Tuberculosis is a major burden of disease worldwide. Although drug regimens for the treatment of tuberculosis are available, they are complex and lengthy. Efforts are being made to understand and develop novel drugs. The Parish Lab has identified a common mechanism of drug resistance in the type VII secretion system (T7SS) of Mycobacterium tuberculosis. Mutations in several components of the Esx3 T7SS system confer resistance to aminothiazole (AmT), 8-hydroxyquinoline (8HQ) and nitrothiazole salicylamide (NTS) compounds. The Esx3 T7SS is unlikely the cellular target for these compounds as four other genes had mutations, one of the four genes not being a part of the ESX-3 complex. Therefore, resistance is believed to arise from a distinct mechanism. Rv0474 is a transcriptional regulator identified to mediate resistance to AmT and 8HQ. It is a copper-responsive regulator with a copper binding motif. I am studying the Rv0474 homolog (MSMEG_0918) in Mycobacterium smegmatis, a fast-growing species used as a model organism. MSMEG_0918 lacks the copper binding motif, permitting determination of whether copper binding activity is required to confer resistance. I am using CRISPRi knock down technique to generate strains with reduced expression of MSMEG_0918, along with constructing overexpression strains. Knock down and overexpression strains allow us to determine growth, AmT/8HQ sensitivity and copper sensitivity. We propose that if copper binding activity is involved in AmT/8HQ resistance, overexpression or downregulation of MSMEG_0918 would have no effect on compound activity, because it lacks this activity. This work will determine whether MSMEG_0918 plays an equivalent role to Rv0474 in conferring resistance to AmT and 8HQ compounds. This research will provide knowledge to further global efforts in developing an adequate and effective treatment necessary for tuberculosis. 

Induced pluripotent stem cells (iPSCs) can be directed to create 3D mini retinas in vitro known as retinal organoids. Retinal organoids recapitulate the developmental timeline of the human fetal retina and have the potential to serve as disease models for a multitude of retinopathies. However, we have observed that retinal organoids contain a disorganized inner nuclear layer and discontinuous lamination hindering their ability to be considered as a comprehensive disease model. The protein β-catenin is produced from the canonical WNT signaling pathway and has been shown to be essential for retinal lamination in mice due to its role in cellular adhesion. I investigated the effects of a canonical WNT signaling pathway agonist, CHIR,  in order to improve the disorganization and lamination of retinal organoids so that we can develop a comprehensive disease model for different retinopathies. We have used three different stem cell lines to construct retinal organoids that I cultured with the addition of the WNT pathway agonist, CHIR. I performed immunohistochemistry staining followed by microscopy analysis and have obtained data that shows an increase in lamination and cellular organization with the addition of the WNT signaling pathway agonist. Our data suggests that the WNT signaling pathway plays a role in maintaining organization and lamination in the developing human fetal retina. 

Human adenoviruses (AdVs) infect and cause disease in multiple organ systems, and certain human AdV serotypes are associated with particular diseases; however, the basis for AdV tissue tropism is unknown. To better understand this problem, I am using mouse adenoviruses (MAdV). Like human AdVs, MAdV serotypes are associated with distinct tissue tropisms: MAdV-1 infects macrophages whereas MAdV-2 infects intestinal epithelial cells. By swapping genes between MAdV-1 and MAdV-2, I hope to uncover the genetic basis of tissue tropism in MAdVs. Importantly, these studies will be aided by the availability of a cell culture system that recapitulates the cellularity of the intestinal epithelium and supports MAdV-2 replication, but not MAdV-1 replication. Due to genetic conservation, principles of MAdV tissue tropism are likely to apply to HAdVs. A major determinant of viral cell tropism is receptor usage, which may also play a primary role in tissue tropism. For both MAdV species, the trimeric fiber protein that extends from the icosahedral capsid is thought to be the viral attachment protein that binds to a host receptor. Although the MAdV receptors are not completely known, they are distinct for MAdV-1 and MAdV-2. To gain insight into tissue tropism, I have created a chimeric MAdV-1-M2f virus, where the fiber gene of MAdV-2 has been inserted in place of the native fiber gene in the MAdV-1 genome. A prior student in the Smith lab created the inverse chimera. If replication is fiber dependent, the chimeric MAdV-1-M2f virus should be capable of infection. By studying these chimeric viruses, I will uncover whether the fiber/receptor interaction plays a central role in determining tissue tropism.

Castration-resistant prostate cancer (CRPC) development is a major obstacle in conventional androgen deprivation therapies for prostate cancer. Progression of CRPC often includes formation of androgen-independent androgen receptor (AR) splice variants (AR-Vs), which are resistant to AR targeting therapies. Plymate laboratory recently found that bumped kinase inhibitors (BKIs), originally developed as antiparasitic agents, are capable of suppressing growth of cellular and pre-clinical models of CRPC and also activate cellular energy sensor adenosine monophosphate-activated kinases (AMPK) in AR-V-dependent CRPC cell line LNCaP95. AMPK activation results in growth inhibition in multiple models of cancer. Therefore, I tested the hypothesis that AMPK activation accounts for BKI’s antiproliferative activity. Co-treatment with pharmacological AMPK inhibitor Compound C (CC) did not affect antiproliferative activity of BKI-1553 in LNCaP95. BKI-1553 does not affect proliferation of another prostate cancer cell line PC3 whereas it increased AMPK enzymatic activity in both LNCaP95 and PC3. On the other hand, AMPK activator A-769662 inhibited proliferation of both LNCaP95 and PC3. These results suggest that BKI-induced AMPK activation is not robust enough to suppress proliferation of prostate cancer cell line. This study warrants further investigation of mechanism of action of this drug to fully understand its anti-CRPC action.

Skin can detect a wide range of stimuli through the touch system, which is mediated by specific cells and structures. Merkel cells, one of these specialized types of skin cells, sense gentle touch and texture. In mammals, Merkel cells are identified by the expression of the transcription factors Sox2 and Atoh1. Recently, the Rasmussen lab identified Merkel cells in the zebrafish skin that share many characteristics of mammalian Merkel cells, including expression of Sox2 and Atoh1a. Interestingly, a paper by Konig in 2018 also described a novel cell type that expresses serotonin (5-HT), calretinin, and synaptic vesicle glycoprotein 2 (SV2), termed “HCS” cells, within the zebrafish skin. These researchers proposed that HCS cells are a different sensory population than Merkel cells, because of differing visual characteristics between these cells. However, the paper did not present any conclusive evidence, making the relationship between HCS cells and Merkel cells still uncertain. I hypothesized that Merkel cells and HCS cells are actually the same population of cells. I tested my hypothesis using antibody staining and confocal imaging of zebrafish skin. I found that Atoh1a-positive Merkel cells express serotonin and SV2, demonstrating that Merkel cells in zebrafish are the same cells as HCS cells. Overall, my results resolve the identity of recently described sensory cell types in the zebrafish skin. In future research, I hope to use zebrafish Merkel cells as a promising model to better understand touch system development and regeneration.