No description available.

No description available.

No description available.

No description available.

The human wildtype p53 tumor suppressor gene contains a polymorphism at amino acid residue 72 which results in either an arginine (p53 Arg-72) or proline (p53 Pro-72) at this codon. In the present study I have examined this polymorphism at the molecular level to determine whether differences exist in the biochemical functions of these two p53 variants. No differences were observed in their sequence-specific DNA binding abilities, nor in their ability to be targeted by HPV-18 E6 oncoprotein for degradation by ubiquitination in vitro. However, differences were observed in the ability of these two variants to function as transcriptional activators: p53 Pro-72 was more transcriptionally active than p53 Arg-72. I propose that the polymorphism at codon 72 may affect the structure of the N-terminal transactivation domain of the p53 protein, which would then have an effect on the ability of these variants to interact with transcription factors in order to initiate transcription of target genes and function as a tumor suppressor.

Reactivation of master regulators of epithelial to mesenchymal transition (MR-EMT) represents the molecular basis for tumour cell plasticity, malignant transformation and metastases. However, the current evidence on the specific role of MR-EMT in melanomagenesis has not been fully addressed. The purpose of this investigation was to assess the expression and regulation of these factors in malignant melanoma and to evaluate their prognostic and clinical significance. In vitro experiments indicated that a switch in MR-EMT protein expression ZEB2/SNAI2 to ZEB1/TWIST1 is RAS-RAF-MAPK signalling dependent. In addition, evidence supported a MR-EMT interactome, in which transcriptional repression of ZEB2 by Fra-1 resulted in upregulation of ZEB1, independently of miR-200 family. Further in vitro and immunohistochemical (IHC-P) analyses showed that E-cadherin and VDR protein levels were significantly reduced by the presence of ZEB1 in melanoma cells and archive tissues. Motility assays demonstrated that ZEB1 but not ZEB2 enhances cell migration. IHC-P analyses of ZEB2/SNAI2 (n=142/28) showed a statistically significant gradient of stronger staining at superficial sites compared to the deep sites in a select cohort of independent and matched melanoma tumour samples. In contrast, ZEB1 (n=142) and TWIST1 (n=133) showed higher staining in deep sites of primary melanomas and metastases. Trend analyses showed a significant MR-EMT switch in this progression series from high levels of ZEB2/SNAI2 in naevi towards high ZEB1/TWIST1 expression in melanomas. In primary melanomas these factors were also significant in Kaplan Meier survival curves and after two step cluster analysis the combined profile of ZEB1[superscript high]/TWIST1[superscript high]/ZEB2[superscript low] predicted the worse prognosis (P=0.001). Multivariate Cox regression analyses of IHC-P staining indicated that only the gain of ZEB1 (P<0.002, n=98) and superficial TWIST1 (P=0.012) were associated with poor metastasis-free survival and independent of breslow depth. In conclusion, the reversible switch between ZEB1/TWIST1 and ZEB2/SNAI2 is controlled by RAS-RAF-MAPK pathway activity and constitutes an independent factor of poor prognosis in patients with malignant melanoma.

Condensed tannins are wide-spread polyphenols with diverse ecological functions, including defense against herbivores and microbial pathogens. In poplar, condensed tannin synthesis is induced by a variety of biotic and abiotic stresses. The objective of this study was to determine the function of the R2R3 MYB transcription factor MYB115 in the regulation of condensed tannin synthesis. MYB115 was shown to be induced by wounding along with tannin biosynthetic genes and shows sequence similarity to characterized regulators of tannin synthesis in grape and persimmon suggesting that it functions in the regulation of condensed tannin synthesis. To analyze the function of MYB115, transgenic plants overexpressing MYB115 were generated and showed enhanced accumulation of condensed tannins and higher expression of flavonoid biosynthetic genes involved in condensed tannin biosynthesis compared to wild-type control plants. In promoter activation assays, MYB115 activated the promoter of a tannin-specific biosynthetic enzyme, anthocyanidin reductase. This suggests that MYB115 acts as a regulator of condensed tannin synthesis. MYB115 overexpressors showed additional changes to phenolic metabolism, including changes in levels of phenolic glycosides and hydroxycinnamic acids. These results indicate an important role of MYB115 in the regulation of the condensed tannin pathway in poplar. / Graduate / 0817 / 0307 / 0487 / frankla@uvic.ca

