Pharmacology & Toxicology Department Theses and Dissertations
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About the programs
The advanced degree programs at the Indiana University of Medicine Department of Pharmacology and Toxicology prepare scientists for careers across the spectrum of biomedical research. The Master of Science (M.S.) degree is a thesis research degree that gives a student the intellectual background to understand and participate in ongoing research projects. The Doctor of Philosophy (Ph.D.) degree is offered for the student who wants to pursue an independent career in research. Students with the Ph.D. degree are prepared for an academic career combining research with teaching or for a career in industrial pharmaceutical research. A combined M.D./Ph.D. degree is open to qualified individuals who ultimately seek to direct biomedical research with a clinical emphasis.
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Recent Submissions
Item Identification of a Hypothalamic Neural System That Can Reduce Body Weight and Adipose Mass in Diet-Induced Obesity(2024-11) Basu, Rashmita; Flak, Jonathan N.; Linnemann, Amelia K.; Witczak, Carol A.; Sheets, Patrick L.; Jerde, Travis J.Dynamic hypothalamic circuits balance energy intake with expenditure to protect individuals from obesity. Lasting negative energy balance, however, triggers a compensatory decrease in energy expenditure, hindering progressive weight loss. While we understand some key players underlying energy balance, the detailed neural underpinnings remain unclear. Here I will delineate the functional efferent circuitry from the ventromedial hypothalamic nucleus (VMN) that facilitates weight loss and prevents rebound weight gain. VMN neurons have long been linked to a role in energy balance. Both vesicular communication by VMN steroidogenic factor 1 (SF1) neurons and pituitary adenylate cyclase activating peptide (PACAP) release from VMN neurons are essential for maintaining body weight and activating VMNSf1 neurons curbs diet-induced obesity without altering food intake. However, the exact pathway of this VMN signal is unclear because the VMN does not directly communicate with preganglionic sympathetic neurons, indicating signal transmission through an efferent node. Of the few brain sites they communicate with, VMNSf1 neurons sends the densest projections to the caudal preoptic area (POA) and the anterior bed nucleus of stria terminalis (BNST). Stimulating VMNPACAP axonal fibers in the caudal POA, but not anterior BNST, induced thermogenesis in brown and beige adipose tissues in both sexes of mice. To identify caudal POA populations in body weight regulation, I activated excitatory (glutamatergic) and inhibitory (GABAergic) caudal POA cells in diet-induced obese male mice and found that both glutamatergic and GABAergic caudal POA neurons can reduce diet-induced obesity through separate means. While there is intra-POA communication, my data supports efferent communication with separate downstream circuits by glutamate and GABA caudal POA cells in ameliorating diet-induced obesity. Because the POA and BNST are extremely complex regions with diverse functions, I then employed deep transfer learning to pinpoint obesity and diabetes risk-associated cell subsets in the POA and BNST. Using single nuclei RNA sequencing on >200,000 nuclei from both sexes of mice, I identified 6 specialized sets of caudal POA and BNST neuronal subtypes that increased in obese and glucose-intolerant mice on a high-fat diet. Targeting these newly identified pathways and neuron subtypes could lead to future obesity and diabetes therapeutics.Item The Impact of Abstinence from Chronic Alcohol Consumption on the Mouse Striatal Proteome: Sex and Subregion-Specific Differences(2024-10) Duffus, Brittnie-lee Marie; Atwood, Brady; Oblak, Adrian; Mao, Yao-Ying; Baucum, AJ; Fischer, KathrynAlcohol misuse is the third leading preventable cause of death in the world. The World Health Organization currently estimates that 1 in 20 deaths are directly alcohol related. One of the ways in which consuming excessive levels of alcohol can both directly and indirectly affect human mortality and morbidity, is through chronic inflammation. Recently, studies have suggested a link between increased alcohol use and the incidence of neuroinflammatory-related diseases. However, the mechanism in which alcohol potentially influences neuroinflammatory processes is still being uncovered. We implemented an unbiased proteomics exploration of alcohol-induced changes in the striatum, with a specific emphasis on proteins related to inflammation. The striatum is a brain region that is critically involved with the progression of alcohol use disorder. Using mass spectrometry following voluntary alcohol self-administration in mice, we show that distinct protein abundances and signaling pathways in different subregions of the striatum are disrupted by chronic exposure to alcohol compared to water drinking control mice. Further, in mice that were allowed to experience abstinence from alcohol compared to mice that were non-abstinent, the overall proteome and signaling pathways showed additional differences, suggesting that the responses evoked by chronic alcohol exposure are dependent on alcohol use history. To our surprise we did not find that chronic alcohol drinking or abstinence