstrated two novel roles for glia at the NMJ. First is that glia engulfing activity is important for proper synaptic growth. Second is that the secretion of glial-derived molecules are required to orchestrate synaptic development. This further supports that glia are critical active players in maintaining a functional nervous system.

Global biodiversity is declining as a direct result of anthropogenic climate change. Ectothermic species have become focal organisms for studying the ecological effects of altered climates due to the clear relationship between environmental temperatures and ectotherms’ basic physiological functions. Historically, examinations of these effects have focused heavily on heliothermic lizards, and most others have tended to focus on single populations or sympatric species within a single community. Addressing the longterm energetic implications of environmental temperature variation will provide valuable insight into the cascading physiological effects that certain populations or species may experience as a result of altered climates. In this study, we used thermal and behavioral data collected between 2010 and 2017 from four distinct populations of Pacific rattlesnakes (Crotalus oreganus) on the Central Coast of California. Two of these populations occupy thermally mild, coastal habitats while the other two occupy more thermally dynamic, inland habitats. Using operative temperature models, surgically implanted temperature loggers, and radiotelemetry, we collected data on the thermal microhabitats available within each of these study sites as well as field active body temperatures for 85 individual snakes. With the addition of a lab-derived preferred body temperature range, we determined the thermal quality of each site and the thermoregulatory accuracy of snakes from each population. Field behavioral observations, gathered from snakes at all four sites simultaneously during the year 2017, revealed how snakes utilize the thermal landscape and adjust thermoregulatory behavior to mitigate the effects of different climates. Although overall thermal quality was best at coastal sites, thermal quality of the microhabitats within each site varied greatly. Consistent with findings in other squamate reptiles, inland snakes thermoregulated more accurately, despite being in more thermally constrained environments. Despite the fact that coastal snakes had lower mean field active body temperatures, the preferred body temperatures of snakes were the same across all four sites. However, field active body temperatures were consistently lower than the preferred range, suggesting there are additional variables that influence thermoregulatory behavior. Using established equations estimating the resting metabolic rates of snakes based on body mass and temperature, we calculated resting metabolic rate and annual baseline maintenance energy expenditure for each population. Coastal snakes, which had lower field active body temperatures, had overall lower metabolic rates than inland snakes, but upon correcting for mass, snakes at neither coastal nor inland sites differed in metabolic rates. Therefore, the majority of the differences observed in metabolic rates are driven by body size and not field-active body temperature. Inland snakes need, on average, approximately 1.6x more food annually than coastal snakes. Due to overall low resting metabolic rates, this translates to snakes at all sites needing less than one ground squirrel (their most common food item on the Central Coast) per year to fuel basic physiological functions. Finally, we used conservative predictive climate change models allowing either 1°C or 2°C increases to predict changes in the thermal quality of each site and ensuing changes in snake metabolic rates and maintenance energy expenditure. Due to the relatively high preferred body temperature of C. oreganus, thermal quality of the environment will actually increase under these climate models; due to an increase in ambient temperature, the proportion of hourly temperatures that fall within the preferred body temperature range will increase. If snake body temperature were to increase as the climate warms, a theoretical increase in body temperature of 1 and 2°C would have a low impact on the overall energetic needs of snakes, still allowing them to meet baseline maintenance energetic needs with only one large meal a year. Furthermore, we expect small increases in ambient temperature to have little impact on rattlesnakes because they are fairly precise thermoregulators, maintaining fairly constant body temperatures regardless of their thermal surroundings. Overall, our results show that studying the thermal ecology of multiple populations of a single species can reveal fine-scale information about the relationship between the thermal landscape and both ectotherm behavior and physiological processes. Additionally, our findings show that some species of large-bodied reptiles may be robust to modest thermal perturbations under conservative climate change predictions.

