Ion channels are proteins that facilitate ion diffusion across cell membrane. Nevertheless, various groups of ion channels can act as surface receptors and play important roles in signal transduction. Transient Receptor Potential Melastatin 7 (TRPM7) ion channel has been implicated in diverse cellular functions including actomyosin cytoskeletal remodeling and anoxic neuronal death. However the mechanisms behind TRPM7’s physiological roles remain undetermined. TRPM7 possesses unusually long intracellular domains and a functional C-terminal alpha kinase domain that may contribute to regulation of channel activity and signal transduction. We therefore identified proteins that interact with TRPM7 C-terminus. Pull-down assays coupled with LC-MS/MS revealed that cytoskeletal proteins (actin and tubulin) and synaptic vesicle proteins (VAMP2 and SNAP25) associate with the TRPM7. In addition, we further found that TRPM7 does not directly bind microtubules or single dimeric tubulin subunits. Thus one or more microtubule binding proteins is involved in the association between TRPM7 and microtubules.

The vertebrate hindbrain is divided early in embryogenesis along its anterior-posterior axis into eight segments known as rhombomeres. This provides an excellent model for studying early segmentation and region-specific transcriptional domains. MafB, a basic domain leucine zipper transcription factor, is the first gene known to be expressed in the presumptive rhombomere 5 and 6 domain (r5-r6). MafB expression is directly activated by the homeodomain protein vHnf1. vHnf1 and MafB share an anterior expression limit at the r4/r5 boundary but vHnf1 expression extends beyond the posterior limit of MafB and, therefore, cannot establish the posterior expression boundary of MafB. Through the use of in situ hybridization, immunofluorescence, and chromatin immunoprecipitation analyses, I have determined that the caudal-related homeodomain protein Cdx1 establishes the posterior boundary of MafB by directly inhibiting expression beyond the r6/r7 boundary. My results indicate that MafB is one of the earliest direct targets of Cdx1.

The bacterial protein H-NS acts to silence horizontally acquired genes. H-NS physically interacts via its N-terminus with two paralogous proteins, Hha and YdgT. Deletion of hha and ydgT results in derepression of a subset of H-NS repressed genes. I compared expression of hha/ydgT-dependent genes in Salmonella strains lacking hns and hha/ydgT/hns. Deletion of all three genes does not result in greater gene expression than deletion of hns alone, indicating that Hha and YdgT cannot act to repress genes in the absence of H-NS. Further, I used site-directed mutagenesis to generate H-NS proteins incapable of binding Hha. Complementing an hns deletion with an Hha-blind H-NS molecule, H-NS I11A, recapitulated the pattern of gene expression in the hha/ydgT strain. Indicating that elimination of the Hha-H-NS interaction is sufficient to result in derepression of hha/ydgT repressed genes. Hha and YdgT repress gene expression by acting through H-NS and cannot act independently of H-NS.

Paediatric undifferentiated soft tissue sarcomas (USTSs) are a diagnostically challenging group of neoplasms. We hypothesized that USTSs contain distinct subgroups that can be identified based on their morphology, genomic aberrations and expression profiles. We sought to characterize genomic aberrations within primitive round cell (PRC) sarcomas which may underlie aberrant expression patterns. Using molecular and cytogenetic analyses, we identified 5 of 18 CIC-DUX4-positive PRC sarcomas. The consistent involvement of the CIC-DUX4 fusion in a subset of PRC sarcomas suggests a central role for the fusion transcript in such tumours. These analyses also identified a cohort of CIC-DUX4-negative USTSs with no established genetic markers. We performed integrative copy number and expression profiling, and identified significant genomic and transcriptomic changes. We propose that these genes are involved in biological pathways that are important to the initiation and progression of undifferentiated sarcoma, and may provide novel insights into the biological events responsible for sarcomagenesis.

Distal regulatory elements (DREs) have been identified upstream of the hematopoietic regulator Lim domain only 2 (Lmo2) gene in the human and mouse genomes. In this thesis I have investigated how these elements regulate Lmo2 transcription in erythroid cells. My results show that strong chromatin-chromatin interactions exist between the DREs and the Lmo2 gene promoter in erythroid cells. These interactions are absent from kidney cells that do not express Lmo2. Within the distal chromatin interaction cluster encompassing three of the DREs increased DNase I sensitivity, presence of high levels of H3K4me1, and binding of multiple transcription factors, p300, cohesin (RAD21) and CTCF are observed. CTCF bound regions are located between the farthest DRE and the neighboring Caprin1 promoter suggesting that CTCF insulates Caprin1 from the DREs. Hence, my data suggests that these DREs function through a chromatin looping mechanism supported by cohesin associated with CTCF and transcription factor bound regions.