altered protein abundance or pathways associated with inflammation, but rather affected proteins and pathways associated with neurodegeneration and metabolic, cellular organization, protein translation, and molecular transport processes. These outcomes suggest that in this drinking model, alcohol-induced neuroinflammation in the striatum is not a primary outcome controlling altered neurobehavioral function, but these changes are rather mediated by altered striatal neuronal structure and cellular health.Item eIF3a Regulates De Novo Fatty Acid Synthesis as an Alternative Mechanism in Cisplatin Response in Non-Small Cell Lung Cancer Cells(2024-08) Gu, Boqing; Jerde, Travis; Lu, Tao; Safa, Ahmad R.; Zhang, Jian-Ting; Wek, Ronald C.eIF3a is known to modulate DNA damage repair and cancer chemotherapy resistance partially via translational regulation of Raptor and its downstream mTOR pathway activity. Fatty acid synthase (FASN) has recently been reported to exert negative feedback on the mTOR signaling pathway, and FASN overexpression is associated with reduced chemotherapy efficiency in multiple cancer types. Here, we show that eIF3a exerts additional regulation on mTOR signaling pathway and chemotherapy resistance in non-small cell lung cancer by inhibiting FASN-mediated de novo lipid synthesis. Through genetic and chemical manipulations, we demonstrate that eIF3a physically interacts with the 5’-UTR of FASN mRNA to prevent FASN protein synthesis. Furthermore, FASN downregulation by eIF3a results in accumulation of malonyl-CoA, a substrate for fatty acid synthesis, which in turn directly inhibits mTOR activity of mTORC1 complex, decreasing NER protein level and cellular sensitivity to cisplatin in an eIF3a-dependent manner in addition to eIF3a-regulated expression of Raptor subunit in mTORC1. Taken together, our findings reveal a direct translational control of FASN-mediated fatty acid metabolism, suggesting a multi-level eIF3a regulatory paradigm on NER protein synthesis and activity during cancer cell response to cisplatin treatment.Item The Role of T Cells in Toxoplasma gondii-Induced Prostatic Hyperplasia(2024-08) Schmidt, Tara D.; Jerde, Travis; Arrizabalaga, Gustavo; Fehrenbacher, Jill; Relich, Ryan; Schmidt, NathanChronic inflammation is the most common histological feature in Benign Prostatic Hyperplasia (BPH), and T cells are a key component of immune infiltrate. Advanced BPH is commonly associated with the formation of nodules, but it remains unclear whether a link exists among T cell infiltration, nodular development, and BPH progression. Using a Toxoplasma gondii (T. gondii) model and human specimens, we characterize the subtypes of T cells present during prostatic hyperplasia and their association with nodular development of the prostate. Using flow cytometry, we found that infecting male mice with T. gondii resulted in an increase of both CD4+ and CD8+ T cells in the prostate that was most prominent at 14 days post-infection. Next, we established the presence of microglandular hyperplasia (MGH) and glandular nodule formation at this timepoint through hematoxylin and eosin (H&E) staining. Immunofluorescence revealed that CD8+ cells were found proximal to forming glandular nodules relative to non-nodular glands. We also found that more CD8+ cells localized to non-nodular glands in nodular BPH tissue versus non-nodular BPH tissue. Finally, we discovered a higher prevalence of CD8+ cells in T. gondii IgG+ patients than in IgG- patients. All T. gondii IgG+ patients exhibited nodular BPH, whereas all but one IgG- patient exhibited non-nodular BPH. This study is the first to identify the subsets of T cells in T. gondii-infected mouse prostates. Additionally, the locality of CD4+ and CD8+ T cells to nodular and non-nodular glands within our mouse model and human BPH prostate tissue has never been analyzed. Translationally, CD8+ T cells may enhance nodular BPH progression, and T. gondii infection may promote this CD8+ T cell-mediated response. Future work will focus on dissecting the molecular pathways induced by secreted factors from these CD8+ T cells that may contribute to epithelial cell proliferation and re-activation of glandular patterning in BPH.Item Characterization of a Novel Hunk Inhibitor in HER2+ Breast Cancer(2024-07) Dilday, Tinslee Y.; Yeh, Elizabeth; Fehrenbacher, Jill; Brustovetsy, Nickolay; Safa, Ahmad; Sankar, UmaHuman Epidermal Growth Factor Receptor 2 (HER2)-targeted agents have proven to be effective, however, the development of resistance to these agents has become an obstacle in treating HER2+ breast cancer. Prior evidence implicates Hormonally Upregulated Neu-associated Kinase (HUNK) as an anti-cancer target for primary and resistant HER2+ breast cancers. An inhibitor Staurosporine (STU) has been identified as a HUNK inhibitor in HER2+ breast cancer. While STU was determined as a promising tool for inhibiting HUNK, it is a broad-spectrum kinase inhibitor and has not moved forward clinically. Therefore, identifying a more selective inhibitor of HUNK could be critical for targeting HUNK in HER2+ breast and understanding mechanisms by which HUNK promotes resistance to HER2-inhibitors. Specifically, HUNK has been implicated in promoting autophagy as a resistance mechanism in HER2+ breast cancer. Previously, we have identified that HUNK binds and phosphorylates an