<p>A saugeye is the progeny of a female walleye (<i>Sander vitreus)</i> and male sauger (<i>Sander canadensis)</i>. In the United States, hybrid saugeyes are considered important for recreational fisheries and as a potential food source. Saugeyes grow exceptionally faster than their non-hybrid parents and are more tolerant of a broader range of water conditions. They are also of interest to anglers due to their increased growth rate and ease to catch. Rather unexpectedly, biologists have recently observed fish that they believe to be saugeye in the Fort Wayne Rivers even though only walleye have been stocked in the area. The fish in Hurshtown Reservoir are believed to be walleye and the identification of those in the Three Rivers is unknown. A potential source for saugeye in the Fort Wayne Rivers is St. Marys State Fish Hatchery in Ohio. This research aims to determine if the fish found in the Fort Wayne Rivers are walleye or saugeye using microsatellite analysis. Microsatellites at seven loci were genotyped for 20 reference walleye, sauger, and saugeye as well as 21 unknown fish caught near Fort Wayne. Of the fish caught near Fort Wayne, three are from Hurshtown Reservoir and 18 are from the Three Rivers. Assignment tests of genotypes were completed using model and non-model based cluster analysis. Genotypic variation clearly resolved the two parent species from their hybrid offspring. Sixteen of eighteen <i>Sander</i> (unknown species) caught in Fort Wayne Rivers between 2018 and 2019 were determined to be first generation saugeye. The other two were walleye found in the Maumee River downstream of Hosey Dam. The three <i>Sander</i> caught in Hurshtown Reservoir were verified to be walleye. Sauger have never been stocked in the Fort Wayne Rivers and connecting waterways. Therefore, it is not likely that the saugeye found in the analysis are from natural reproduction. It is speculated that saugeye are swimming to Fort Wayne from hatcheries within the Maumee watershed. There are many potential sources for walleye in the Fort Wayne Rivers. </p>

<p>The production of developmentally competent in vitro derived embryos is necessary to decreasing both economic and emotional losses. Epigenetic abnormalities/insults have been shown to occur at a higher incidence in in vitro embryos. An increased prevalence of epigenetic derived disorders such as Parkinson’s disease, Prader-Willi syndrome, and α-thalassemia as well as elevated preimplantation embryo arrest and reduced developmental rates are theorized to be caused by errors in the mediation of chromatin remodeling. Chromatin remodeling refers to the restructuring of packaged DNA so that transcription factors are either given more or less access to specific sequences. This can be done by covalent modification through histone methylation, acetylation, and phosphorylation as well as noncovalent modifications which employ ATP dependent chromatin remodeling complexes. The purpose of this thesis was to characterize two structurally integral core subunits, BAF155 and BAF170, of the SWI/SNF chromatin remodeling complex in porcine oocytes and preimplantation embryos. </p> <p>The first study concentrated on the transcript abundance of BAF155 and BAF170 in porcine oocytes and embryos. First, BAF155 and BAF170 transcript sequences were identified in porcine muscle and heart tissues. Those sequences were used to create quantitative polymerase chain reaction (qPCR) primers. mRNA from pools of GV oocytes (100-800) was converted to cDNA for transcript abundance measurements. However, transcript abundance remained too low for either BAF155 or BAF170 to be accurately quantified. </p> <p>The second study focused on developmental competency of embryos post interfering RNA (RNAi) knockdown of BAF155, BAF170, or both BAF155/BAF170 combined. After 7 days of culture, an analysis of variance (ANOVA) was performed to determine differences in mean nuclei numbers and morphological blastocyst percentages across the three groups. No significant difference was seen between means of treatment groups vs. both control groups. Significant differences were seen between siRNA and Non-Injected groups as well as Non-Injected and Scramble RNA groups. However this indicates that loss of BAF155, BAF170, or a combination of the two transcripts is not the driving force of the significant differences, rather the microinjection itself caused the differences.</p> <p>The third study examined the process by which BAF155 and BAF170 proteins are imported from the cytoplasm into the nucleus. It was hypothesized that karyopherin α 7 (KPNA7), a nuclear importer known to be prevalent in the porcine oocyte and early embryo, is the main importer of both subunits. A dominant-negative KPNA7 construct missing the importin beta binding (IBB) domain was microinjected into parthenogenetically activated embryos to outcompete competent wild-type KPNA7. No change in protein localization was seen at the 4-cell stage of development (48 hours post-injection) for either BAF155 or BAF170. To reinforce these results, an RNAi targeting KPNA7 was also microinjected into parthenogenetically activated embryos. Again, no change was shown in protein localization at the 4-cell stage (48 hours post-injection), indicating that KPNA7 was not the main nuclear importer of either BAF155 or BAF170.</p> <p>Further study is necessary to determine transcript abundance and the mechanism of nuclear import of both BAF155 and BAF170.</p><div><br></div>

