Serotonin-specific reuptake inhibitor (SSRI) treatment of depressed patients displays a three week latency in amelioration of symptoms. Delayed desensitization of 5-hydroxytryptamine 1A (5-HT1A) autoreceptors is believed to be in part responsible for this latency and may involve receptor dephosphorylation. We hypothesized the 5-HT1A secondary intracellular (i2) loop interacts with receptor regulatory molecules. Yeast two-hybrid screen of a human cDNA library using 5-HT1A-i2 peptide as bait identified a novel 5-HT1A-i2 effector protein phosphatase 2A catalytic subunit (PP2AC). The 5-HT1A-i2-PP2AC interaction was quantified using yeast mating/beta-galactosidase assay. Furthermore, co-immunoprecipitation of tagged recombinant 5-HT1A receptor and endogenous PP2AC verified the interaction. Treatment with 5-HT1A receptor agonist enhanced the interaction. Mutation of critical receptor residues reduced or eliminated binding of PP2AC. Our results indicate a novel interaction between the 5-HT1A receptor and PP2AC and future studies will provide new insight into the role of protein phosphatase-receptor interactions in regulating 5-HT1A receptor desensitization, and resensitization.

Transcription-coupled nucleotide excision repair (TC-NER) removes bulky DNA lesions from the template strand at actively transcribed genes. The RNA polymerase II (RNAPII) holoenzyme complex forms a stable ternary complex at the site of DNA damage which may block access of DNA repair proteins to the site of DNA lesions. Therefore, there is considerable interest in understanding how repair is coupled to transcription. Based on elegant in vitro studies, it has been hypothesized that transcription elongation factor IIS (TFIIS), by catalyzing the reverse translocation of RNAPII, may allow access of DNA repair proteins to sites of DNA damage. Here, we tested this hypothesis by assessing TC-NER capacity in cells in which TFIIS expression has been reduced by RNA interference. Surprisingly, we found that decreased TFIIS levels did not affect the repair of transcription-blocking DNA lesions and did not affect the sensitivity of targeted cells to UV light or cisplatin. These results do not support a role for TFIIS in TC-NER. We conclude conservatively that TFIIS levels are not limiting for TC-NER.

Methylation of arginine residues is a post-translational modification mediated by specific enzymes known as the Protein Arginine Methyltransferase (PRMT) family. In this thesis, we present our research on PRMT8, an enzyme catalyzing the formation of asymmetric dimethylarginine (aDMA), which displays a unique distribution at the plasma membrane of neuronal cells of the central nervous system (CNS). An ontogenic analysis of PRMT8 in mouse tissues revealed that its expression in the brain is induced during the perinatal stage and is maintained in adulthood. The P19 cell line was identified as a valid model for endogenous PRMT8 and showed the induction and requirement of PRMT8 to achieve neuronal processes. A P19 PRMT8 knock-down cell line does not survive neuronal differentiation while, in contrast, no cell death is observed when the muscle lineage is induced. Taken together, these findings suggest an important role for PRMT8 in neuronal differentiation and/or function in the CNS.

The activity of the ATP-sensitive potassium (KATP) channel, whose sensitivity to ATP and ability to permit K+ flux together allow the channel to couple the metabolic state of a cell to its membrane excitability, is important in several tissues for the maintenance of glucose homeostasis and cytoprotection. In skeletal muscle specifically, the channel has been shown to be involved in cell volume regulation, modulation of glucose uptake, and the prevention of fiber damage and contractile dysfunction during fatigue. The extent of the cytoprotective capabilities of the KATP channel vary tremendously between muscles with different muscle fiber type composition. Semi-quantitative measurement of Kir6.2 subunit content showed that variances exist in KATP channel content between fiber types and between different muscles. These differences may be related to the extent of importance of the channel's function in a specific muscle. The highest Kir6.2 content was observed in the most glycolytic fiber types and in the more glycolytic muscles studied, and it is these muscles and fiber types which appear to be most dependent on functional KATP channels for their cytoprotective role.

