Master of Science in Pharmaceutical Science (MSP), University of Toledo, 2020, Pharmaceutical Sciences (Pharmacology/Toxicology)

Autosomal dominate polycystic kidney disease (ADPKD) poses a high risk for catastrophic renal cysts, hypertension, and fatal cardiac complications among afflicted patients. Due to its nature as a ciliopathy, researchers are searching the ciliary proteome for viable treatment targets.Muscarinic acetylcholine receptors (AChMRs) are widespread throughout the body and play various homeostatic roles; AChM3Rs in particular are predominately expressed in smooth muscles including the endocrine glands, vascular system, and heart. AChM3Rs also are localized to primary cilia where they play roles in cholinergic vasodilation responses; due to their localization to primary cilia, it was hypothesized that AChM3Rs are involved in the pathogenesis of ADPKD. In this study, a morpholino oligonucleotide (MO) targeting chrm3b, the zebrafish homologue to CHRM3, was used to knockdown AChM3R expression and observed the resultant phenotypes in developing embryos. Based on previous work and ADPKD zebrafish MO studies performed by others, a loss of primary cilia structure was expected to occur, resulting in multisystemic effects such as kidney cysts and cardiovascular aberrations as a result of the MO. While heart aberrations were indeed observed, along with developmental delays and increased mortality, no direct evidence that iv primary cilia were affected by the MO was observed. Nodal cilia, however, were visually absent or decentralized (scattered and few in number) within the neural tube/floorplate after injection, resulting in altered cerebrospinal fluid flow. Additionally, the disruption of neural tube nodal cilia was accompanied by a number of phenotypes including abnormal body curvature, fluid retention in the brain, kidney damage, and large gaps in the extracellular matrix between adjacent fibers in their myotome/muscle tissues. Because nodal cilia in injected groups were still present and functional elsewhere, these results imply a link between the nodal cili (open full item for complete abstract)

Committee: Wissam Aboualaiwi Dr. (Committee Chair); Williams Frederick Dr. (Committee Member); William Messer Dr. (Committee Member); Frank Hall Dr. (Committee Member) Subjects: Pharmacology

Master of Science, University of Akron, 2017, Biology

Mammalian leptin (LEP) is a pleiotropic peptide hormone best characterized for its roles related to obesity and diabetes. However, the molecular function of the leptin signal transduction pathway in non-mammals is less clear. Comparative studies that address leptin signaling in non-model organisms are integral components of the leptin phylogenetic history, and there is little evidence addressing the functional disparities between the teleost leptin paralogues and mammalian leptins. To demarcate genes and biochemical pathways regulated by leptin signaling in developing zebrafish, microarray gene expression data were generated with total RNA isolated at 48 hours post fertilization from leptin-a morpholino oligonucleotide “knockdown”, recombinant leptin-a “rescue”, and wild type embryos. Expression estimates were computed for 26,046 genes across 16 microarray samples. Differentially expressed genes (DEG), (KEGG) pathways, and Gene Ontologies (GO) were evaluated for three contrasts (Morphant:Control, Rescue:Morphant, Rescue:Control). Signaling pathways that respond to leptin-a knockdown and rescue are analogous to gene targets of the mammalian LEP system (“GnRH”, “MAPK”, “Adipocytokine”, “Phosphatidylinositol”, “mTOR”, “ErbB”, “FoxO”, and “Notch”). A subset of differentially expressed transcription factors in leptin-a morphants are homologous to putative regulators of LEP expression in mammals (cebpb, creb5, fosl1a, mybl1, pax5, pou3f1, pparg, stat1a). “Neuroactive ligand-receptor interaction” as well as cAMP-responsive hormone signaling pathways responded to leptin-a. Consistent with leptin-a as an endocrine regulator, agouti-related peptide-2 (agrp2), cocaine-and-amphetamine-related-transcript (LOC557301), gonadotropin-releasing hormone 2 (gnrh2), and melanocortin receptor 5a (mc5ra) were dysregulated in rescue embryos. Further, “Notch signaling” and “Spinal cord/CNS development” were enriched in morphants whereas rescue arrays were comparable to wild type e (open full item for complete abstract)

Committee: Richard Londraville (Advisor) Subjects: Bioinformatics; Biology; Comparative; Developmental Biology; Endocrinology

