- Pharmacology & Toxicology Department Theses and Dissertations
ItemInvestigation of the Knockout of LMF1 on the Transcriptome of Toxoplasma gondii(2024-01) Thibodeau, Katherine E.; Arrizabalaga, Gustavo; Absalon, Sabrina; Fehrenbacher, Jill; Flak, Jonathan; Schmidt, NathanToxoplasma gondii is an obligate intracellular apicomplexan parasite that infects one third of the global population. There are limited treatments for Toxoplasmosis, however a potential drug target for Toxoplasma is its mitochondrion. While much is known about the function of this organelle in Toxoplasma, little is known about the mechanisms that regulate mitochondrial structure and division. The shape of the mitochondrion changes throughout the life cycle of the parasite. When inside a host cell, the mitochondrion is in a lasso shape, stretching around the periphery of the parasite, while in extracellular parasites it is collapsed towards the apical end of the parasite. While in a lasso shape the mitochondrion shows areas of contact with the parasite pellicle. We have determined that the proteins LMF1 (associated with the outer mitochondrial membrane) and IMC10 (inner membrane complex) interact and form a reversible tether that maintains the lasso shape of the mitochondrion. When either of these proteins are knocked out, the mitochondrion collapses. To elucidate the biological relevance of the interaction between the mitochondrion and the pellicle we explored the consequence of disrupting the interaction on the transcriptome of the parasite. RNA sequencing of the LMF1 knockout strain showed a disruption in the expression of genes involved in nucleotide metabolism and Coenzyme A biosynthesis, which might be an adaptation mechanism to the disruption of mitochondrial morphology. Current work focuses on investigating the connection between mitochondrial tethering and these pathways as well as a potential role for the mitochondrion/pellicle connection in metabolite transport. ItemA Machine Learning-Based Histopathological Image Analysis Reveals Cancer Stemness in TNBCs with 17p Loss(2023-05) Dong, Tianhan; Huang, Kun; Safa, Ahmad R.; Jerde, Travis J.; Lu, Tao; Lu, XiongbinArtificial intelligence and machine learning based methods have incorporated scientific research into clinical decision, leading to great improvement in clinical diagnosis and therapeutics. Here we developed a Convolutional Neural Network based model to identify cancer stem-like cells (CSCs) on H&E-stained histopathological images. Combined with cancer genomics profiles, our analysis revealed that triple negative breast cancers (TNBCs) with heterozygous deletion of chromosome 17p (17p-loss) correlate with higher cancer stemness potential compared to TNBCs with neural copy numbers of 17p (17p-intact). 17p-loss TNBC cells also have an increased percentage of CSCs and are resistant to chemotherapies compared with the 17p-intact TNBC cells. Moreover, we built a bioinformatics pipeline to screen compounds that target the stemness of 17p-loss cancer cells, one of which is FK866. FK866 promoted the antitumor activity of doxorubicin in the treatment of 17p-loss TNBCs. Our study provides a powerful computational tool for cancer image analysis as well as a feasible approach for precision cancer medicine. ItemThe Role of Inflammation in Mediating Different Cognitive and Behavioral Functions(2023-03) El Jordi, Omar; Atwood, Brady K.; McKinzie, David L.; Oblak, Adrian L.; Block, Michelle L.; Sheets, Patrick L.Inflammation, specifically brain inflammation, can be both neuroprotective as well as detrimental in multiple neuropathic diseases. We investigated the inflammatory profile in two different receptor systems, opioids, and estrogens. Microglia, brain immune cells, can influence the neuron’s microenvironment and regulate neuronal activity via multiple signaling mediators. We investigated the role of microglia in opioid withdrawal in an oxycodone dependence paradigm. We found that microglial reduction had no effect on opioid withdrawal symptoms, glial activation markers, body temperature dysregulation, or select inflammatory cytokines. Interestingly, we found them involved in the acquisition of analgesic tolerance potentially mediated by the chemokine KC/GRO. This suggests that microglia modulate neuron adaptations to repetitive opioid dosing through chemokines or alternatively through direct interaction (cell-to-cell), indirect interaction through cytokines, or neurotransmitters that were not measured. In a different experiment, we investigated the effect of Raloxifene, a selective estrogen receptor modulator, in a model of lower back pain/spinal damage precipitated by intervertebral disc degeneration. Raloxifene’s therapeutic effect at the molecular