Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Although there are many therapies against cancer, they involve devastating side effects. Hence, we need to understand the nature of cancer cells and the unique markers that exist within these cells that allow them to evade the immune system. These cell properties could be exploited to our advantage. CD47 is a cell membrane receptor protein widely expressed in most cells and is a versatile and crucial target in the tumor microenvironment for creating novel therapeutic approaches for cancer treatment. However, few studies have examined CD47 in glioma. CD47 is found on the surface of multiple cell types, and it usually protects the cells from being removed by phagocytes. It has been found that most cancer cells have high CD47 expression that prevents them from being engulfed by macrophages or activated microglial cells, essentially acting as a “don't eat me signal.” A second protein expressed on cancer cells, calreticulin (CALR), facilitates cell removal by phagocytes, serving as an “eat me signal.” In this project, we compared CD47 expression in glioma cancer stem cells (CSCs), which are negative for Hoechst 33342 nuclear staining (H-), and non-stem glioma cells (Hoechst-positive, H+) of the C6 cell line derived from a rat astrocytoma. We examined the colocalization of CALR with CD47 in both C6 cell types using immunocytochemistry and compared CALR and CD47 gene expression reported in the NCI-60 database of multiple human cancers. We found a significant difference in CD47 expression, with more CD47 in the H+ cells than the H- cells, which could imply that GSCs are more susceptible to CD47 immunotherapy. The highest expression of CD47 ( 50% above signal range) appeared to be in exosomes related to both cell types. We found a positive correlation between CD47 and CALR distribution in the H+ cells (p = 0.0204) and in both H+ and H- cells combined (p = 0.0121), suggesting that the cells might protect themselves from CALR-induced phagocytosis by increasing CD47. We al (open full item for complete abstract)

Committee: Michael Geusz PhD (Committee Chair); Julia Halo PhD (Committee Member); Paul Morris PhD (Committee Member) Subjects: Biology; Medicine

Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

An important mechanism that cancer cells use to survive adverse conditions is upregulating their unfolded protein response (UPR) to secrete more chaperone proteins including calreticulin (CALR). CALR is a resident endoplasmic reticulum protein that has various functions spanning from the nucleus, cytoplasm, and up to the cell membrane. Evidence indicates that cancer cells and especially cancer stem cells (CSCs) have adapted an elevated UPR causing CALR to be produced abundantly such that some of the protein relocates to the cell membrane. At the cell membrane CALR functions as an extracellular signal to immune cells that triggers removal of the cell by microglia and macrophages through phagocytosis. This project tested whether CSCs of gliomas have higher surface CALR expression than non-stem-like cancer cells that form most of the tumor. CSCs previously identified in the C6 rat glioma cell line were further characterized by immunocytochemistry. Based on previous reports from our lab and others, we identified the CSCs in C6 rat cell cultures along with immunofluorescence imaging of cell-surface CALR. Significantly higher surface CALR expression was present in CSCs within the cell clusters forming early tumorspheres. We also extracted RNA-Seq results from the IVY Glioblastoma Atlas Project database describing gene expression for CALR and genes coding for 25 proteins closely related by function to CALR. We compared this activity across five glioblastoma tumor structures and found elevated gene expression in the Pseudopalisading Cells Around Necrosis and Microvascular Proliferation. The known prevalence of CSCs in these tumor structures and the transcriptome data agree with our C6 stem cell results indicating elevated CALR in glioma CSCs. When we induced epithelial-mesenchymal transition (EMT) in C6 cells, an event leading to invasive CSCs, surface CALR was significantly elevated in the resulting cells. We also examined by immunofluorescence the expression of ATF4, a tr (open full item for complete abstract)

Committee: Michael Geusz Ph.D. (Committee Chair); Carol Heckman Ph.D. (Committee Member); Vipa Phuntumart Ph.D. (Committee Member) Subjects: Biology; Biomedical Research; Health; Immunology; Medicine; Neurosciences

Master of Science (MS), Bowling Green State University, 2019, Biological Sciences