autophagy inhibitory protein, Rubicon, at Serine (S) 92 in 293T cells. This phosphorylation event causes Rubicon to switch to being an autophagy promoter. However, the role that Rubicon S92 plays in HER2+ breast cancer has yet to be examined. In this study, a novel inhibitor of HUNK is characterized alongside Rubicon S92 phosphorylation. This study establishes that HUNK-mediated phosphorylation of Rubicon at S92 promotes tumorigenesis in HER2/neu+ breast cancer. HUNK inhibition prevents S92 Rubicon phosphorylation in HER2/neu+ breast cancer models and inhibits both autophagy and tumorigenesis. This study characterizes a downstream phosphorylation event as a measure of HUNK activity and identifies a novel HUNK inhibitor that has meaningful efficacy toward HER2+ breast cancer.Item Dissecting the Role of Novel O-GlcNAcylation of NF-κB in Pancreatic Cancer(2024-06) Motolani, Aishat Abiola; Lu, Tao; Safa, Ahmad; Dong, Charlie; Pollok, Karen; Corson, TimothyPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited PDAC cellular proliferation, migration, and anchorage-independent growth compared to WT-p65. We also show that inhibition of NF-κB O-GlcNAcylation may mitigate gemcitabine resistance and enhance its efficacy in PDAC cells. Collectively, our study uncovers a novel aspect of NF-κB regulation, which could aid future therapeutic development by targeting O-GlcNAc transferase (OGT) in pancreatic cancer.Item Elucidating the Influence of Microglia on Retinal Ganglion Cells in a Human Pluripotent Stem Cell Model(2024-06) Harkin, Jade; Meyer, Jason; Sheets, Patrick; Landreth, Gary; Block, Michelle; Sharma, Tasneem; Gomes, CatiaGlaucoma is a complex disease that leads to irreversible blindness, characterized by the loss of retinal ganglion cells (RGCs), which are the cells that transmit visual information from your eye into your brain. Evidence suggests that microglia, the resident immune cells in the central nervous system, may have a detrimental role in the onset and the progression of glaucoma. Microglia become activated in response to damage, pathogens and toxins and are initially thought to be beneficial to RGCs. However, when these cells are activated for excessive periods of time, they are thought to be harmful to RGCs. Thus, we sought to develop novel human pluripotent stem cell (hPSC)-derived microglia, astrocyte and RGC co-cultures to determine how microglia activation modulates RGC phenotypes in a human cellular model. Healthy and LPS-activated microglia were first co-cultured with RGCs for up to 3 weeks and the effects of microglia upon RGCs were assessed. Additionally, healthy and LPS-activated microglia were also co-cultured with astrocytes and RGCs for up to three weeks to assess if LPS-treated microglia can activate astrocytes and the effects this would have on RGCs. Results showed that when co-cultured with RGCs alone for 1 week, microglia activation is initially beneficial to RGCs. However, when co-cultured with RGCs for 3 weeks, microglia activation leads to RGC damage. Consequently, when astrocytes are present, microglia activation is harmful to RGCs in both short-term and long-term co-cultures, suggesting an additional role for microglia modulation of astrocytes, further leading to neurodegeneration. Taken together, our results have allowed for the precise study of how individual cell types are adversely affected in disease-relevant states, how microglia can directly influence RGCs, and how multiple co-cultures of human microglia, astrocytes and RGCs allows for a more sophisticated investigation of cellular interactions in disease states relevant to glaucoma.Item HUNK as an Immune Regulator of Triple Negative Breast Cancer(2024-05) Ramos Solis, Nicole; Yeh, Elizabeth; Arrizabalaga, Gustavo; Fehrenbacher, Jill; Cook-Mills, Joan; Jerde, Travis J.Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Unlike other breast cancer types, TNBC tumors do not respond to endocrine therapy, and standardized treatment protocols for TNBC are currently unavailable. TNBC is recognized as a more metastatic, aggressive, and immunogenic subtype of breast cancer, rendering it to be more receptive to immunotherapy. Among the immune cell populations abundant in TNBC tumors, tumor-associated macrophages (TAMs) are particularly more prevalent and are particularly known to play a role in cancer metastasis. This work focuses on and investigates the involvement of the protein kinase HUNK in tumor immunity. With the use of gene expression analysis, such as NanoString's nCounter PanCancer Immune Profiling panel, we found that targeting HUNK is associated with alterations in the IL-4/IL-4R cytokine signaling pathway. Experimental analysis and work demonstrated that HUNK kinase activity regulates IL-4 production in mammary tumor cells, and this regulation is dependent on STAT3. Furthermore, in vivo, analysis shows that HUNK-dependent control of IL-4 secretion from tumor cells leads to the polarization of macrophages into an M2-like phenotype, and consequently, IL-4 induction promotes cancer metastasis and prompts macrophage's metastatic capacities. These findings underscore HUNK as a potential therapeutic target for mitigating TNBC metastasis by modulating the TAM population.Item Investigation 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.Item A 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.