level, verified by collaborators and evident by regeneration of healthy intervertebral disc tissue and reduction of pain signaling molecules (Substance P), did not translate to behavior. Our behavioral measures evaluating motor and psychological deficit, and ataxia were not altered by treatment. Interestingly, we found that hedonic behavior improved by the end of the treatment suggesting that Raloxifene may have a therapeutic effect on adverse sensory signals such as back pain resulting in reward-seeking behavior. ItemTreatment During Abstinence from Methamphetamine in a Rat Model of Methamphetamine Use Disorder(2022-12) Baek, James Jaewoo; Sheets, Patrick L.; Yamamoto, Bryan K.; Atwood, Brady K.; Fehrenbacher, Jill C.; Ma, Yao-Ying; Yoder, Karmen K.Methamphetamine (METH) is a psychostimulant with high abuse potential. Currently there are no pharmacological treatments specific for relapse to METH use disorder. Chronic METH abuse has been associated with changes to the dopamine and glutamate neurotransmitter systems, as well as inflammation. Phosphodiesterase-4 inhibitors are known to affect cAMP involved in dopaminergic and glutamatergic neurotransmission, as well as having anti-inflammatory action. In pre-clinical models, phosphodiesterase inhibitors can reduce behaviors associated with the self-administration of drugs of abuse if given directly before tests of relapse-like behavior. However, they have not been examined in the more clinically relevant context as a treatment for use during abstinence from drugs of abuse. To address this gap, a METH self-administration model in the rat was used in which roflumilast, a phosphodiesterase 4 inhibitor, was administered during the abstinence period before a relapse test. The overarching hypothesis was that roflumilast inhibited inflammation associated with METH self-administration abstinence to reduce subsequent relapse-like behaviors. A detailed behavioral analysis showed that the chronic treatment with roflumilast during 7 days of forced abstinence reduced relapse-like behavior to METH seeking and METH taking. Roflumilast treatment during 7 days of forced abstinence did not affect subsequent sucrose seeking and sucrose taking behaviors. Biochemical analyses of proteins related to dopamine and glutamate neurotransmission did not reveal changes in these neurotransmitter systems, nor was there evidence of overt inflammation. These data suggest that roflumilast may be a treatment for METH use disorder that is effective when taken during abstinence, but further studies related to the mechanism of action of roflumilast are needed. ItemFunction of a Unique Dually Localized EF-Hand Domain Containing Protein, TgEFP1, During the Lytic Cycle of the Human Parasite Toxoplasma Gondii(2022-08) Dave, Noopur Kirti; Arrizabalaga, Gustavo; Absalon, Sabrina; Fehrenbacher, Jill; Gilk, Stacey; Jerde, Travis; Mastracci, TeresaThe pathogenesis associated with toxoplasmosis is attributed to repeated rounds of the parasite lytic cycle, which has been shown to be regulated by calcium fluxes. However, little is known about the calcium homeostatic mechanisms utilized by T. gondii. Recently, our lab has identified a novel protein-TgEFP1 (TGGT1_255660), which is predicted to bind Ca2+ through its two EF-hand domains. Interestingly, TgEFP1 showed a unique dual localization at the PLV/ELC and the PV of the parasite. Previous work showed that the PLV/ELC harbors other ion binding and conducting proteins that are important for parasite survival and propagation. However, the function of this compartment in the parasite is unknown. Therefore, I hypothesize that the PLV/ELC, through the function of TgEFP1, plays a key role in calcium homeostasis of T. gondii. To test this hypothesis, we sought to characterize the function of TgEFP1 during the parasite lytic cycle and determine TgEFP1 interacting proteins that also localize to the PLV/ELC. Partial permeabilization and ultrastructure expansion microscopy techniques confirmed the dual localization of TgEFP1 at the PLV/ELC and the PV. TgEFP1 knockout parasites exhibited several phenotypic defects including a faster lytic rate, shorter intracellular cycle, and were more sensitive to calcium ionophore treatment. Signal peptide deletion led to a mislocalization of TgEFP1 as cytosolic puncta, while mutations at key calcium coordinating residues lead to exclusive localization of TgEFP1 at the PV. Lastly, immunoprecipitation assays followed by LC-MS/MS identified a novel lectin-like protein- TgLectin (TGGT1_258950) as a direct interactor of TgEFP1-HA. Collectively, these findings support that through the function of TgEFP1, the PLV/ELC, plays a key role in calcium-dependent processes during the lytic cycle of the parasite. ItemTargeting