Telomeres form a nucleoprotein cap at the end of eukaryotic chromosomes. The telomere protein constituents repress the DNA damage response (DDR) and facilitate maintenance of terminal sequences by a specialized ribonucleoprotein complex called telomerase. In turn, factors involved in the DDR guarantee telomerase acts only in telomere homeostasis, and not at double-strand breaks (DSBs). Thus, the three pathways surrounding telomeres display incredible overlap and are immensely complex. Here, I report a novel regulatory pathway that limits telomerase action during DNA damage. Duplication of the telomerase RNA subunit (TER) in Arabidopsis has given rise to a TER that is not required for telomere homeostasis. Indeed, this TER, termed TER2, is a competitive inhibitor of TER1 RNP complexes. Exposure to genotoxic agents results in TER2 upregulation and a subsequent inhibition of telomerase activity. Using data from the 1,001 Arabidopsis genomes project, I determine that the TER duplication and inhibitory nature of TER2 is likely derived from a transposon-like element within TER2. This element is found throughout Brassicaceae, with at least 32 members in Arabidopsis lyrata. These findings highlight the complex and diverse mechanisms by which an organism will regulate telomerase action. Here I characterize two members of the A. thaliana POT1 gene family. Contrary to POT1a, these proteins appear to have derived unique ways to perform their roles in chromosome-end protection. POT1b may protect telomeres as part of a TER2 telomerase RNP complex, as telomere defects only appear in the absence of both POT1b and TER2. POT1c is also appears to provide for chromosome end protection and appears to compete with POT1a to regulate telomerase access to the G-overhang. Together, these proteins represent part of a critical telomere capping complex distinct from CST. Additionally, I describe a means for elucidating factors that regulate telomere addition at DSBs. This incredibly detrimental process, termed de novo telomere formation (DNTF), is toxic, and thus this work describes the first in depth characterization of DNTF in multicellular eukaryotes. In summary, my work describes several novel regulatory and protective mechanisms for keeping telomeres and DSBs distinct.

Doctor of Philosophy (PhD) / Aged care is a critical public policy issue in Australia. The growing significance of the sector raises important and pressing questions about many aspects of care itself, the size of the labour force and employment relations. Answering these questions is vital, with demand for labour in the sector already outstripping supply and with demand certain to grow substantially. The implications of this labour shortfall for the sector have already been the subject of a number of key government reports. Although these reports have begun to construct a more detailed picture of the issues facing aged care workers and employers, significant gaps remain, most notably any explicit examination of approaches to the management of labour or the importance of labour law in determining these approaches. Despite the obvious importance and critical social and economic significance of the ageing population, we do not sufficiently understand many of the critical labour market features, workplace characteristics or management strategies which are evident in the aged care sector. This study seeks to build knowledge of employment and labour management in this growing and crucial sector at a decisive moment in history. It deepens our understanding of these issues and processes through a study of three residential aged care providers in New South Wales during the period from 2005 to 2009. The thesis specifically examines employer strategy in relation to the management of labour in the three cases. Further, it investigates the impact of the regulatory environment on these approaches. In doing so, the case studies reveal the intricate web of internal and external, direct and indirect, formal and informal regulation which shapes the management of labour within the sector. The complexity of the regulatory web in aged care demands the use of an explanatory framework which recognises that labour-management approaches are influenced by constraints not traditionally associated with the direct, legal regulation of employment relations. Consequently, regulation theory is applied here as an organising framework and as an interpretive prism for the research. This allows for an explicit acknowledgment of the importance of non-legal, informal and indirect regulation ‘at work’ in this sector. The study finds that in the period under review labour law was not the primary determinant of labour-management approaches in aged care. The case studies presented here show that it was, in fact, a second order consideration for aged care providers struggling with what they saw as insufficient funding, onerous ‘paperwork’ and staff recruitment and retention difficulties – in short a range of other regulatory influences. This study also shows that, despite the constraints imposed by these other regulatory modes, employers remained free to exercise their prerogative within the workplace; this, in turn, is revealed as a form of internal regulation in aged care.

