Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "August 2003." Typescript. Vita. Includes bibliographical references (leaves 158-174). Also issued on the Internet.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 126-149). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Molecular Biology) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 126-147). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Proliferation and differentiation are essential processes for the growth and development of higher eukaryotic organisms. Regulation of gene expression is essential for control of cell division and differentiation. Normal eukaryotic cells have a limited proliferative capacity, and ultimately undergo cellular senescence and apoptosis. Terminal differentiation of cells is associated with loss of proliferative capacity and acquisition of specialized functions. Proliferation and differentiation are processes required for the creation and maintenance of diverse tissues both during embryonic development and postnatal life. The cell cycle is the process by which cells reproduce, and requires duplication and segregation of hereditary material. Loss of cell cycle control leads to genetic instability and cancer. Expression of replication-dependent histone genes is tightly coupled to DNA synthesis, thus making histone genes a good model for studying cell cycle regulation. The HiNF-D complex interacts with all five classes (H1, H2A, H2B, H3 and H4) of histone genes in a cell cycle-dependent manner. The CCAAT displacement protein (CDP)/Cux and the tumor suppressor pRB are key components of the HiNF-D complex. However, the molecular interactions that enable CDP/Cux and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters are poorly understood. Transient transfection assays show that CDP/Cux represses the histone H4 promoter and that the pRB large pocket domain functions with CDP/Cux as a co-repressor. Direct interaction between CDP/Cux C-terminus and the pRB pocket domain was observed in GST pull-down assays. Furthermore, co-immunoprecipitation assays and immunofluorescence microscopy established that CDP/Cux and pRB form complexes in vivo and associate in situ. pRB interaction and co-repression with CDP/Cux is independent of pRB phosphosphorylation sites, as revealed by GST pull-down assays and transient transfection assays using a series of pRB mutant proteins. Thus, several converging lines of evidence indicate that complexes between CDP/Cux and pRB repress cell cycle-regulated histone gene promoters. CDP/Cux is regulated by phosphorylation and acetylation at the C-terminus, which contains two repressor domains and interacts with histone deacetylase HDAC1. In vivo function of the CDP/Cux C-terminus in development and gene regulation was assessed in genetically targeted mice (Cutl1tm2Ejn, referred to as Cutl1ΔC). The mice express a mutant CDP/Cux protein with a deletion of the C-terminus including the homeodomain. Indirect immunofluorescence microscopy showed that the mutant protein exhibited significantly reduced nuclear localization in comparison to the wildtype protein. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Cutl1 ΔC -/-) mice, indicating the functional loss of CDP/Cux in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wildtype and Cutl1 ΔC -/- MEFs in culture. However, the histone H4.1 (murine FO108) gene containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice exhibit stunted growth (20-50% weight reduction), a high postnatal death rate of 60-70%, sparse abnormal coat hair and severely reduced fertility. Hair follicle deformities and severely diminished fertility in Cutl1 ΔC -/- mice suggest that CDP/Cux is required for normal development of dermal tissues and reproductive functions. Together the data presented in this dissertation provide new insight into the in vivo functions of CDP/Cux in the regulation of histone gene expression, growth control and differentiation.

The nucleolus is a plurifunctional organelle with dynamic protein exchange involved in diverse aspects of cell biology. Additionally, the nucleolus has been shown to have a role in the replication of numerous viruses, which includes HIV-1. Several groups have reported HIV-1 vRNA localization within the nucleolus. Moreover, it has been demonstrated the HIV-1 Rev protein localizes to the nucleolus and interacts with nucleolar proteins, including NPM1. Despite evidence for a nucleolar involvement during replication, a functional link has not been demonstrated. I investigated whether introncontaining vRNAs have a Rev-mediated nucleolar localization step prior to export. Furthermore, I examined whether NPM1 mediates Rev nucleolar localization, participates in Rev function, and/or post-transcriptional events during viral replication. I used coupled RNA fluorescence in situhybridization and indirect immunofluorescence to visualize intron-containing vRNA relative to the nucleolus in the absence or presence of Rev expression. An RNAi-based approach was used to examine the role of NPM1 in Rev function and viral replication in cell lines and primary human macrophages. My research findings support a model for a Rev-independent nucleolar localization step of introncontaining vRNA prior to export. Intriguingly, my results also suggest NPM1 does not participate in Rev nucleolar localization or Rev-mediated vRNA export, as previously proposed. Rather, my findings support a novel role for NPM1, the cytoplasmic localization and utilization of a select class of Rev-dependent vRNAs. Collectively, my findings provide novel insight for a functional role of the nucleolus and NPM1 in HIV-1 replication, which enhances our current understanding of HIV-1 biology.