<p>During gestation, the maternal liver undergoes various adaptive changes to cope with the increasing physiological and metabolic demands from both maternal and fetal compartments. Among these changes are robust growth and changes in transcriptome profile. However, how these events happen, and other aspects of this physiological phenomenon remains unexplored. Therefore, we aimed at further understanding how maternal liver responds to pregnancy. We used BrdU labeling combined with a virus-based tracing approach to quantify the percentage of maternal hepatocytes undergoing DNA synthesis and division over the course of gestation in mice. </p> <p>We found that ~50% maternal hepatocytes entered S-phase but, unexpectedly, did not undergo cytokinesis. This strongly suggests that maternal hepatocytes in fact undergo endoreplication instead of hyperplasia, as believed previously. Pericentral Axin2⁺ hepatocytes were reported to behave as liver stem cells responsible for liver homeostasis and turnover. We generated an <i>in vivo</i> fate-tracing mouse model to monitor the behavior of these cells in the maternal liver. Our results showed that they did not proliferate during pregnancy, homeostasis, and following partial hepatectomy. Curiously, we uncovered that, hepatocytes exhibit developmental phenotypes at mRNA level pre-pregnancy and at both mRNA and protein level during pregnancy. In the non-pregnant state, hepatocytes reserved mRNA expression of liver progenitor marker genes <i>Cd133</i> and <i>Afp</i>, which are localized in the nuclei, without protein translation. During gestation, maternal hepatocytes displayed cytoplasmic translocation of <i>Cd133</i> and <i>Afp</i> transcripts, concomitant with corresponding protein expression. </p> <p>Overall, all maternal hepatocytes became CD133⁺, and a subset of them express AFP. Additionally, in non-pregnant livers, mRNA of <i>Epcam</i>, another liver progenitor marker, was expressed within majority of hepatocytes, whereas its protein was solely translated in the pericentral region. In contrast, by end-gestation, EPCAM protein expression switched to the periportal region. These observations indicate that maternal hepatocytes exhibit heterogeneous developmental phenotypes, partially resembling fetal hepatocytes. It is intriguing why mature hepatocytes dedifferentiate into a progenitor state in response to pregnancy. AFP is considered to be produced primarily from fetal liver and thus is used to evaluate fetal development health. </p> A potential clinical relevance of our data is that we identified maternal liver as a new source of AFP. The hippo signaling pathway has been shown to potently control liver growth and hepatocyte heterogenicity. Surprisingly, we found that pregnancy neither altered the expression nor activities of the components of this pathway and its effector YAP1/TAZ. This finding indicates that pregnancy-induced maternal liver growth is not driven by hippo-YAP1 pathway. However, we demonstrate that the presence of YAP1 is essential for CD133 protein expression in maternal hepatocytes. Collectively, we revealed that, as pregnancy advances, maternal hepatocytes likely undergo endoreplication and display developmental phenotypes. Mechanistically, YAP1 dictates the expression of CD133, contributing to the pregnancy-dependent phenotypic changes of maternal hepatocytes.

<p>  </p> <p>Down syndrome (DS) is caused by the partial or complete trisomy of human chromosome 21 (Hsa21) and can result in skeletal deficits, including lower bone mineral density (BMD) and increased risk of fracture and osteoporosis or osteopenia earlier than the general population. Mouse models of DS have been developed to understand the genetic mechanisms resulting in these phenotypes, but models differ due to the complex genetic nature of DS and differing genome structures between humans and mice. Ts65Dn mice have been a popular model of DS as they contain ~50% of Hsa21 orthologous genes on a freely segregating minichromosome, but there is speculation that the phenotypes are exaggerated by non-Hsa21 orthologous trisomic genes also present. To address this issue, the Ts66Yah mouse model was developed to remove the non-Hsa21 orthologous trisomic genes. In this study, male and female Ts66Yah mouse femurs were evaluated during bone accrual and peak bone mass to investigate structural differences using micro-computed tomography. Additionally, the role of trisomic <em>Dyrk1a</em>, a Hsa21 gene previously linked to bone deficits in Ts65Dn mice, was evaluated through genetic and pharmacological means in Ts66Yah femurs at postnatal day 36. Ts66Yah mice were found to have little or no trabecular deficits at any age evaluated, but sex-dependent cortical deficits were present at all ages investigated. Reducing <em>Dyrk1a</em> copy number in Ts66Yah mice significantly improved cortical deficits but did not return cortical bone to euploid levels. Pharmacological treatment with DYRK1A inhibitor L21 was confounded by multiple variables, making it difficult to draw conclusions about DYRK1A inhibition in this manner. Overall, these results indicate trabecular deficits associated with Ts65Dn mice may be due to the non-Hsa21 orthologous trisomic genes, and more Hsa21 orthologous trisomic genes are necessary to produce trabecular deficits in DS model mice. As more mouse models of DS are developed, multiple models need to be assessed to accurately define DS-associated phenotypes and test potential treatments.</p>