The process of tumourigenesis in breast cancer involves a number of cellular proteins that work in concert to allow for cellular survival, proliferation, angiogenesis and continued tumour growth. The transcription factor NF-kappaB, which is often constitutively expressed in mammary carcinoma, signals through a number of downstream targets, one of which is cIAP2, to promote cellular growth and evasion of apoptosis. It is unclear if the NF-kappaB in cancer is needed for mammary stem cell or tumour-initiating cell survival. In the first part of this two-part study, it was shown that MMTV-ErbB2 mice lacking cIAP2 expression developed tumours with increased latency compared to MMTV-ErbB2 mice with cIAP2 expression. These tumours exhibited no change in cellular mitotic index or levels of tumour-associated macrophage infiltration, but did exhibit an increase in the number of apoptotic cells and an increase in angiogenesis. Preliminary data also suggests that tumours in knockout mice also exhibit a decrease in ErbB2 transgene expression levels, and an associated decrease in the expression of phospho-Akt, which may be associated with an increase in transgene silencing with age. It is proposed that cIAP2 knockout tumours have circumvented the absence of this survival gene in part by signaling angiogenesis, but the exact mechanism driving tumour formation remains unclear. In the second part of this study, mouse mammary epithelial stem and progenitor cells from dissociated mammary glands of NF-kappaB EGFP reporter mice were analyzed for NF-kappaB expression by flow cytometry and by fluorescent staining for nuclear p65 (NF-kappaB). Mouse mammary repopulating units (MRUs) and mammary colony forming cells (Ma-CFCs) were analyzed; these cell types have been previously described. MRUs have been characterized as basal-like mammary cells, and Ma-CFCs express markers characteristic of luminal mammary cells. It was found that only 6% of MRUs exhibited NF-kappaB expression, whereas 100% of the Ma-CFCs contained NF-kappaB expression. Analysis of tumours from MMTV-ErbB2 mice expressing the NF-kappaB EGFP reporter transgene was also completed. Sorting MMTV-ErbB2 induced mammary tumour cells based on CD44 surface staining (a previously reported tumour initiating cell marker) was unsuccessful, suggesting that CD44 may not be a putative marker for the cancer stem cell. These data suggest that NF-kappaB is activated during differentiation in the luminal Ma-CFC progenitor population, but not in the stem cell enriched populations.

The commitment of cells to skeletal muscle differentiation is regulated by the myogenic regulatory factors Myf5, MyoD, myogenin and MRF4. Myf5 is the earliest of the MRFs expressed in an embryo. An array of transcriptional factors and signals present in dermomyotome and myotome such as Wnt, Shh, Six1/4, Eya1/2 and Pax3/7 regulate the expression of Myf5. Also, the HAT activity of coactivator p300 is also needed for Myf5 expression. However, the exact function of the p300 HAT activity that is required for expression of Myf5 has not been determined. The spatio-temporal expression of Myf5 is also regulated by a large number of enhancers spread over 140 kb upstream of the transcription start site. The early epaxial enhancer regulates the expression at the earliest time point known. We hypothesized that HAT activity of p300 may be involved in direct regulation of Myf5. We used the embryonal carcinoma P19 cells to study skeletal myogenesis and the chemical inhibitor curcumin to study the role ofp300 HAT activity. Curcumin was able to inhibit commitment into skeletal myogenesis by downregulating expression of Myf5 and MyoD. Furthermore we show that p300 is present at the early epaxial ehancer and that the function ofp300 there may be histone acetylation. Therefore we provide evidence that p300 may be directly involved in regulation of Myf5 expression.

Herpes simplex virus 1 (HSV-1) infection is the main cause of vision loss in the developed world due to the fact that after initial infection, the virus establishes latency and can be reactivated; resulting in a very low success rate of treatment by transplantation (22%). We are developing corneal substitutes suitable for HSV-1 transplantation by incorporating anti-viral drugs such as acyclovir (ACV), into corneal substitutes that could potentially suppress the viral reactivation. Specifically, we examined the feasibility of preventing viral reactivation during surgery, by sustained delivery of ACV introduced during corneal transplantation surgery, through encapsulation of the drug within silica (SiO2) nanoparticles (NP) incorporated into biosynthetic alternatives to donor corneas. We show that incorporation of NPs did not affect optical clarity of the collagen-based corneal substitutes nor their biocompatibility. NP-encapsulation effectively sustained ACV release from the biosynthetic implants over 10 days, compared to free ACV incorporated directly into the hydrogel constructs. The NP-enabled sustained release resulted in effective prevention of virally-induced cell death, not observed with the free drug. This early model demonstrates the feasibility of using biomimetic corneal substitutes that incorporate a drug release system (e.g. silica nanoparticles encapsulating ACV) as future alternatives to human donor tissue grafts, for transplantation of HSV-infected corneas.