No description available.

ABSTRACT I CHARACTERIZATION OF A NOVEL NUCLEAR VARIANT OF BMP2Bone morphogenetic protein 2 (Bmp2) is a signaling protein that was first detected by its ability to induce cartilage and bone formation. It has since been implicated in broad variety of developmental, patterning, and disease processes. To date, Bmp2 has only been known to function as an extracellular signaling molecule. However, we have obtained clear evidence for a nuclear form of Bmp2. This nuclear variant, nBmp2, contains a bipartite NLS that overlaps the site of proteolytic cleavage. The NLS remains intact and functional when translation of Bmp2 initiates from a downstream alternative start codon. The resulting protein lacks the signal peptide and is therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing and secretion. Instead, the uncleaved protein containing the intact NLS is translocated to the nucleus. Preliminary functional analyses in zebrafish indicate that nBmp2 is critical for proper heart development. To determine if this function is conserved in mammals, we have also generated mice harboring a null allele for nBmp2. ABSTRACT II COORDINATE REGULATION OF COL11A2 AND COL27A1 BY THE TRANSCRIPTION FACTOR LC-MAF During skeletal development, long bones of the body develop from a cartilage template that is progressively replaced by bone. This process of endochondral ossification requires precisely coordinated expression of extracellular matrix proteins such as the cartilage-specific collagens. In this study, enhancer/reporter assays demonstrated that the transcription factor Lc-Maf inhibits the transcriptional activity of a cartilage-specific Col11a2 enhancer element while a cartilage-specific COL27A1 enhancer element was strongly activated by Lc-Maf. Site-directed mutagenesis identified the binding region within the COL27A1 enhancer, and it was found to be unlike any known consensus Maf family binding site. The in vivo significance of these results was examined using immunohistochemistry and in situ hybridization in mouse limbs undergoing endochondral ossification. Taken together, these results suggest that Lc-Maf participates in the developmental transition from proliferating to hypertrophic chondrocytes during endochondral ossification by coordinately downregulating Col11a2 and upregulating Col27a1 collagen gene expression.

One of the major unresolved questions in cancer biology is why the majority of tumour cells express mutant p53 proteins. p53 is considered the prototype tumour suppressor protein, whose inactivation is the most frequent single genetic event in human cancer (Bourdon et al., 2005). Genetically-engineered p53-null knockout mice acquire multiple tumours very early on in life and human Li-Fraumeni families who carry germline mutations in p53 are highly cancer-prone (reviewed in Vousden and Lane, 2007). p53 mutant proteins have been found to acquire novel functions that promote cancer cell proliferation and survival, yet exactly why mutant p53s acquire oncogenic activity is still poorly understood. Mutant p53 has also been found to complex with wildtype p53, thus acting in a dominant negative way. However, this inhibition is incomplete since many cancers with mutant p53 alleles also have a loss of the second wild-type p53 allele and thus only express the mutant p53 (Baker et al., 1989). An N-terminal truncated p53 isoform, p47, arising from alternative splicing of the p53 gene (Ghosh et al., 2004) or by alternative initiation sites for translation (Yin et al. , 2002), has been described. Alternative splicing was found to be universal in all human multi-exon genes (Wang et al., 2008) and therefore determining the role of the p47 isoform with respect to the p53 gene is essential. Evidence in this study suggests that mutant p53 (p53RI75H) has a similar structure and function as p47, including the ability to complex with and impair both p53 and p73. Therefore, in addition to expressing a tumour suppressor protein, the p53 gene can also express an onco-protein (p47). This study therefore argues that tumours select for mutant p53 because it has gained the ability to function like p47, a wild-type p53 isoform.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.