Ovarian cancer is a common cancer in women and has a poor prognosis due to its late diagnosis and drug resistance. Research on ovarian cancer cell lines, such as SKOV3 and PEO4, in 3D cultures has become crucial to mimic in vivo conditions accurately. CellMate uGel is a promising platform for 3D cultures, yet the slow growth rate of cells within these models presents a significant challenge, often requiring extended periods for culture preparation. Current 3D culture models for ovarian cancer exhibit slow cellular proliferation which leads to prolonged preparation times. This inefficiency hinders timely experimental outcomes and research progress. This study aims to improve the growth rates of ovarian cancer cell lines (i.e., SKOV3 and PEO4) in 3D cultures by incorporating extracellular matrices, specifically collagen and Matrigel, into CellMate 3D u-Gels. By improving the growth rates, the time required for cell culture preparation is reduced and the efficiency of these models is increased for research purposes. Cell proliferation was assessed using the PrestoBlue ATP assay. SKOV3 and PEO4 cell lines were cultured in CellMate 3D u-Gels with and without the addition of collagen and Matrigel. Growth rates were analyzed to determine the impact of these extracellular matrices on the cells. Preliminary results indicate that the inclusion of Matrigel significantly enhances the growth rates of both cell lines tested in 3D cultures. Those with collagen had negative growth rates on the fluorescence assays though further testing is needed to determine why.

Infertility affects 10-15% of females in the U.S. with issues around ovulation as the most common cause of infertility. Successful reproduction requires ovarian follicular development, ovulation, mature oocytes, embryo development, and a receptive endometrium. The immune system, particularly CD4+ regulatory T cells (Tregs), plays a critical role in reproduction. Tregs mediate immune tolerance to the implanting oocyte. G-protein-coupled receptors control signaling pathways within T cells. Regulators of G-protein signaling (RGS) proteins turn off this signaling. One such RGS protein, RGS2, is crucial in reproductive physiology, including maturation of oocytes and embryo implantation. Systemically, RGS2 controls T cell activation, proliferation, and function. Previous studies have investigated the role of RGS2 and CD4 T cells in ovarian and uterine function in reproduction separately, but the role of RGS2 specifically in CD4 T cells in reproduction has not been studied. This research aims to investigate the role of RGS2 specifically in CD4 T cells on ovarian and CD4 T cell function in reproduction. A mouse model will be used in which RGS2 is knocked out only in CD4 T cells. Female CD4 RGS2 knockout and littermate control mice will undergo oocyte maturation and induction of ovulation. Oocytes will be enumerated, and ovary and uterus will be evaluated for CD4 T cell populations via flow cytometry. Sex hormones, estrogen and progesterone, will be determined via ELISA. The long-term goal of this study is to better understand the signaling pathways that contribute to infertility in females to identify potential therapeutic targets for future treatments.

CD4 T cells are essential for the adaptive immune response, recruiting and activating other immune cells to eliminate pathogens. However, without proper regulation, CD4 T cells can cause autoimmune diseases. Their functions are controlled by G-protein-coupled receptors (GPCRs), with Regulator of G-protein signaling (RGS) proteins acting as brakes to rapidly adjust GPCR signaling. One such family member, RGS2, has been shown to regulate T cell responses. Studies in mice have shown that a global loss of RGS2 impairs T cell proliferation and cytokine production. These findings suggest that RGS2 expression in T cells is necessary for T cell proliferation and cytokine production, both of which are crucial for an appropriate immune response. Previous studies used mice lacking RGS2 in all cells in the mouse, thus this study aims to determine the role of RGS2, specifically in CD4 T cells. Given previous studies, it is hypothesized that the loss of RGS2 in CD4 T cells will impair their function and proliferation. To test this, a mouse model will be used where RGS2 is knocked out only in CD4 T cells. CD4 T cells will be isolated from the spleens of these mice and their littermate controls. After isolation, the CD4 T cells will be activated in vitro, and their cytokine production and proliferation will be analyzed using ELISAs and flow cytometry. This study aims to provide deeper insights into the regulation of CD4 T cell signaling, highlighting potential areas for future research to optimize T cell responses.

Cutaneous leishmaniasis (CL) is a chronic parasitic infection that can result in difficult to treat chronic non-healing ulcers (CNUs). CNUs are common in CL and treatment is further complicated by subsequent infections by biofilm-forming multidrug resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive, multidrug resistant pathogen that forms biofilms on skin surfaces and exposed wounds and can lead to additional complications including sepsis. Our research group is investigating the role of MRSA biofilms in CL treatment and further exploring the pathology of Leishmania and MRSA coinfections in chronic non-healing wounds. My project is to prepare biofilm cultures, in both abiotic cultures and cocultures with 3T3 fibroblasts, and test conditions for biofilm formation in preparation for imaging by both confocal microscopy (CFM) and scanning electron microscopy (SEM). Using a crystal violet biofilm assay to quantify cellular and non-cellular biofilm components, we are screening culturing conditions (temperature, time, and nutrition) for imaging biofilm by both CFM and SEM in preparation for imaging in murine and patient tissues. Through SEM imaging, we have noted that in tested conditions (37 Celsius, 10 percent oxygen), MRSA forms discreet microcolonies across abiotic and 3T3 cocultures. Furthermore, these colonies form extrapolymeric substance (EPS) only after the colony dimensions exceed 15 microns in diameter, and EPS production is in greater quantity when cocultured with 3T3 fibroblasts vs. abiotic culture. We expect that our cultures will replicate biofilms observed in ex vivo samples and be used to further validate microscopy techniques for the rapid identification of biofilm.