Spinal Muscular Atrophy results from loss of the survival of motor neuron (SMN) gene, leading to a deficiency in SMN protein and selective degeneration of motor neurons. We have found that coactivator-associated arginine methyltransferase 1 (CARM1) regulates the switch from proliferation to differentiation, downstream of neurotrophic signaling, in motor neuron-like cells. Here, through down-regulation of CARM1 levels, methylation of one of its substrates, HuD, is reduced, resulting in an increase in p21 mRNA. Methylation of HuD by CARM1 negatively regulates its direct interaction with p21 mRNA, the first demonstration that CARM1 can directly influence the RNA binding activity of a substrate. We have also identified a novel interaction between HuD and SMN. In MN-1 differentiation, we propose that SMN functions as an adaptor module through arginine methylation-regulated interactions with RNA binding proteins, including HuD, and that the formation of distinct mRNP complexes on mRNA are regulated either similarly or differentially by PRMTs and/or SMN. These findings may help to elucidate the specific role of arginine methylation and SMN in regulating differentiation of motor neurons and provide crucial insights into the cell-specific pathophysiology of spinal muscular atrophy.

Retinoic acid (RA) plays an integral role during embryonic development, in addition to being a potential chemotherapeutic and chemopreventative agent in various cancer models due to its growth inhibitory and pro-differentiation properties. RA signaling occurs in a ligand-dependent manner through a family of nuclear receptors consisting of the RARs and RXRs. RA elicits growth inhibitory effects in transformed wild type keratinocytes, however the target genes that mediate this outcome remain largely unidentified. In order to isolate genes that could mediate the latter effects, a suppressive subtractive hybridization experiment was conducted by our lab and revealed growth arrest specific 3 (gas3) as a candidate gene. Gas3 is expressed in the skin and is induced by RA as early as two hours post-treatment in tissue culture models. We investigated a possible relationship between gas3 and epidermal tumorigenesis and found that this gene behaves in a context dependent, strain-specific manner as both an RA-mediated tumor suppressor and an oncogene. To further investigate this, I first demonstrated that RA-induced growth arrest in keratinocytes is mediated in part by gas3. Additionally, previous work from our lab demonstrated that gas3-null mice from an FVB/NJ background evaded tumor formation following the two-stage carcinogenesis protocol. I demonstrate that this occurs as a result of an increase in apoptotic response following chemical mutagenesis in gas3 mutants. Conversely, I found that Gas3 mutants on a C57Bl/6 background behave as wild type animals with respect to carcinogenesis, suggesting that genetic modifiers between these two strains impact on Gas3 function with respect to tumorigenesis. Finally, I found that p21 activity is affected by the loss of gas3, which may begin to elucidate how gas3 impacts on carcinogenesis.

ZMAN1 protein was identified in the brain, the gills, the liver, the kidney and the muscle tissue of the adult zebrafish. It is ubiquitously present in the brain and gill tissues. Using immunofluorescence on adult and embryonic cell lines, ZMAN1 was observed at the nuclear periphery. Moreover, cytoplasmic localization of ZMAN1 during mitotic phases indicates that ZMAN1 is not associated to mitotic chromosomes. ZMAN1 appeared at the surface of the new nuclear envelope at the beginning of nuclear reassembly. ZMAN1 distribution in blastula embryos was in the cytoplasm at different mitotic stages except for interphase, telophase and cytokinesis where it was targeted to the nuclear envelope. The maternally synthesized ZMAN1 was localized to the tail/posterior trunk of mid-pharyngula embryos and showed a very low expression and distribution patterns in the muscle tissue suggesting down-regulation of ZMAN1 at the adult stage. I also detected phenotypical abnormalities in ZMAN1 morphants, displayed a curved tail, dysfunctional tail vasculature and an enlarged pericadium, suggesting a possible function of ZMAN1 in blood vessels and heart morphogenesis. Altogether, my data suggest that ZMAN plays a role in early developmental processes and during the adult life of zebrafish.