Würzburg, Universiẗat, Diss., 2002.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

No description available.

The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Though it is primarily known as the site of ribosomal biogenesis, the nucleolus is also capable of orchestrating the immobilization of a broad range of proteins under specific environmental conditions. This process, known as nucleolar sequestration, contributes to cell viability under stress. Despite the importance of this post-translational regulatory pathway, very little is known about the mechanisms that govern it. Here, we show that heat shock and acidosis, two physiological stimuli associated with nucleolar sequestration, induce the expression of long noncoding RNA (lncRNA) from stimulus-specific loci of the ribosomal intergenic spacer (IGS). These lncRNAs, in turn, immobilize proteins encoding a nucleolar detention sequence (NoDS) within a compartment of the nucleolus termed the detention centre (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate (ANS)-positive hydrophobic signature. Its formation is accompanied by a redistribution of nucleolar factors and an arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA, by environmental signals, operates as a molecular switch that regulates the structure and function of the nucleolus.

No description available.

Overwhelming evidence indicates that aspirin (ASA) and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity against colorectal cancer (CRC). Although the underlying mechanisms have yet to be fully elucidated, the host laboratory have shown that nucleolar sequestration of the NF-κB component RelA is critical. In the course of these studies, it was noted that alongside effects on the NF- κB pathway, ASA has a profound effect on nucleoli, including a dramatic increase in nucleolar size. These data were particularly interesting as, in addition to its role in ribosome biogenesis, the nucleolus is known to act as a stress sensor and play a key role in the regulation of cell growth and apoptosis. Indeed, this organelle has been identified as a potential target for anti-tumour agents. However, how stress causes changes to nucleolar function, and how these are translated into changes in cell phenotype, remain unclear. Therefore, the aim of my thesis was to fully characterise ASA effects on nucleoli and to determine whether these effects contribute to the anti-tumour activity of this agent. I found that ASA induced an atypical form of nucleolar stress that was associated with enlargement of the organelle, relocalisation of nucleolar markers to the periphery, depletion of the critical component of the Pol I transcription factor complex, TIF-IA, and inhibition of rRNA transcription. These effects were independent of the p38 and JNK2 MAP kinase pathways. However, they were mimicked by inhibition of CDK4, which had previously been shown to lie upstream of ASA effects on the NF-κB pathway. These data describe a novel mechanism by which ASA, and CDK4 inhibition, may inhibit the growth of colon cancer cells. In addition to this candidate approach, I used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based quantitative proteomics to obtain a global overview of ASA effects on nucleoli of colon cancer cells. Firstly, a protocol was successfully developed to isolate pure nucleoli from SW480 CRC cell lines. This protocol was then applied to SILAC labelled cells treated with ASA for three time-points (0, 6, 10 h). In collaboration with R.T Hay and M. Tatham (University of Dundee), proteomic analysis was then carried out by tandem-mass spectrometry. These data confirmed that ASA has a significant effect on the nucleolar proteome. They also revealed that ASA induces a distinct type of nucleolar stress that is associated with the accumulation of chaperones, translational regulators and members of the ubiquitin-proteasome system (UPS) in this organelle. These data were reminiscent of studies previously published on the effect of proteasome inhibition on nucleoli. I therefore used SILAC-based proteomics to compare ASA effects on nucleoli to those induced by the proteasome inhibitor, MG132. I found that similar sub-groups of proteins accumulate in nucleoli in response to both agents and that ASA induced proteotoxic stress in a similar manner to MG132. Fluorescence correlation spectroscopy in collaboration with R. Duncan and K. Martin (Heriot-Watt University) demonstrated the relative reduction in mobility of nucleolar DsRed-RelA, indicating that, similar to MG132, ASA induces formation of nucleolar aggresomes. Mechanistic studies suggested that blocking ASA-mediated proteotoxic stress blocked the apoptotic effects of the agent. Taken together, these data define a distinct type of nucleolar stress that may be involved in the cells response to proteotoxic stress and be required for the anti-tumour activity of ASA.

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 114-122).

Filamin A is a well-characterised cytoskeletal protein which regulates cell shape and migration by cross-linking with actin. Filamin A mutations cause a number of human developmental disorders, many of which exhibit skeletal dysplasia. However, the molecular mechanisms by which Filamin A affects skeletal development are unknown. The transcription factor Runx2 is a master regulator of osteoblast and chondrocyte differentiation. Data presented in this thesis show that Filamin A forms a complex with Runx2 in osteoblastic cell lines. Moreover, it is demonstrated that Filamin A is present in the nucleus in several cell lines, including those of osteoblastic origin. The data presented show that the Filamin A/Runx2 complex suppresses the expression of the gene encoding the matrix-degrading enzyme, matrix metalloproteinase-13 (MMP-13), which is an important osteoblastic differentiation marker. ChIP assays were employed to demonstrate that endogenously expressed Filamin A associates with the promoter of the MMP-13 gene. In addition, Filamin A is not only located in the nucleus but also in the nucleolus, an important nuclear compartment involved in ribosomal RNA (rRNA) transcription. Ribosomal DNA promoter-driven reporter assays, Filamin A-knockdown experiments and exogenous Filamin A transfections demonstrated that Filamin A and Runx2 can repress ribosomal gene expression activity. Importantly, Filamin A is recruited to the human ribosomal DNA promoter, suggesting its direct involvement in the regulation of rRNA transcription. These findings reveal a novel role of Filamin A in the direct regulation of ribosomal gene expression. Finally, by using microarray technology, changes in gene expression were identified when Filamin A was downregulated. Some of the differentially expressed genes were known orchestrators of bone development. The data presented in this thesis strengthen the link between Filamin A and bone development and provide a molecular rationale for how Filamin A, acting as a regulator of gene expression, might influence osteoblastic differentiation.

The nucleoplasmin (NPM) family of acidic histone chaperones and the FK506-binding (FKBP) peptidyl proline isomerases are both linked to chromatin regulation. In vertebrates, NPM and FKBP domains are found on separate proteins. In fungi, NPM-like and FKBP domains are expressed as a single polypeptide in nucleoplasmin-like FKBP (NPL-FKBP) histone chaperones. Saccharomyces cerevisiae has two NPL-FKBPs: Fpr3 and Fpr4. These paralogs are 72% similar and are clearly derived from a common ancestral gene. This suggests that they may have redundant functions. Their retention over millions of years of evolution also implies that each must contribute non-redundantly to organism fitness. The redundant and separate biological functions of these chromatin regulators have not been studied. In this dissertation I take a systems biology approach to fill this knowledge gap. First, I refine the powerful synthetic genetic array (SGA) method of annotating gene-gene interactions, making it amenable for the analyses of paralogous genes. Using these ‘paralog-SGA’ screens I define distinct genetic interactions unique to either Fpr3 or Fpr4, shared genetic interactions common to both paralogs, and masked genetic interactions which are direct evidence for processes where these enzymes are functionally redundant. I provide transcriptomic evidence that Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. I identify an important role for Fpr4 at the 5’ ends of protein coding genes and the non-transcribed spacers of ribosomal DNA. Finally, I show that yeast lacking Fpr4 exhibit a genome instability phenotype at rDNA, implying that this histone chaperone regulates chromatin structure and DNA access at this locus. Collectively, these data demonstrate that Fpr3 and Fpr4 operate separately, cooperatively and redundantly to regulate a variety of chromatin environments. This work is the first comprehensive and comparative study of NPL-FKBP chaperones and as such represents a significant contribution to our understanding of their biological functions. / Graduate