Animals carry lipids such as hydrocarbons, fats, and sterols throughout their circulatory systems bound to a carrier protein known as lipophorin. The lipophorin receptor has been characterized in locusts, mosquitoes and cockroaches yet little is known about it in Drosophila melanogaster. An antibody against the eleven variants of the lipophorin receptor was developed and tested. Although this was the main feature of the work, several preliminary experiments using RNA interference were conducted to determine the effects of lipophorin receptor. Flies whose lipophorin receptor proteins were knocked down were found to have no major differences in locomotor activity in total darkness suggesting that their circadian rhythms are unaffected. The same flies were found to have extensive differences in their cuticular hydrocarbon profiles as compared with wild‐type flies. Whole‐mount tissue staining of Drosophila adult brains revealed that several cells in the central nervous system are immunoreactive with the anti‐Lipophorin receptor antibody.

Staphylococcus aureus secretes two cysteine proteases, Staphopain A (scpA) and Staphopain B (sspB). We hypothesized that ScpA will exhibit a distinct activation mechanism, and a different or complementary substrate specificity compared to SspB. A Cys>Ala active site substitution led to the accumulation of unprocessed 40-kDa proScpA, confirming that ScpA undergoes autocatalytic activation. A temporal analysis of ScpA expression revealed that activation was initiated by processing at Lys171 and Glu176, producing an intermediate that was rapidly converted to several isoforms of mature protease by processing after Thr202, Lys209, Thr214 and Asn216. Consistent with broad specificity, mature ScpA was sensitive to autocatalytic degradation. ScpA demonstrated activity towards elastin, fibrinogen and indicated evidence for binding to heparin. Elastinolytic activity was uniquely associated with strains belonging to CC30, and was correlated with ScpA expression. Therefore, although ScpA and SspB share both sequence and structural similarity, they exhibited very different substrate specificities and activation mechanisms.

Replication of HIV-1 requires nuclear export and translation of the incompletely spliced 4 and 9 kb classes of HIV-1 mRNA, which encode the structural and enzymatic proteins of the virus. HIV-1 Rev binds to the Rev-responsive element (RRE) contained in the introns of incompletely spliced HIV-1 mRNAs and mediates their nuclear export via the Crm1 pathway. Sam68C, is a C-terminal deletion mutant of the endogenous human protein Sam68, and has been shown to be a potent inhibitor of Rev-dependent reporters. In this study we have performed deletion analysis of Sam68C, and determined the minimal mutant required for inhibition of Rev-dependent expression is Sam6814(45-54)-300. Sam68C inhibition is specific to RRE/Rev/Crm1 transported mRNAs: the Rev/Crm1 exported reporter construct GagRRE is inhibited while the Tap/p15 transported GagCTE reporter construct is not. Previous work from our lab showed that Sam68C co-localized with the Rev-exported mRNAs in perinuclear bundles. Further investigation has shown that Sam68C inhibition of incompletely spliced HIV-1 mRNAs is independent of the perinuclear bundling of the viral mRNA. We go on to show that Sam68C specifically inhibits the translation of the incompletely spliced HIV-1 mRNAs. Translational inhibition by Sam68C is correlated with a loss of PABP1 binding with no attendant change in abundance, polyadenylation or polyadenosine tail length of the affected mRNAs. The selective inhibition of Crm1 exported HIV-1 mRNAs by Sam68C suggests that it is able to recognize unique characteristics of these viral mRNPs. We show that Rev and the RRE are required, but individually neither is sufficient for complete Sam68C inhibition. Study of the incompletely spliced HIV-1 mRNP revealed that the nuclear cap binding complex, CBP20/80, is not exchanged for eIF4E. Additionally, in cells expressing the HIV-1 provirus, CBP80 relocalizes to the cytoplasm and co-sediments with polysomes. This supports the hypothesis that incompletely spliced HIV-1 mRNAs are translated in an eIF4E-independent, CBP20/80-dependent fashion. This property of the 9kb and 4kb HIV-1 mRNAs could be utilized to develop new therapeutic approaches to controlling HIV-1 infection.

Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.

Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.