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

Fusarium verticillioides (Teleomorph Giberella moniliformis) is an ascomycete fungus responsible for ear and stalk rots of maize. Most importantly, it produces a group of mycotoxins called fumonisins upon colonization of maize kernels. Fumonisin B1 (FB1), the most prevalent fumonisin in nature, was first identified in 1988 and has been found to be toxic to human and animals. The gene cluster for FB1 biosynthesis and some environmental conditions responsible for the toxin production are known, but gaps in our understanding of the signaling pathways leading to FB1 biosynthesis still remain. MADS-box transcription factors (TF) are known to regulate diverse cellular functions in all eukaryotes, and in silico analyses revealed two genes, MADS1 and MAD2, in F. verticillioides. Reverse genetics studies indicated that MADS1 and MADS2 positively regulate sexual mating and FB1 biosynthesis but not pathogenicity in F. verticillioides. Furthermore, MADS1 was found to act as a broad regulator of polyketide-derived secondary metabolism. Additionally, population diversity studies were conducted in 164 F. verticillioides cultures isolated from 65 maize-producing counties in Texas. The result showed a fluid population with no particular niches formed. F. verticillioides strains were also isolated from counties that have previously tested negative for FB1 contamination in maize. The presence of the pathogen represents a risk for future FB1 contamination events if suitable conditions were to arise. My research revealed new genetic components involved in F. verticillioides secondary metabolite biosynthesis and provided a better understanding of the pathogen population fluidity in Texas.

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other CP2 family members, CRTR-1 expression is regulated developmentally. Major sites of expression in the embryo include the pluripotent inner cell mass (ICM) of the pre-implantation blastocyst and the developing kidney. It is also expressed in embryonic stem (ES) cells, which are derived from the ICM of blastocysts, and is downregulated as these cells differentiate into early primitive ectoderm-like (EPL) cells. This expression pattern suggests that CRTR-1 plays a role in early pluripotent populations. This thesis aims to characterize the transcription factor CRTR-1 at the molecular level and analyses the role of sumoylation on CRTR-1 function to develop a better understanding of the molecular role of CRTR-1 in ES cells. Luciferase reporter assays show that CRTR-1 is able to regulate the activities of other CP2 family members: CP2, NF2d9 and altNF2d9. It enhances CP2- and NF2d9-mediated activation but suppresses altNF2d9-mediated activation. To map the functional domains in the CRTR-1 protein, transactivation studies using CRTR-1 deletion mutants fused to the GAL4 DNA binding domain and a GAL4-responsive reporter system were performed. These studies map repressor activity to amino acids 48-200, but fail to identify a transactivation domain within the CRTR-1 protein. In order to understand the mechanisms by which CRTR-1 regulates the transcriptional activities of CP2 family members, a number of approaches are taken, including co-immunoprecipitation to show that CRTR-1 interacts with other CP2-like proteins, EMSA which demonstrate that CRTR-1 forms DNA binding complexes with CP2 family members, and subcellular protein localisation studies which reveal the ability of CRTR-1 and other family members to shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. In addition, the subcellular localisation of CRTR-1 appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells. Co-expression of CRTR-1 with CP2 or NF2d9 results in the re-localisation of CRTR-1 to the cytoplasm in ES cells. The sumoylation enzymes Ubc9 and PIAS1 have previously been identified as CP2-interacting proteins (Kang et al., 2005a). Given the identification of two potential sumoylation sites within CRTR-1, FK³⁰ QE and LK⁴⁶ ⁴AE, and the ability for sumoylation to regulate transcription factor function, the possibility that CRTR-1 is regulated by sumoylation is investigated in this thesis. Immunoprecipitation experiments show that CRTR-1 is modified by SUMO-1 and that lysine 30 is the critical residue for this modification. Mutation of lysine 30 to alanine, which abolishes CRTR-1 sumoylation, results in enhancement of transactivation by CRTR-1, suggesting that sumoylation of CRTR-1 blocks maximal activation. Unexpectedly, however, overexpression of Ubc9, PIAS1, or SUMO-1 results in enhancement of CRTR-1 transcriptional activity, indicating that a more complex mechanism of regulation of CRTR-1 activity is likely. This thesis presents several novel properties of CRTR-1 and other CP2 family members, including the ability of CRTR-1, previously characterized as a repressor, to activate transcription. It is also the first demonstration that CP2 proteins are regulated by sumoylation and that they shuttle between the nucleus and cytoplasm via a CRM1-dependent mechanism. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374290 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009