Soluble Epoxide Hydrolase to Treat Choroidal Neovascularization(2022-05) Park, Bomina; Corson, Timothy W.; Bhatwadekar, Ashay D.; Jerde, Travis J.; Lu, Tao; Nass, Richard M.Neovascular or “wet” age-related macular degeneration (nvAMD) is a leading cause of blindness among older adults, affecting millions of people worldwide. Choroidal neovascularization (CNV) is a major pathological feature of nvAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible loss of vision. Currently, the effort to treat nvAMD is hampered by resistance and refractory responses to the current standard of anti-angiogenic care, anti-vascular endothelial growth factor biologics. Thus, there is a critical need to develop novel therapeutic strategies. Previously, we discovered an anti-angiogenic small molecule SH-11037, and identified soluble epoxide hydrolase (sEH) as a target of SH-11037 through a forward chemical genetics approach. sEH, encoded by the EPHX2 gene, is a lipid-metabolizing enzyme that hydrolyzes epoxy fatty acids into corresponding diols. I hypothesized that sEH is a key mediator of CNV. Given that the kinetic mechanism of sEH inhibition by SH-11037 and the cellular role of sEH in CNV are poorly understood, the objectives of my thesis project were to elucidate drug-target interactions through enzyme kinetics, investigate sEH mediated mechanisms that regulate CNV, and preclinically validate sEH as a therapeutic target. I discovered that SH-11037 is a mixed inhibitor of sEH with a binding affinity for both the enzyme and enzyme-substrate complex. I examined retinal spatial expression of sEH at both the protein and mRNA levels through immunohistochemistry and RNAscope in situ hybridization and investigated the efficacy of adeno-associated virus (AAV) serotype 8 vector expressing shRNA against Ephx2, in the mouse laser-induced (L-) CNV model with features of nvAMD. My study revealed sEH protein and mRNA overexpression in the retinal pigment epithelium (RPE), vasculature and photoreceptors under the disease state. The delivery of AAV8-Ephx2 shRNA, which has tropism towards RPE and photoreceptor cells, significantly reduced CNV. In addition, gene expression analysis showed normalized Vegfc and CNV-related inflammatory markers upon sEH knockdown. Thus, my study demonstrated sEH overexpression in disease-relevant cell types, highlighted a functional role of sEH in AMD pathophysiology, and provided a novel context to target these cell types for developing pharmacotherapies. ItemA New Mechanism of Serotonin Transporter Regulation by Simvastatin and the Isoprenylation Pathway(2021-07) Deveau, Carmen Marie; Yamamoto, Bryan K.; Sheets, Patrick L.; Sullivan, William J.; Atwood, Brady K.; Brustovetsky, NickolayThe serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function. To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5- HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase (CAMKII). Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs. ItemThe Role of Heme Synthesis in Endothelial Mitochondrial Function and Ocular Angiogenesis(2020-08) Shetty, Trupti; Corson, Timothy W.; Bhatwadekar, Ashay D.; Hoffmann-Longtin, Krista J.; Jerde, Travis J.; Lu, Tao; Sullivan, William J.Abnormal blood vessel growth from pre-existing blood vessels, termed pathological angiogenesis, is a common characteristic of vascular diseases like proliferative diabetic retinopathy (PDR) and wet age-related macular degeneration (wet AMD). Retinal detachment, hemorrhage, and loss of vision are only some of the debilitating consequences of advanced pathological angiogenesis. Current therapeutics targeting these blood vessels are ineffective in many patients. We previously identified a novel angiogenesis target, ferrochelatase (FECH), from the heme synthesis pathway, and found that FECH inhibition is antiangiogenic in cell and animal models. Heme synthesis occurs in mitochondria, where FECH inserts Fe2+ into protoporphyrin IX (PPIX) to produce heme. However, the relationship between heme metabolism and angiogenesis is poorly understood. I sought to understand the mechanism of how FECH and thus, heme is involved in endothelial cell function. First, I determined the energetic state of retinal and choroidal endothelial cells, previously uncharacterized. I found that mitochondria in endothelial cells had several functional defects after heme inhibition. FECH loss changed the shape of mitochondria and depleted expression of genes maintaining mitochondrial dynamics. FECH blockade elevated oxidative stress and depolarized mitochondrial membrane potential. Heme depletion had negative effects on cellular metabolism, affecting oxidative phosphorylation and glycolysis. Mitochondrial complex IV of the electron transport chain (cytochrome c oxidase) was decreased in cultured human retinal endothelial cells and in murine retina ex vivo after FECH inhibition. Supplementation with heme partially rescued phenotypes of FECH blockade. Additionally, I discovered that partial loss-of-function Fech mutation in mice caused PPIX accumulation with no change in normal vasculature, as assessed by fundoscopy. