2017 Denman Undergraduate Research Forum Accepted Student Abstracts Laboratory/Cellular Health Sciences

2017 Denman Undergraduate Research Forum Accepted Student Abstracts Laboratory/Cellular Health Sciences Category: Laboratory/Cellular Health Sciences Title: The nuclear pore complex protein NupA is required for normal localization of the mRNA export factor Gle1 in Aspergillus nidulans Student Presenter: Leymaan Abdurehman Faculty Advisor: Osmani, Stephen Abstract: Mitosis is a fundamental process for the development of multicellular organisms. While significant insights have been obtained into mitotic mechanisms, the disassembly and reassembly of Nuclear Pore Complexes (NPCs) is not well understood. NPCs carry out transport across the nuclear envelope. During mitosis in Aspergillus nidulans, NPCs are disassembled during mitotic entry. Some NPC proteins locate to mitotic structures and operate transport functions in interphase. NupA was identified as a novel NPC protein that co-purified with the conserved NPC protein Nup2 during interphase and mitosis. During mitosis, both proteins locate to mitotic chromatin with unknown functional significance. Deletion of NupA leads to activation of the Spindle Assembly Checkpoint (SAC) as well as late mitotic defects. NupA was found to be important for normal localization of NPC proteins Mad1 and Ndc1. We are interested in determining whether additional NPC proteins localization depends on NupA. As a candidate, we chose Gle1, an essential NPC protein involved in mRNA export. To delete NupA, a gene replacement construct was generated by fusion PCR and transformed into A. nidulans. Since NupA is an essential gene, we analyzed the phenotype of its deletion using the heterokaryon rescue technique. NupA deletion was confirmed through diagnostic PCR with primers flanking the deletion construct. Live cell confocal microscopy indicated that the localization of Gle1-GFP was affected in the absence of NupA. While Gle1-GFP locates exclusively at the nuclear periphery in WT cells, it additionally located in the cytoplasm without NupA after mitosis in G1. Since Gle1 is an important mediator of mRNA export, we speculate that mRNA export might be affected in NupA-deleted cells. Our studies suggest that NupA has important roles in correct localization of NPC proteins particularly after mitosis. We speculate that NupA performs mitotic functions on chromatin that are required for normal localization of NPC proteins. In the future, it will be important to examine whether the localization of other NPC proteins also depend on NupA and Nup2 and if their chromatin location is required for NPC segregation during mitosis. Category: Laboratory/Cellular Health Sciences Title: Targeting MDSC enhances the anti-tumor effects of checkpoint inhibitor therapy in a mouse model of melanoma Student Presenter: David Abood Faculty Advisor: Carson III, William Abstract: Myeloid derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that expand in tumor-bearing hosts. MDSC play a critical role during tumor evasion and have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species (ROS and RNS) and release of immune-suppressive cytokines. Our group discovered that MDSC express a kinase known as Bruton's tyrosine kinase which can be inhibited with the kinase inhibitor ibrutinib which is FDA-approved to treat leukemias. Tumors express immune-checkpoint molecules including programmed death ligand 1 (PD-L1) to escape immune destruction. PD-L1 binds to its receptor, programmed cell death protein 1 (PD-1), which inhibits the function of T cells. Anti-PD-L1 monoclonal antibodies are in clinical use and their administration prevents the engagement of PD-L1 to its receptor, allowing an effective immune response to proceed in upwards of 30% of patients. We hypothesized that inhibition of MDSC with ibrutinib would enhance the activity of a PD-L1 blocking antibody (Ab). We therefore investigated the effect of ibrutinib in combination with an anti-PD-L1 antibody on tumor growth and the levels of MDSC using a mouse model of melanoma. C57BL/6 mice were inoculated with 1x105 B16F1 melanoma cells. Mice were treated with either vehicle control, ibrutinib (12.5 mg/kg/day) via drinking water, anti-PD-L1 antibody (25 µg) injected three times a week, or a combination. Tumors were measured three times a week using digital calipers. Ibrutinib or anti-PD-L1 Ab treatment alone had a modest effect on tumor growth compared to control-treated mice while the combination treatment led to a 32% reduction in tumor volume. Mice treated with ibrutinib, anti-PD-L1 Ab or the combination had average spleen MDSC levels of 6.57%, 5.64%, and 5.46% respectively, compared to the vehicle at 8.58%. These results show that ibrutinib can enhance the anti-tumor effects of