Pharmacology & Toxicology Department Theses and Dissertations

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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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AP2IX-4, a cell cycle regulated nuclear factor, modulates gene expression during bradyzoite development in toxoplasma gondii
(2017-01-10) Huang, Sherri Y.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Lu, Tao; Takagi, Yuichiro; Zhang, Jian-Ting
Toxoplasma gondii is a ubiquitous, protozoan parasite contributing significantly to global human and animal health. In the host, this obligate intracellular parasite converts into a latent tissue cyst form known as the bradyzoite, which is impervious to the immune response. The tissue cysts facilitate wide-spread transmission through the food chain and give rise to chronic toxoplasmosis in immune compromised patients. In addition, they may reactivate into replicating tachyzoites which cause tissue damage and disseminated disease. Current available drugs do not appear to have appreciable activity against latent bradyzoites. Therefore, a better understanding of the molecular mechanisms that drive interconversion between tachyzoite and bradyzoite forms is required to manage transmission and pathogenesis of Toxoplasma. Conversion to the bradyzoite is accompanied by an altered transcriptome, but the molecular players directing this process are largely uncharacterized. Studies of stage-specific promoters revealed that conventional cis-acting mechanisms operate to regulate developmental gene expression during tissue cyst formation. The major class of transcription factor likely to work through these cis-regulatory elements appears to be related to the Apetala-2 (AP2) family in plants. The Toxoplasma genome contains nearly 70 proteins harboring at least one predicted AP2 domain, but to date only three of these T. gondii AP2 proteins have been linked to bradyzoite development. We show that the putative T. gondii transcription factor, AP2IX-4, is localized to the parasite nucleus and exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had negligible effect on tachyzoite replication, but resulted in a reduced frequency of bradyzoite cysts in response to alkaline stress induction – a defect that is reversible by complementation. Microarray analyses revealed an enhanced activation of bradyzoite-associated genes in the AP2IX-4 knockout during alkaline conditions. In mice, the loss of AP2IX-4 resulted in a modest virulence defect and reduced brain cyst burden. Complementation of the AP2IX-4 knockout restored cyst counts to wild-type levels. These findings illustrate the complex role of AP2IX-4 in bradyzoite development and that certain transcriptional mechanisms responsible for tissue cyst development operate across parasite division.
Biochemical and pharmacological characterization of the Atg8 conjugation system in toxoplasma gondii
(2017-06-28) Varberg, Joseph M.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Mosley, Amber; Safa, Ahmad; Vasko, Michael R.
Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes.
Elucidating the Role of the Essential Kinase TgGSK in the Human Parasite Toxoplasma Gondii
(2025-02) Krueger, Amanda; Yeh, Elizabeth; Arrizabalaga, Gustavo; Sullivan, William; Nass, Richard; Aoki, Scott
Toxoplasma gondii is an intracellular parasite that infects nearly a third of the world’s human population. While infection is largely asymptomatic in an immunocompetent host, Toxoplasma infection in immunocompromised or immunosuppressed individuals can lead to toxoplasmosis, which can include brain lesions and lead to death. Similarly, toxoplasmosis can result in birth defects, brain swelling, and blindness of a developing fetus in the case of a congenital infection. With minimal treatments for toxoplasmosis available, it is crucial to study parasite-specific processes that could be potential drug targets for the treatment of toxoplasmosis. Toxoplasma gondii divides through a unique process known as endodyogeny, where two daughter parasites are formed within a mother. In this study, we investigated a novel protein called TgGSK that is crucial for proper parasite division. Experiments reveal that TgGSK changes its localization within the parasite dependent on the stage of division. Knockdown of TgGSK causes abnormal division phenotypes and causes Toxoplasma to be unable to complete its propagation cycle. We determined through microscopy and phosphoproteomics that TgGSK may play its role in parasite division through an interaction with the centrosome, an organelle which is a main feature of cell division in many organisms. Our findings suggest that TgGSK also regulates messenger RNA processing. Finally, our study suggests that TgGSK is regulated and stabilized through acetylation from the GCN5b lysine acetyltransferase complex. Taken together, we have performed an in-depth study of the functional role of the essential protein TgGSK in Toxoplasma gondii. This and future studies have potential to demonstrate that TgGSK is a parasite-specific drug target for the therapeutic treatment of toxoplasmosis.
A forward genetic approach to identifying novel calcium regulators in Toxoplasma Gondii
(2017-07-25) LaFavers, Kaice Arminda; Arrizabalaga, Gustavo; Brustovetsky, Nickolay; Cummins, Theodore; Gilk, Stacey; Sullivan, William J., Jr.
Toxoplasma gondii is an obligate intracellular eukaryotic pathogen that causes severe neurologic disease in immunocompromised adults and congenitally infected neonates. Events critical to the propagation of T. gondii, such as invasion and egress, are regulated by calcium-dependent signaling. In order to identify unique components of the parasite’s calcium signaling networks, members of the Arrizabalaga laboratory have used a forward genetics approach to isolate mutants with altered sensitivity to the calcium ionophore A23187. Exposing extracellular parasites to A23187 induces protein secretion, motility and cytoskeletal rearrangements and prolonged treatment causes exhaustion of factors required for invasion, which results in what is referred to as ionophore induced death (iiDeath). Mutants capable of surviving this treatment were isolated from a chemically mutagenized population. Whole genome sequencing of one such mutant, MBD2.1, identified a nonsense mutation in a protein of unknown function (TGGT1_069070, ToxoDBv7.2) Complementation