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis. My studies identify a novel role of a heme synthesis enzyme in blood vessel formation and provide an opportunity to exploit these findings therapeutically for patients with PDR and wet AMD. ItemEpilepsy Mutations in Different Regions of the Nav1.2 Channel Cause Distinct Biophysical Effects(2020-06) Mason, Emily R.; Cummins, Theodore; Sullivan, William; Brustovetsky, Nickolay; Sheets, Patrick; Hashino, EriWhile most cases of epilepsy respond well to common antiepileptic drugs, many genetically-driven epilepsies are refractory to conventional antiepileptic drugs. Over 250 mutations in the Nav1.2 gene (SCN2A) have been implicated in otherwise idiopathic cases of epilepsy, many of which are refractory to traditional antiepileptic drugs. Few of these mutations have been studied in vitro to determine their biophysical effects on the channels, which could reveal why the effects of some are refractory to traditional antiepileptic drugs. The goal of this dissertation was to characterize multiple epilepsy mutations in the SCN2A gene, which I hypothesized would have distinct biophysical effects on the channel’s function. I used patch-clamp electrophysiology to determine the biophysical effects of three SCN2A epilepsy mutations (R1882Q, R853Q, and L835F). Wild-type (WT) or mutant human SCN2A cDNAs were expressed in human embryonic kidney (HEK) cells and subjected to a panel of electrophysiological assays. I predicted that the net effect of each of these mutations was enhancement of channel function; my results regarding the L835F and R1882Q mutations supported this hypothesis. Both mutations enhance persistent current, and R1882Q also impairs fast inactivation. However, examination of the same parameters for the R853Q mutation suggested a decrease of channel function. I hypothesized that the R853Q mutation creates a gating pore in the channel structure through which sodium leaks into the cell when the channel is in its resting conformation. This hypothesis was supported by electrophysiological data from Xenopus oocytes, which showed a significant voltage-dependent leak current at negative potentials when they expressed the R853Q mutant channels. This was absent in oocytes expressing WT channels. Overall, these results suggest that individual mutations in the SCN2A gene generate epilepsy via distinct biophysical effects that may require novel and/or tailored pharmacotherapies for effective management. ItemGenetic Approach to Discover ARMC4 as a Novel NF-κB Negative Regulator and Tumor Suppressor in Colorectal Cancer(2020-04) Martin, Matthew Peter; Lu, Tao; Safa, Ahmad; Corson, Tim; Jerde, Travis; Pollok, KarenThe nuclear factor κB (NF-κB) plays pivotal roles in inflammatory and immune responses and in cancer. Therefore, understanding its regulation holds great promise for disease therapy. Using validation-based insertional mutagenesis (VBIM), a powerful technique established by us, we discovered armadillo repeat containing protein 4 (ARMC4) as a novel negative regulator of NF-κB in colorectal cancer (CRC). ARMC4 is a rarely studied protein only known to date for its role in primary ciliary dyskinesia (PCD) and mouse spermatogenesis. Thus, my work reveals a completely new facet of ARMC4 function that has never been reported before. We showed that ARMC4 overexpression downregulated the expression of NF-κB-dependent genes, many of which are related to cancer. Additionally, compared to the vector control group, overexpression of ARMC4 in HEK293 cells or CRC HT29, DLD1, and HCT116 cells dramatically reduced NF-κB activity, cellular proliferation, anchorage-independent growth, and migratory ability in vitro, and unsurprisingly, significantly decreased xenograft tumor growth in vivo. In contrast, shARMC4 knockdown cells showed quite opposite effect. Furthermore, co-immunoprecipitation (Co-IP) experiment confirmed that ARMC4 may form a complex with the p65 subunit of NF-κB. Importantly, immunohistochemistry (IHC) data exhibited much lower ARMC4 expression level in CRC patient tumor tissues compared to normal tissues, indicating that ARMC4 may function as a tumor suppressor in CRC. To conclude, my important findings for the first time uncovered the negative regulatory function of ARMC4 in NF-κB signaling, and present ARMC4 as an innovative therapeutic target in CRC treatment.