The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

<p>  G Protein Coupled Receptor (GPCR) signaling plays a key role in intercellular communication and regulates many physiological processes relevant to disease. Approximately 30-40% of all FDA approved drugs target GPCR pathways, but limitations and off-target side effects remain obstacles. Regulator of G protein Signaling (RGS) proteins negatively modulate GPCR signaling by accelerating deactivation of the Gα subunit and thus represent a novel alternative to current approaches. While research on RGS proteins and how they are regulated has expanded rapidly, there are still gaps in knowledge for some members of the RGS family. One example is RGS2, which is selective for Gαq signaling. Lowered RGS2 levels are implicated in numerous diseases, and while the E3 ligase responsible for facilitating degradation of RGS2 has been identified more work needs to be done to viably drug it to enhance RGS2 protein levels. In this thesis, I explore how FBXO44, an E3 ligase substrate recognition component responsible for RGS2 degradation, interacts with RGS2 to explore approaches to inhibit degradation.</p> <p><br></p> <p>While the FBXO44-RGS2 interaction has been demonstrated previously, the degron sequence of RGS2 remained unknown. We hypothesized that FBXO44 binds RGS2 at its Nterminus and investigated this using N-terminally truncated RGS2 constructs. Our results indicated that FBXO44 binds between residues 5 and 16 of RGS2, as removal of these stabilized RGS2 against proteasomal degradation. Based on these results we designed a peptide microarray to identify important residues and properties for FBXO44 in vitro and found that Cys13 is essential for FBXO44 binding.</p> <p><br></p> <p>We also developed and optimized a high-throughput split luciferase screen to identify potential inhibitors of the FBXO44-RGS2 interaction. After forming a cell-line stably expressing tagged FBXO44 and RGS2 and optimizing assay condition, we achieved a robust assay for screening as determined by Z’-factor. <br>  </p>

<p>Sea urchins are fascinating marine creatures belonging to the phylum Echinodermata that serve as an essential ecological component and hold promise as a prospective source of therapeutics. However, sudden environmental changes, such as global warming and marine pollution, are placing significant stress on these organisms. To maintain natural resources and exploit sea urchins commercially, researchers are investigating aquaculture as a solution.</p> <p>This investigation discloses the physiological and immunological effects of physical and chemical stressors on one of the most common edible species of sea urchin, <em>Arbacia punctulata</em>. The study employed an elevated temperature as a physical stressor (1°C/day), lipopolysaccharides (LPS) inoculation as a chemical stressor (4µg/ml/day), and a combination of both LPS and elevated temperature as combined stressors. The results demonstrated a significant alteration in the total and differential coelomocyte count in the LPS-stressed group (p<0.05) and combined stressed group (p<0.05) followed by abnormal behavioral activity compared to those of control. Additionally, exposure to acute LPS exposure (at day 1 and day 3) and combined stressors led to an increase in phagocytic capacity (p<0.05) and lysozyme activity (p<0.05). Chronic exposure to LPS and combined stressors resulted in a decrease in gonadosomatic index (p<0.05, at day 10) and lysozyme activity (at day 7). A significant increase in coelomic fluid (CF) protein (p<0.05)was observed in the temperature-stressed group on days 5 and 10, while the combined stressed group had significantly more CF protein on days 1, 5, 7, and 10. An upregulation of Nf-kB gene expression was also observed (p>0.05) in temperature stressed group.  </p> <p>The study also revealed that sea urchins contain bioactive compounds that protect against external and internal injury, cell death, and body wall extract of sea urchin exhibited high antioxidant activity(p<0.05). Furthermore, it confirmed the antibacterial activity (p<0.05) of sea urchin (<em>Arbacia punctulata </em>and<em> Lytechinus variegatus</em>) body wall and coelomic fluid (cell-free plasma) extracts against ten pathogenic bacteria. The ethyl acetate body wall extract of both sea urchin species demonstrated higher inhibitory activity against the pathogenic bacteria tested. Overall sea urchin has potentials to meet the demand of food and medicine. </p>