Doctor of Philosophy, University of Akron, 2014, Integrated Bioscience

In this dissertation, I tested the hypothesis that leptin A in zebrafish (D. rerio) plays a similar role to mammalian leptin in regulating metabolic rate and immune function, whereas leptins's effects on the zebrafish transcriptome may be distinct. Leptin is now identified in all major vertebrate lineages, but its role in controlling food intake, development, metabolic rate, and fat storage is best studied in mammals. In that group, leptin has pleiotropic effects including those on angiogenesis, bone formation, reproductive status, immune function, and energy expenditure. A homozygous mutation (ob-/ ob-) for leptin is the most common model for leptin study in mammals. The use of leptin-null mutants in non-mammal models is not common. We recently developed a leptin knockdown model in zebrafish and applied a comparative approach to studying some well-characterized mammalian leptin functions in this new system. I tested the impact of leptin expression on metabolism in the developing zebrafish embryo. Leptin knockdown reduced oxygen consumption most prominently during early development (24-48 hours post fertilization, hpf) whereas carbonic acid production was most significantly attenuated later in development (48-72 hpf). Cardiac output was significantly reduced in embryos with reduced leptin expression (leptin morphants); all of these effects could be rescued by co-injection of recombinant fish leptin. The second part of my research focused on the innate immune response. When presented with a bacterial challenge, leptin morphants had reduced macrophage respiratory burst activity and bacterial load clearance was unaffected 12 hours post infection (hpi). By 36 hpi, leptin morphants had significantly increased bacterial burden and reduced survival compared to control embryos. I then focused on the transcriptomic effects of reduced leptin A expression in the developing zebrafish embryo. Microarray analysis identified sensory and development pathways as the most signific (open full item for complete abstract)

Committee: Richard Londraville Dr (Advisor); Qin Liu Dr (Committee Member); Zhong-Hui Duan Dr (Committee Member); Ahmed Mustafa Dr (Committee Member); Brian Bagatto Dr (Committee Member) Subjects: Bioinformatics; Molecular Biology

Master of Science in Pharmaceutical Sciences, University of Toledo, 2010, College of Pharmacy

Cytosolic sulfotransferases are phase II drug metabolizing enzymes responsible for carrying out a sulfonation reaction which adds a sulfonyl group to a hydroxyl or amino group of its substrate. These enzymes are believed to be involved with the regulation of endogenous hormones such as neurosteroids as well as xenobiotics. Considering the increasing rates of environmental estrogen exposures, it is important to understand the role that sulfotransferases may play in the early stages of vertebrate development. SULT2 ST1 is a sulfotransferase identified in zebrafish which resembles the SULT2 A1 gene in humans. To establish a link between sulfotransferase expression and developmental toxicity, the SULT2 ST1 gene was knocked down in the zebrafish model. This was accomplished by microinjection of translation blocking morpholino oligonucleotides into <4 cell stage zebrafish embryos. The phenotypic changes observed in the SULT2 ST1 knockdown fish indicate that the absence of the enzyme may be responsible for underdeveloped fin and systemic failure in embryo development phenotypes. Western Blot was used to confirm a reduction in expressed protein in knockdown animals.

Committee: Frederick Williams PhD (Committee Chair); Ming-Cheh Liu PhD (Committee Member); Steven Peseckis PhD (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science in Pharmaceutical Sciences, University of Toledo, 2010, College of Pharmacy

Sulfonation is an important reaction in regulating the biological activities of a variety of endogenous and environmental compounds and is catalyzed by cytosolic sulfotransferases that use PAPS (3'-phosphoadenosine-5'-phosphosulfate) as the sulfonate (SO3-) donor. SULTs are present in mammals and other vertebrates and play an important role in the detoxification of xenobiotics especially environmental estrogens. SULTs are also involved in the biotransformation of endogenous compounds (hormones, steroids) which might be a mechanism for maintaining the homeostasis of these compounds in-vivo. In this study we have attempted to assess the role of the hydroxysteroid sulfotransferase SULT2 ST2 in zebrafish (Danio rerio) development by knocking down the expression of the enzyme using a morpholino. Zebrafish embryos were microinjected with a morpholino which had a sequence complementary to the sequence of the SULT2 ST2 gene. The injections were done when the embryos were in the 1-4 cell stage with three concentrations of the morpholino: 0.5 ng/nL, 1.0 ng/nL and 2.0 ng/nL. The embryos were then observed for survival rates and abnormal phenotypes up to 144 hours post fertilization (hpf). The phenotypes observed were cardiovascular abnormalities such as cardiac edema and irregular heartbeat, abdominal edema, lordosis, notochord deformities, tail deformities and very few cases of craniofacial malformations. At the highest concentration of the morpholino, almost all knockdowns displayed mild to severe cardiac edema which was the specific phenotype at that concentration after the hatching period (48-55 hpf). The extent of knockdown was also determined by western blot experiment and it was found that the knockdown was partial and there was some enzyme present at measurable levels in the embryos. In conclusion, morpholino knockdown of the SULT2 ST2 gene in zebrafish embryos caused several non-specific phenotypes. At the 2.0 ng/nL concentration mild to severe cardiac edema was obser (open full item for complete abstract)

Committee: Frederick Williams PhD (Committee Chair); Ming-Cheh Liu PhD (Committee Member); Steven Peseckis PhD (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science, University of Akron, 2007, Biology

Studying Cadherin4 function using Morpholino Antisense technology

Committee: Qin Liu (Advisor) Subjects: Biology, Neuroscience