The master circadian clock in the hypothalamic suprachiasmatic nucleus of vertebrates receives information from the retina that entrains the clock to cycles of light and darkness in the animal's environment. This timing signal is important for maintaining the appropriate organization of circadian rhythms generated throughout the body, which have many effects on health, development, and aging. One potential influence on entrainment is from retinal light signals acting on SCN cells that have protein expression patterns typical of stem cells. These stem-like cells may have plasticity in their interactions allowing circadian rhythms generated in the SCN to be modified when needed to adapt to changing environmental or internal conditions. Calcium-binding proteins in SCN cells have important functions in the entrainment process, but the role of one of these proteins, calreticulin (CALR), has not been examined in the SCN. This study characterized the spatial pattern of CALR-expressing SCN cells and their distribution among neurons (MAP2-positive), glial cells (GFAP-positive), and stem-like cells (SOX2-positive) by using immunocytochemistry and confocal fluorescence microscopy. Cells expressing significant levels of CALR were found throughout most of the SCN, with fewer in the core region. In the SCN, 86.9% of CALR cells were classified as neurons, 19.1% as astrocytic glial cells, and 60.33% as stem-like cells according to immunofluorescence imaging. To determine whether CALR expression can be induced by a stimulus that can entrain the SCN circadian clock a group of mice were exposed to two hours of light in the early portion of the night and were compared with mice remaining in darkness. Although SCN neurons did not show a significant response to the stimulus, non-neuronal cells including glial cells did, showing a two-fold increase in percentage of CALR-positive cells. We conclude that a substantial number of the CALR cells, 98.6%, express stem-marker SOX2 elevated, sugge (open full item for complete abstract)

Committee: Michael Guesz Dr. (Advisor); Verner Bingman Dr. (Committee Member); Raymond Larsen Dr. (Committee Member) Subjects: Biology; Molecular Biology; Neurosciences

Master of Science (MS), Wright State University, 2019, Microbiology and Immunology

Macrophages are essential phagocytic cells involved in both innate and adaptive immune systems and play vital roles in the host defense and inflammation. Macrophages have a remarkably high capacity to clear unnecessary cellular materials in interstitial environment through a process called “phagocytosis”, which is affected by many factors including suppressors of cytokine signaling (SOCS). SOCSs are a group of intracellular proteins that downregulate the cytokine signals involved in various JAK/STAT pathways through a negative feedback loop. This study focuses on investigating the effects of SOCS1 and SOCS3 on the phagocytic ability of RAW 264.7 macrophages polarized into M2a with IL-4/IL-13 and into M2c with IL-10 to clear fluorescently marked Neuro-2a malignant cells. Calreticulin (CRT) is one of the cellular signals, exposed on the surface of malignant cells, that play vital roles in controlling the phagocytosis process mediate by phagocytes including macrophages. Treatment of M2a (IL-4/IL-13) and M2c macrophages with SOCS1 peptide mimetics had no significant effects on M2a and M2c-mediated phagocytosis of CFSE-stained N2a cells compared with M2a (IL-4/IL-13) and M2c cells without SOCS1 peptide mimetic treatment, while it slightly increased the M2a (IL-4/IL-13), but not M2c, macrophage-mediated phagocytosis of N2a cells that had their calreticulin receptors blocked. When N2a cells were blocked with anti-CRT antibody, M2a and M2c-mediated phagocytosis was decreased. Interestingly, treatment of M2c with SOCS3 peptide mimetics coincided with blockage of CRT signals on N2a cells caused a significant increase in the phagocytosis of N2a cells mediated by M2c polarized macrophages. These data help to get a deeper understanding of macrophage functions in cleaning the microenvironment from apoptotic and malignant cells and even wound healing. Overall, this study sheds more light on how SOCS1/ SOCS3 can be used along with manipulation of “eat-me” signals exposed on m (open full item for complete abstract)

Committee: Nancy Bigley Ph.D. (Advisor); Dawn Wooley Ph.D. (Committee Member); Marjorie Markopoulos Ph.D. (Committee Member) Subjects: Immunology; Microbiology