Molting in crustaceans is a highly complex physiological process involving negative regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone (MIH) which negatively regulates synthesis of the molting hormones, ecdysteroids, by the YO. Analysis of gene expression in the XOs and YOs has led to the development of a proposed molecular signaling pathway which regulates ecdysteroidogenesis and subsequent molting in crustaceans. In this study, changes in protein abundance in the YO were characterized over the course of a molt cycle (intermolt, early premolt, mid premolt, and late premolt) induced by multiple leg autotomy (MLA) in the blackback land crab, Gecarcinus lateralis. In all, 457 distinct protein spots were detected in the molting gland using two-dimensional gel electrophoresis, of which 230 (50%) changed significantly in abundance over the course of the molt cycle (one-way permutation ANOVA, p≤0.05). Changes in protein abundance were most notable between the intermolt and the three premolt stages, indicative of a biological ‘on-off’ switch in the Y-organ. Several hemolymph species proteins, including hemocyanin, cryptocyanin, and transglutaminase, were identified which characterized physiological changes associated with molting beyond the Y-organ. An abundance of cytoskeletal proteins were identified which correspond with glandular hypertrophy and are indicative of vesicular-mediated exocytosis, possibly of ecdysteroids. Further, several proteins involved in the immune, proteostasis, and oxidative stress response are characteristic of supporting the dynamic and demanding cellular changes associated with ecdysteroidogenesis and the transition of the Y-organ from the basal to the highly active state. Many proteins involved in energetic pathways including glycolysis, the citric acid cycle, amino acid metabolism, and one-carbon metabolism changed in abundance in response to both the higher energy demands and the requirement for precursors of macromolecular synthesis of the YO over the molt cycle. Taken together, these changes in diverse physiological pathways represent the complexity involved with regulation of the Y-organ, even with just the single proposed physiological purpose of ecdysteroidogenesis.

Limited breeding success in captive breeding programs has necessitated the development of assisted reproductive technologies (ART) to preserve and increase genetic variation and population numbers of both captive and wild amphibian groups. ART has been shown to be successful in numerous anuran species, and current studies focus on the application of ART in ex-situ populations. The focus of this project is to show that linking in-situ and ex-situ amphibian populations through sperm cryopreservation, genome banking, and in-vitro fertilization is possible, with the goal of increasing gene diversity throughout groups in order to produce self-sustaining, wild populations in the future. Specific objectives include developing a sperm-cryopreservation methodology using sperm from the model species Anaxyrus fowleri, applying this protocol to the cryopreservation of spermatozoa from two other threatened anurans to determine protocol transmissibility, and linking in-situ and ex-situ populations of an endangered species using cryopreserved sperm form wild males to produce viable offspring.

The prevalence of non-alcoholic fatty liver disease (NAFLD) in children has increased over the past decades, creating a need for animal models that recapitulate the features of the pediatric disease. Iberian pigs have a leptin-resistant phenotype characterized by hyperleptinemia, hyperphagia, and extreme adipogenesis. We hypothesized that neonatal Iberian pigs fed a high fat high-fructose (HFF) diet will develop a pattern of liver injury resembling pediatric NAFLD. In addition, we sought to determine if a mixture of probiotics would prevent the disease. Animals were fed 1 of 4 diets containing (g/kg body weight × d) 0 g fructose, 11 g fat and 199 kcal (CON-N; n=8), 22 g fructose, 16 g fat and 300 kcal (HFF2-N; n=8), CON + probiotic (CON-P; n=6), or HFF2 + probiotic (HFF2-P; n=6) every 6 h for 70 d. The probiotic mixture (6.2 × 104 cfu/mL) contained Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum and Bacillus amyloliquefaciens. Body weight was recorded every 3 d. Serum markers of liver injury and dyslipidemia were measured on d 40 and 65 at 2 h post feeding. Fasting leptin, insulin, glucose and homeostatic model assessment (HOMA) values were assessed on d 70. Liver and skeletal muscle (longissimus dorsi) were collected on d 70 for histology, triacylglyceride (TAG) quantification, relative gene expression, and Western blot analysis. Metabolomic analysis was performed on liver tissue and plasma. Body weight was not significantly greater in HFF fed pigs compared to CON. Leptin, alanine and aspartate aminotransferases, alkaline phosphatase, lactate dehydrogenase and total bilirubin were increased (P ≤ 0.001), and high and low density lipoproteins decreased (P ≤ 0.05) in HFF2-N and HFF2-P. Livers in HFF2-P and HFF2-N had higher relative weight and TAG (P ≤ 0.001), micro and macrovesicular steatosis, ballooning degeneration, Mallory-denk bodies, inflammation and necrosis, increased gene expression of TNFα, TGFβ, IL1α and PPARγ (P ≤ 0.001), and decreased ChREBP (P ≤ 0.001). A probiotic affect was seen as pigs fed CON-P and HFF2-P had higher insulin and HOMA values were increased (P ≤ 0.05). Western blot analysis showed dysregulation of autophagy in liver of pigs fed CON-P and HFF2-P, and in skeletal muscle of pigs fed CON-N and HFF2-N. Metabolomic analysis demonstrated dysregulation of one-carbon metabolism, the tricarboxylic acid cycle (TCA), the urea cycle, and amino acid metabolism of pigs fed HFF2 diets compared to CON diets. In conclusion, Iberian pigs fed a HFF diet recapitulate many pediatric NAFLD-associated features, in the absence of obesity and independently of probiotic supplementation, suggesting a potentially suitable model for pediatric NAFLD research. Furthermore, probiotic supplementation did not ameliorate the onset of NAFLD when fed in conjunction with a HFF diet.