of MBD 2.1 with a wild-type copy of TGGT1_069070 restored sensitivity to iiDeath treatment. Endogenous tagging of this locus revealed that the encoded protein is secreted from a unique parasite secretory organelle known as the dense granule into the parasitophorous vacuole, leading to its designation as TgGRA41. Complete knockout of TgGRA41 recapitulates the resistance to iiDeath observed in MBD2.1 but also exhibits a dramatic decrease in propagation in tissue culture not seen in the original mutant. The knockout shows defects in multiple steps of the lytic including compromised invasion efficiency and premature egress of parasites from host cells. Cytosolic calcium measurements of extracellular parasites show enhanced uptake of calcium in the knockout strain as compared to parental and complemented, suggesting that the loss of TgGra41 results in calcium dysregulation. Together, these results provide a novel insight into the role that the parasitophorous vacuole of T. gondii plays in calcium homeostasis and calcium-dependent signaling processes.
Function 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, Teresa
The 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.
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.
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, Nathan
Toxoplasma 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.
Investigations into the function of Elp3 in Toxoplasma gondii
(2017-05-04) Padgett, Leah Rausch; Arrizabalaga, Gustavo; Jerde, Travis; Mosley, Amber; Nass, Richard M.; Sullivan, William J., Jr.
The parasite Toxoplasma gondii causes life-threatening infection in immunocompromised individuals. Our lab has determined that Toxoplasma Elongator protein-3 (TgElp3) is required for parasite viability. While catalytic domains are conserved, TgElp3 is the only component of the six-subunit Elongator complex present in Toxoplasma; moreover, TgElp3 localizes to the outer mitochondria membrane (OMM). These unusual features suggest that TgElp3 may have unique roles in parasite biology that could be useful in drug targeting. The goals of this thesis were to determine the function of TgElp3 and how the protein traffics to the OMM. In other species, Elp3 mediates lysine acetylation of histones and alphatubulin, and its radical S-adenosyl methionine (rSAM) domain is important for the formation of tRNA modifications, which enhance translation efficiency and fidelity. Given its location, histones would not be an expected substrate, and we further determined that tubulin acetylation in Toxoplasma is mediated by a different enzyme, TgATAT. We found that overexpression of TgElp3 at the parasite’s mitochondrion results in a significant replication defect, but overexpression of TgElp3 lacking the transmembrane domain (TMD) or with a mutant rSAM domain is tolerated. We identified one such modification, 5-methoxycarbonylmethyl-2thiouridine (mcm5S2U) that is likely mediated by TgElp3. These findings signify the importance of TgElp3’s rSAM domain for protein function, and confirms TgElp3 activity at the OMM is essential for Toxoplasma viability as previously reported. To determine how TgElp3 traffics to the OMM, we performed a bioinformatics survey that discovered over 50 additional “tail-anchored” proteins present in Toxoplasma. Mutational analyses found that targeting of these TA proteins to specific parasite organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence.
Lysine acetyltransferase Gcn5-B regulates the expression of crucial genes in Toxoplasma and its function is regulated through lysine acetylation
(2014-04-02) Wang, Jiachen; Sullivan, William J., Jr.; Queener, Sherry F.; Arrizabalaga, Gustavo; Nass, Richard M.; Lu, Tao
Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.
Micro-RNA regulation of hepatic drug metabolism : age-related changes in micro-RNA expression and genetic variants in micro-RNA target sites
(2017-08-31) Burgess, Kimberly Sherrelle; Skaar, Todd C.; Arrizabalaga, Gustavo; Cummins, Theodore; Desta, Zeruesenay; Nass, Richard; Zhang, Jian-Tian
Developmental changes in the liver significantly impact drug disposition. Due to the emergence of microRNAs as important regulators of drug disposition, we hypothesize that age-dependent change in microRNA expression and genetic variants in microRNA target sites contribute to variability in drug disposition. In human liver tissues, expression of 533 microRNAs and over 14,000 genes were measured. In all, 114 microRNAs were upregulated and 72 downregulated from fetal to pediatric, and 2 and 3, respectively, from pediatric to adult. Among these microRNAs, 99 microRNA-mRNA interactions were predicted or have previously been validated to target drug disposition genes and over 1,000 significant negative correlations were observed between miRNA-mRNA pairs. We validated these interactions using various cell culture models. Genetic variants in the promoter and coding regions of drug disposition genes have also been shown to alter enzyme expression and/or activity. However, these variants do not account for all variability in enzyme activity. Emerging evidence has shown that variants in the 3’UTR may explain variable drug response by altering microRNA regulation. Five 3’UTR variants were associated with significantly altered CYP2B6 activity in healthy human volunteers. The rs70950385 (AG>CA) variant was associated with decreased CYP2B6 activity among normal metabolizers. In vitro luciferase assays confirmed that the CA allele altered miR 1275 targeting of CYP2B6 mRNA. Due to the large number of 3’UTR variants predicted to alter microRNA regulation, a high-throughput method, PASSPORT-seq, was developed to test over 100 3’UTR variants simultaneously in different cell lines. Thirty-eight variants resulted in FDR-significant altered expression between wild-type and variant sequences. Our data suggest a mechanism for the marked changes in hepatic gene expression between the fetal and pediatric developmental periods, support a role for these age dependent microRNAs in regulating drug disposition, and provide strong evidence that 3’UTR variants are also an important source of variability in drug disposition.

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Browsing Pharmacology & Toxicology Department Theses and Dissertations by Author "Arrizabalaga, Gustavo"

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