anti-PD-L1 Ab in the setting of melanoma. Category: Laboratory/Cellular Health Sciences Title: Effects of sucrose and omega-3 dietary modification on lipid peroxidation in liver of mouse models of breast cancer and chemotherapy Student Presenter: Xin Ni Au Faculty Advisor: Orchard, Tonya Abstract: Will dietary sucrose and long-chain omega-3 fatty acids (EPA+DHA) affect lipid peroxidation in the liver of mouse models of chemotherapy or breast cancer? Doxorubicin, a widely used chemotherapeutic agent, generates free radicals which may increase lipid peroxidation and oxidative stress. Research suggests that sucrose increases oxidative stress; conversely EPA+DHA limit oxidative stress and lipid peroxidation in the brain of mice treated with doxorubicin. It is unknown how the combination of sucrose and omega-3 fatty acids impacts lipid peroxidation in metabolically important tissues such as the liver in the presence of chemotherapy or mammary tumors. Livers from mouse models of breast cancer (Tumor model: n=32; 8/diet) and chemotherapy (Chemo model: n=40;10/diet) were analyzed for 4-hydroxynonenal (4-HNE), a lipid peroxidation marker, using an enzyme-linked immunoabsorbent assay. In both experiments, 8-9 week old female C57BL/6 mice were ovariectomized and randomized to diets one week later. Tumor model mice were fed low sucrose diets, with either 0% or 2% kcal from EPA+DHA. Two weeks later, mice were injected with metastatic mammary tumor cells or control; after 21 days, tissues were collected. Chemo model mice were fed low sucrose, 0% or 2% kcal EPA+DHA diets, or high sucrose, 0% or 2% kcal EPA+DHA diets. Mice were injected with doxorubicin based chemotherapy or saline two and four weeks later; tissues were collected 11 days after second injection. Mean liver 4-HNE in tumor mice (0.0649ug HNE-BSA/ugProtein;SEM 0.0066) vs. control (0.0613ug HNE-BSA/ugProtein;SEM 0.0066) was not significantly different (p=0.71) and did not differ by diet (p=0.54). Similarly, mean liver 4-HNE in chemo mice (0.0817ug HNE-BSA/ugProtein;SEM 0.0114) vs. control (0.0877ug HNE-BSA/ugProtein;SEM 0.0117) was not significantly different (p=0.72) and did not differ by diet (p=0.42). In conclusion, we found no differences in liver lipid peroxidation in chemotherapy-treated or tumor mice vs. controls when fed varying levels of sucrose and EPA+DHA. Category: Laboratory/Cellular Health Sciences Title: The role of CCL2 chemoattractant in lumbar myeloid cell trafficking and locomotor recovery following thoracic spinal cord injury Student Presenter: Kimberly Berry Faculty Advisor: Basso, Michele Abstract: Spinal cord injury (SCI) induces inflammation and damage across the cord that hinders neuronal plasticity and locomotor recovery. Previous studies of spinal cord response to injury show increased levels of chemoattractants (CCL2) and cell adhesion molecules (ICAM-1), chemotactic agents that induce the movement of specific cell types. Peripheral myeloid cells invade the lumbar region after thoracic SCI, rendering the lumbar microenvironment refractive to rehabilitation. We hypothesized that removal of CCL2 via genetic knockout (KO) will decrease myeloid cell infiltration into the lumbar spinal cord after SCI, to promote a more permissive microenvironment for rehabilitation interventions compared to typical, wild-type SCI (WT). We also hypothesized that downhill treadmill training 14-21 days post-injury would improve functional recovery in both KO and WT models. We used four groups with SCI at T9: CCL2KO exercised (n=8) or unexercised (n=8), and WT exercised (n=8) or unexercised (n=4). We quantified monocyte infiltration (IBA-1/p2ry12) and ICAM-1 expression via immunohistochemistry to identify co-regulatory features between chemoattractants and cell adhesion molecules in the trafficking of peripheral myeloid cells into the lumbar cord. Locomotion was assessed via Basso Mouse Scale (BMS) and 2D kinematics. Contrary to our hypothesis, the CCL2KO resulted in lower BMS scores than WT with and without training (untrained, p Category: Laboratory/Cellular Health Sciences Title: Optimization of bioartificial muscle to discover the mechanotransduction pathways of muscle atrophy in microgravity Student Presenter: Perry Blough Faculty Advisor: Lee, Peter Abstract: Of the many physiological changes seen after spaceflight in astronauts, skeletal muscle atrophy is among the most pronounced and damaging to long-duration spaceflight missions. However, the exact mechanism of spaceflight induced skeletal muscle atrophy is not completely understood. Under the funding of NASA and the Ohio Space Grant Consortium, our project is to launch tissue- engineered skeletal muscle constructs termed BioArtificial Muscle (BAM) into space to study the

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