PhD / Self-regulation is a vital function to humanity, and is an important factor in the dominant paradigm of consumer research, whereby consumer decisions are characterised by the battle between long- and short-term interests. The current research examined the relative effectiveness of two self-regulatory strategies: stopping an already-commenced consumption episode, or to not commence one at all. Traditional economic theories, including the principle of diminishing marginal utility, would predict that not starting is harder to accomplish; whereas a proposal by Thaler (1983) suggests that not starting is in fact the optimal strategy. Two studies were conducted whereby participants were asked to either perform a less-favoured task and resist from starting a more-favoured one (Not Start), or to cease performing a more-favoured task to complete the less-favoured task (Stop). Study 1 found that Stop was more difficult than Not Start, which tentatively supported Thaler’s argument; however there was an explanation which could not be ruled out, namely the psychological distance of the anticipated second task. Study 2 addressed this issue by manipulating that factor by incorporating it into the experimental design. It was found that Not Start became as depleting as Stop when psychological distance of the second task was reduced. This research contributed to the literature by establishing a boundary condition upon the strength model of self-regulatory resource depletion, and adds to the discussion on the descriptive validity of the principle of diminishing marginal utility.

Research Doctorate - Doctor of Philosophy (PhD) / Pre-term birth is still the leading cause of perinatal mortality and morbidity. CRH is a hormone that is involved in the timing of labour, therefore investigation of its regulation is of importance in understanding human parturition. The CRE is a central regulatory element on the CRH promoter and in investigating proteins that bind to this element a novel protein was discovered. CREAP or cAMP Regulatory Element Associated Protein, was initially discovered by its ability to bind to the CRE. Its sequence encodes a unique set of modular domains including two zinc fingers, two leucine zippers, two coiled-coils and an RS-rich domain. These domains point to functions in both DNA binding/transcription and RNA splicing, with the leucine zippers being characteristic of bZIP transcription family and the RS domain characteristic of the SR Protein family of splicing factors, to represent a new protein family. In this thesis, molecular reagents were produced for the study of CREAP together with a polyclonal antibody. This antibody was used in western blotting to detect a 58 kDa full-length CREAP protein and a shorter 25-30 kDa truncated splice variant. CREAP was localised to the nucleus and to intranuclear splicing speckles, with co-localisation and co-immunoprecipitation with the splicing factor SC35, strongly suggesting a role in splicing. To test the transcriptional activity of CREAP, specifically if it regulates CRH expression, luciferase reporter studies were conducted. However, CREAP showed negligible effect on CRH or CRE promoter activities suggesting that it is not involved in CRH regulation. CREAP did however react with a large number of transcription factors in an in vitro assay, mostly from the bZIP and zinc finger families. siRNA mediated knockout of CREAP was conducted and the effect on genome-wide expression analysed using a microarray. CREAP knockdown caused an over-representation of genes from the protein transport, metabolism, signal transduction and transcription factor processes. Overall, CREAP appears to be a multifunctional protein that is ubiquitously expressed, and is involved in both splicing and transcriptional processes.

The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.

The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.

Research Doctorate - Doctor of Philosophy (PhD) / Pre-term birth is still the leading cause of perinatal mortality and morbidity. CRH is a hormone that is involved in the timing of labour, therefore investigation of its regulation is of importance in understanding human parturition. The CRE is a central regulatory element on the CRH promoter and in investigating proteins that bind to this element a novel protein was discovered. CREAP or cAMP Regulatory Element Associated Protein, was initially discovered by its ability to bind to the CRE. Its sequence encodes a unique set of modular domains including two zinc fingers, two leucine zippers, two coiled-coils and an RS-rich domain. These domains point to functions in both DNA binding/transcription and RNA splicing, with the leucine zippers being characteristic of bZIP transcription family and the RS domain characteristic of the SR Protein family of splicing factors, to represent a new protein family. In this thesis, molecular reagents were produced for the study of CREAP together with a polyclonal antibody. This antibody was used in western blotting to detect a 58 kDa full-length CREAP protein and a shorter 25-30 kDa truncated splice variant. CREAP was localised to the nucleus and to intranuclear splicing speckles, with co-localisation and co-immunoprecipitation with the splicing factor SC35, strongly suggesting a role in splicing. To test the transcriptional activity of CREAP, specifically if it regulates CRH expression, luciferase reporter studies were conducted. However, CREAP showed negligible effect on CRH or CRE promoter activities suggesting that it is not involved in CRH regulation. CREAP did however react with a large number of transcription factors in an in vitro assay, mostly from the bZIP and zinc finger families. siRNA mediated knockout of CREAP was conducted and the effect on genome-wide expression analysed using a microarray. CREAP knockdown caused an over-representation of genes from the protein transport, metabolism, signal transduction and transcription factor processes. Overall, CREAP appears to be a multifunctional protein that is ubiquitously expressed, and is involved in both splicing and transcriptional processes.

München, Techn. University, Diss., 2007.

Zugl.: Bochum, University, Diss., 2005.

Universiẗat, Diss., 2007--Giessen.