Amidst the amphibian extinction crisis, in situ and ex situ amphibian species conservation initiatives utilize assisted reproductive technologies for optimal genetic management of captive and wild populations. Development of effective, simple, low-cost methods for obtaining gametes for artificial fertilization, sperm biobanking, or natural breeding alleviates obstacles for institutions or programs to apply reproductive technologies. Objectives herein include investigating the efficacy of alternative hormones for inducing spermiation, an alternative hormone administration route for inducing ovulation, exploring the physiological effects of hormone therapy, and utilizing sperm from deceased animals for biobanking.

Cocaine is a plant-based illicit drug commonly involved in substance use disorder. Although cocaine overdose and cocaine use disorders cause adverse health consequences to individuals and the economic burden on their family and society, there are no FDA (Food and Drug Administration) approved medications for treatment. Recently, it has been recognized that delivery of cocaine hydrolase (CocH) is a promising therapeutic strategy. Human butyrylcholinesterase (hBChE), the primary enzyme involved in cocaine metabolism in human, have advantages over other candidates for the development of CocH. Previous studies in our laboratory have designed and characterized hBChE mutants that have ~4,000-fold improved catalytic efficiency against naturally occurring (-)-cocaine as compared to the wild-type hBChE. Besides the catalytic efficiency, the biological half-life is another essential factor that influences the desired therapeutic value in the long-term treatment of cocaine use disorder. In order to provide prolonged effects to reduce administration frequency in clinical use, efforts have been made to increase the retention time of CocHs in blood circulation by fusing CocHs with other thermostable proteins or their mutants, including human serum albumin (Albu) or the Fc region of the human IgG (Fc). In this dissertation, we demonstrated the clinical potential and the benefits of long-lasting CocHs for cocaine overdose treatment. We used rodent models to show the ability of AlbuCocH1 to block or reverse manifestations of toxic effects of cocaine. In addition, a concomitant LC-MS/MS-based analysis was conducted to investigate the pharmacokinetic profile of a lethal dose of cocaine with the presence of AlbuCocH1. These experimental data demonstrated AlbuCocH1 as an effective cocaine detoxification agent by accelerating the metabolism of cocaine. In order to examine the potential therapeutic value of Fc-fused CocHs in the treatment of cocaine use disorder, we conducted a series of behavioral experiments in rats to evaluate the effectiveness and duration of Fc-fused CocHs in blocking or attenuating cocaine-induced psychostimulant and discriminative stimulus effects. In addition, the intravenous self-administration model was used to investigate the long-term effectiveness of Fc-fused CocHs in blocking or attenuating the reinforcing effects of cocaine. It has been shown that a single dose of E30-6-Fc (3 mg/kg) was able to effectively alter the cocaine dose-response curve and attenuate the reinforcing efficacy of cocaine for at least a month in both male and female rats. In summary, AlbuCocH1 (TV-1380), which failed to meet the primary efficacy endpoint in clinical trials for facilitating abstinence in cocaine-dependent subjects with a weekly dosing schedule (due to the short biological half-life), is more suitable to be developed as a cocaine detoxification agent. On the contrary, the newly designed Fc-fused CocH (e.g. CocH3-Fc, E30-6-Fc) with higher catalytic efficiency and longer biological half-life will be beneficial for long-term abstinence management in cocaine-dependent individuals.

Equine arteritis virus (EAV) establishes persistent infection in the stallion reproductive tract, and the carrier stallion continues to shed virus in semen for weeks to years or lifelong. The objective of this study was to elucidate the intra-host evolution of EAV during persistent infection in stallions. Seven EAV seronegative stallions were experimentally infected with EAV KY84 strain and followed for 726 days post-infection, and sequential clinical samples including semen were collected for virus isolation and next-generation sequencing (NGS). In addition, archived sequential semen samples from two stallions that were naturally infected with EAV KY84 for a long-period (up to 10 years) were also sequenced by NGS. The data demonstrated genetic bottleneck event and selection during acute infection followed by intra-host quasispecies diversification during persistent infection in the stallion reproductive tract. Also, the full-length genome of a novel EAV donkey strain from Chile and a noncytopathic bovine viral diarrhea virus-1 (ncpBVDV-1) strain contaminating rabbit kidney-13 cells were also sequenced by NGS. The EAV donkey strain was genetically distinct but antigenically cross-reacted with EAV antisera, and it was phylogenetically closely related to the South African donkey strain of EAV. Genetic and phylogenetic analyses demonstrated that ncpBVDV-1 belongs to BVDV-1b group.