http://hdl.handle.net/10027/8820 Thu, 20 Jun 2013 07:17:16 GMT 2013-06-20T07:17:16Z http://hdl.handle.net/10027/9777 Sequences in the NCAM Ig5-­‐FN1 Tandem Affect Interactions with the Polysialyltransferases Sequences in the NCAM Ig5-FN1 Tandem Affect Interactions with the Polysialyltransferases Matthew Gregory Thompson, Ph.D. Department of Biochemistry and Molecular Genetics University of Illinois at Chicago Chicago, Illinois (2012) Dissertation Chairperson: Karen J. Colley, Ph. D. The Neural Cell Adhesion Molecule (NCAM) carries a unique post-translational modification, polysialic acid, which modulates the its adhesive and signaling functions and consequently plays roles in nervous system development and plasticity, neuronal regeneration and cancer metastasis. The addition of polysialic acid is carried out by the polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST. Polysialic acid is found on a limited number of proteins and previous work indicates that its addition requires a protein-protein interaction between the polysialyltransferase and substrate prior to modification of the substrate glycans. To evaluate the basis for this unique specificity, the interaction between NCAM and the polysialyltransferases was examined in this work. In this study I investigated sequences in NCAM’s first fibronectin type III repeat (FN1) and fifth immunoglobulin like (Ig5) domain and their role in polyST recognition and interaction. Results from these investigations revealed key residues in both the FN1 and Ig5 domain that affects the ability of the polysialyltransferases to interact with NCAM to allow subsequent NCAM polysialylation. Sequences in FN1, most notably a surface acidic patch, are involved in the interaction of the polyST with NCAM. Sequences at the interface of the FN1 and Ig5 domain are critical in maintaining the Ig5-FN1 relationship to allow NCAM polysialylation. Finally using a non-polysialylated NCAM homolog, the Olfactory Cell Adhesion Molecule (OCAM) to create chimeric proteins, and evaluating their polysialylation, revealed that specific residues in OCAM Ig5 domain block its polysialylation and the analogous residues in NCAM Ig5 are permissive for polysialylation. Taken together these results demonstrate for the first time that the polysialyltransferases recognize NCAM by interacting with residues in both the Ig5 and FN1 domains, and suggest new strategies for controlling the extent of NCAM polysialylation during development and in disease. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9777 2013-02-21T06:00:00Z http://hdl.handle.net/10027/9775 Role of DDB2 in Senescence and EMT DDB2 (Damaged DNA Binding Protein 2) is a DNA repair protein initially identified as a key player in nucleotide excision repair. Several recent studies indicated potential tumor suppressor functions of DDB2. Towards that, I identified two new tumor suppressor functions of DDB2: inhibition of epithelial to mesenchymal transition (EMT) and induction of senescence. My studies revealed that DDB2 deficient cells do not undergo premature senescence after culture shock, exogenous oxidative stress, oncogenic stress or DNA damage. The deficiency in senescence was resulted from lack of ROS accumulation. Further investigation revealed that DDB2 causes ROS accumulation by epigenetically inhibiting expression of two important anti-oxidant genes, MnSOD and Catalase. These findings were further substantiated in mice by using chemical carcinogen induced liver fibrosis model and UV induced skin carcinogenesis model. As an extension of my work on delineating tumor suppressive role of DDB2, I investigated the role of DDB2 in colon cancer because reduced DDB2 expression is correlated with aggressive progression of colon cancer. Moreover, loss of DDB2 increases tumorigenecity of colon cancer cells in vitro and in vivo. A closer analyses attributed this increased tumorigenecity phenotype to a regulation of EMT and resistance to anoikis. Further findings indicated that the EMT regulation by DDB2 is related to transcriptional repression of VEGF, Snail1 and Zeb1. Concordantly, DDB2 deficient colon carcinoma cells are more metastatic in nature. Thus, loss of DDB2 expression results in a deficiency in senescence and increased EMT leading to an aggressive cancer progression. Hence, targeting DDB2 expression would be beneficial from therapeutic perspective. Towards that, I have used a naturally occurring compound Phenethyl Isothiocyanate (PEITC), currently in clinical trial, to elevate expression of DDB2 and inhibit tumorigenesis in mice. Together, my observation provides new insights on the role of DDB2 as a tumor suppressor through its regulation of senescence and metastasis related to EMT. These functions of DDB2 involve its role in transcription repression. Also, my work shows how DDB2 can be therapeutically targeted for the treatment of colon cancer. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9775 2013-02-21T06:00:00Z http://hdl.handle.net/10027/9773 Developmental and Pathological Effects of Psychosine on Oligodendrocytes of the Twitcher Mouse Psychosine is a lipid raft-associated cytotoxic sphingolipid that accumulates in individual nervous system affected with Krabbe disease, a deficiency of lysosomal galactosylceramidase enzyme. High levels of psychosine are believed to cause oligodendrocytes (OLs) cell death and leading to the progressive demyelination observed in these patients. The twitcher mouse (TWI) is a natural occurring mutant for this disease, which accumulates psychosine ~150 fold respect control levels and undergoes demyelination and death by the sixth week of postnatal life. Using transgenic labeling of OLs and stereological quantification, myelinating OLs are reduced in TWI spinal cord at P15 and P30. Accordingly in vivo apoptotic analysis revealed an increased OL cell death in TWI spinal cord at P15. However, at demyelination stage, the majority of apoptotic cells are activated microglia/macrophage, indicating the important involvement of immune cells in the pathology of demyelination. During prenatal and early postnatal development, psychosine has already modestly but significantly increased in TWI spinal cord. At P7, we found an unexpected increase of OLs in the cervical region of the mutant spinal cord. We hypothesized that at low concentration, psychosine might facilitate the generation of oligodendrocyte progenitors (OPCs) by modifying the lipid-raft platform for raft-associated signaling pathways. We further examined for changes in oligodendrocyte patterns and the raft-associated sonic hedgehog (SHH) pathway of TWI in the embryonic life (E10.5 to E15.5). Our results indicate distinct changes in abundance and distribution of OPCs in the embryonic spinal cord, which are concurrent with increasing levels of SHH downstream effectors. These findings are the first to show physiological levels of psychosine might affect the embryonic development of the nervous system in mutant. The relevance of these changes is still unclear but they may have profound and long lasting effects on the onset and progression of the disease. Meanwhile, our results also suggest high levels of psychosine may block the myelin formation OLs. But demyelination in mutant CNS may not be a direct result of OL cell death but be caused by immune cells reactions. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9773 2013-02-21T06:00:00Z http://hdl.handle.net/10027/9772 Effects of A Gap Junction Inhibitor on Stem Cell Retention and Efficacy During Early Myocardial Ischemia Bone marrow-derived mesenchymal stem cell (BM-MSC) replacement therapy is beneficial to the heart following ischemia but a significant loss of these cells within hours of administration could diminish their effect. Early coupling between BM-MSC and ischemic cardiomyocytes through gap junction (GJ) may play a detrimental role in stem cell survival and retention in the acute phase of cell therapy. We seeded 1×105 HL-1 atrial myocytes and place them in either normoxic or ischemic condition for four hours. Then, 2×104 lineage negative murine BM-MSC were seeded over the HL-1 monolayer and the cocultures were returned to incubation either in their previous conditions (normoxia, ischemia) or switched from ischemic to normoxic condition (ischemia–reperfusion) for an additional two hours after which they were lifted, labeled with fluorescent markers for dead and apoptotic cells, and subjected to flow cytometry analysis. Ischemia induced a greater proportion of dead BM-MSC over the two-hour coculture compared to the normoxic group. Ischemia–reperfusion resulted in significantly higher apoptotic but fewer dead BM-MSC. The presence of non-selective GJ inhibitor carbenoxolone (CBX) in the coculture significantly reduced the number of dead and apoptotic cells in ischemia and ischemia-reperfusion groups, respectively. We then created ischemia in mice by LAD ligation for 90 minutes after which the suture was removed to allow reperfusion. The animals were then given an intramyocardial injection of either 1×105 BM-MSC, 1×105 CBX-treated BM-MSC, CBX or vehicle. At 24 hours after the injury, their hemodynamic parameters, number of transplanted BM-MSC remained in the heart, and infarct size were assessed. Ischemia–reperfusion caused impaired cardiac function which can be attenuated by BM-MSC injection. CBX-treated BM-MSC further enhanced the cardiac function while the CBX alone did not. Flow cytometry analysis of whole heart digests demonstrated significantly increased number of BM-MSC in the hearts that had been given CBX-treated BM-MSCs as compared to those that received untreated BM-MSCs. However, no significant change in infarct size was observed in the heart at this specific time point. Our data suggest that early GJ communication may represent a novel paradigm whereby ischemic cardiomyocytes induce a detrimental effect on newly transplanted stem cells and thus reduce their efficacy. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9772 2013-02-21T06:00:00Z