<p>  </p> <p>Cilia are cell appendages that sense our environment and are critical in cell-to-cell communication. Dysfunction of cilia can result in several disease states including obesity. While cilia in the brain are known to be important for feeding behavior, it is unclear how they regulate energy homeostasis. Classically, cilia coordinate signaling through surface receptors called G-protein coupled receptors (GPCRs). For example, cilia mediated GPCR signaling is critical for both our senses of vision and smell. How cilia regulate the signaling of GPCRs in other areas of the body including the brain is only now emerging. To answer cell biology questions around cilia mediated GPCR signaling in neurons, we developed a system for primary neuronal cultures. We discovered that the cilia mediated hedgehog pathway influences the ability of neurons to respond to GPCR ligands. For the first time, this result highlights the role of the hedgehog pathway in neurons. We continue to explore how cilia integrate the hedgehog pathway and GPCR signaling in the central nervous system, and the potential connections to energy homeostasis. We discovered that hedgehog pathway activity in feeding centers of the brain changes based upon feeding conditions like fasting. We also learned that activating the hedgehog pathway in these brain regions is sufficient to cause obesity in mice. These novel results highlight an unrecognized role for the hedgehog pathway in the regulation of feeding behavior. Overall, this work provides a better understanding of ciliopathy associated obesity and may reveal more common mechanisms of obesity in the general population. In addition, this work implicates the hedgehog pathway in regulating behaviors and new modes of cell-cell communication within the central nervous system.</p>

<p>Cerebral radiation necrosis as a consequence of radiation therapy is often observed in patients several months to years after treatment. Complications include painful headaches, seizures, and in the worst-case death. Radiation necrosis is an irreversible condition with the options available to manage it all having noticeable downsides. As such, there is a critical need for better ways of either preventing the onset of necrosis and/or managing its symptoms. As radiation necrosis cannot be induced in humans for ethical reasons, a mouse model that mirrors the features of radiation necrosis observed in patients would allow for new techniques to be tested before being used in human clinical trials. This thesis will explain how our lab designed a murine model of cerebral radiation necrosis that uses a 320 keV cabinet irradiator to produce radiation necrosis and MRI and histology to evaluate the development of radiation necrosis at multiple time points.</p><p><br></p> <p> </p> <p>Our model required the development of a mouse positioning apparatus that could be used in the cabinet irradiator used as well as the machining of lead shields so that focal semi-hemispheric irradiations could be conducted with other critical structures spared. The MRI scans used as well as the algorithm used to draw radiation necrosis lesions were based off what has been used in previous Gamma Knife models of radiation necrosis. Our initial work showed that since the cabinet irradiator has a relatively flat dose distribution unlike the Gamma Knife, the radiation lesion volumes produced in the former either plateaued or decreased, unlike in the case of the latter where lesion volumes tended to decrease over time. Further work analyzed the effects of fractionation and found minimal sparing using four different fractionation schemes. The effects of strain and sex on the development of radiation necrosis were also analyzed, with strain being found to be a statistically significant parameter while sex was not. Future research should focus on testing the effects of new drugs and techniques for better dealing with radiation necrosis.<b></b></p>

<p dir="ltr">Given the relatively poor visual acuity of compound eyes, many insects have developed alternative means for navigating their environment. For example, insects often rely on chemosensation to find food, mates, and inter- and intraspecific communication. Eusocial insects in particular have developed complex systems of pheromone communication to organize their colonies, enabling them to partition labor for foraging, brood care, and colony defense tasks to different portions of the colony. A variety of genes coding for proteins are involved in detecting these chemicals, including gustatory receptors, ionotropic receptors, and odorant receptors (ORs). Eusocial insects, and especially ants, have evolved an expanded clade of ORs in their genome, likely due to an increased reliance on pheromones compared to other insects. The ability to recognize nestmates from non-nestmates is one of the vital functions performed by these ORs, which detect hydrocarbons present on the cuticle to distinguish friend from foe. However, research into the details of nestmate recognition has been stymied due to difficulties in manipulating OR genes. Despite advances in genetic sequencing and manipulation technologies, strict reproductive divisions within most ant lineages make generating transgenic ants nearly impossible, and so we have been left with limited options to further investigate these receptors. To narrow down the ORs that could be involved in nestmate recognition in the Argentine ant (Mayr, 1868), I took a multi-pronged approach of generating tissue transcriptomes to identify ORs that are selectively upregulated in the antennae, as well as conducting a phylostratigraphic analysis to identify which OR genes arose more recently in the Argentine ant genome. While conducting these analyses, it became necessary to reannotate the set of Argentine ant OR genes, due to current published annotations not containing the full breadth of <i>L. humile</i> ORs. Finally, I orally administered fluorescently-labelled dsRNA to workers, and tracked the extent to which ingested dsRNA is capable of traversing the tissues of ant workers, to investigate whether RNAi is a viable method for investigating gene function for genes showing tissue-selective expression. I discovered a subset of OR genes that are highly expressed in the antennae and confirmed that dsRNA is able to reach the antennae and knock down OR gene expression through ingestion, meaning that RNA interference is a viable method for the practical study of ant OR genes and can be used to further explore how individual ORs regulate nestmate recognition.</p>