Regulation of G2 phase and long-term consequences of DNA damage

Cell proliferation requires the accurate replication of DNA and equal segregation of replicated genes, important for maintaining the integrity of newly formed cells. At the centre of this process is a series of coordinated events termed ‘the cell cycle’, which ensures cell proliferation proceeds with high fidelity. Cell cycle regulation is driven by the activity of cyclin-dependent kinases (Cdks), which require binding to their regulatory subunit cyclin to become activated. However, the activity of Cdk is regulated by several different mechanisms. Transcription and degradation control mechanisms indirectly affect Cdk activity by modulating the expression of several regulatory proteins, including cyclins, while regulatory phosphorylation and dephosphorylation of cyclin-Cdk complexes provide direct control of Cdk activity. Such posttranslational modifications are frequently part of feedback loops, which fine-tune Cdk activity. These mechanisms collectively modulate successive activation of Cdks, and is responsible for timely phosphorylation of Cdk substrates to complete different phases of the cell cycle. This thesis concerns the regulation of G2 phase in the cell cycle, in relation to: 1) the effect of cyclin A2 localisation in G2 phase, 2) the changes in G2 phase regulation in a genetic disorder, and 3) the long-term consequences if G2 phase regulators are completely suppressed. Although Cdk activity is required for well-delineated cell cycle phase transitions, the spatiotemporal regulation of cyclin is important, as it provides unique substrate specificity and accessibility to the Cdk. Nevertheless, the exact mechanisms underlying the activation of cyclin-Cdk complexes remain largely elusive. The first part of this thesis investigates unknown mechanisms of mitotic kinase activation in G2 phase, by assessing the spatio-temporal regulation of cyclin A2 and its function in G2 phase. In paper I, we observe that nuclear cyclin A2 partially translocates to the cytoplasm at S/G2 phase transition. Interestingly, we reveal that cyclin A2-Cdk2 can initiate the activation of Plk1 through phosphorylation of Bora, but only cyclin A2 localised to the cytoplasm can interact with Bora and Plk1. We find no evidence that the change in localisation of cyclin A2 is involved in feedback loops in G2 phase. Thus, our study strongly supports the notion that cytoplasmic A2 functions as a trigger for the activation of mitotic kinases. Although the precise mechanism that changes the localisation of cyclin A2 to the cytoplasm requires further study, we show that cyclin A2 nuclear localisation until S/G2 phase transition is contributed, in part, by the association of cyclin A2 to chromatin during DNA replication. In addition, our work also reveals p21 can restrict cyclin A2 to the nucleus, especially after DNA damage. Together, paper I expands our understanding of the mechanisms of mitotic kinase activation in G2 phase, and identifies future areas of study to fill in our knowledge gaps of how cyclin A2 changes its cellular localisation. Cell cycle dysregulation has been implicated in many genetic diseases and disorders. This highlights the importance of understanding cell cycle regulation in certain disease settings. The second part of this thesis is dedicated to studying the role of a non-coding nuclear RNA gene, RMRP, that is mutated in the rare genetic disorder, cartilage-hair hypoplasia (CHH). CHH cells show proliferation defects, and studies on yeast suggest that RMRP could regulate the accumulation of cyclins. In paper II, we reveal RMRP has pleiotropic effects on several cell cycle regulatory genes, and the mutation of RMRP delays G2 phase progression to mitosis. Furthermore, our work finds evidence of possible impairment in the PI3K-Akt signalling pathway in CHH. These findings contribute to understanding the role of RMRP in cell cycle regulation, particularly in relation to CHH, and indicate a possible pathway for therapeutic interventions. The uncontrolled proliferation of cells with genomic instability can lead to the development of cancer. The cell cycle checkpoint is a mechanism that can restrict cell cycle progression in response to DNA damage and replication blocks. When checkpoint kinases are activated, signals are transmitted to a network of regulatory proteins that increase the inhibitory force and delay cell cycle progression. In the case of persistent DNA damage in G2 phase, p53 and p21- dependent premature activation of APC/CCdh1 mediates cell cycle termination by degrading all cell cycle regulatory proteins. While all these processes ensure genomic integrity, the mechanisms that allow escape from a checkpoint have been the focus of many studies, but whether cell cycle termination in G2 phase can be reversed remains unclear. Therefore, the last part of this thesis investigates the long-term consequences of DNA damage-induced cell cycle termination in G2 phase. Paper III shows that cells can re-initiate S phase after terminating the cell cycle in G2 phase. Interestingly, expression of p21 persists until cells re-initiate DNA replication and increases further once DNA re-replication is complete. This finding supports our observation of repeated cell cycle termination of re-replicated cells. Furthermore, re-replicated cells can progress to mitosis, which creates a heterogenous cell population, and is linked to genomic instability. Thus, resumption of the cell cycle a long period after termination in G2 phase can give rise to multiple cell fates. This shifts our current perception of the long-term consequences of cell cycle termination in G2 phase, from a singular outcome of senescence to that of multiple cell fates, possibly alluding to a mechanism by which cells can undergo oncogenic transformation. In summary, this thesis highlights the importance of the spatio-temporal regulation of cyclin A2 in modulating Cdk to initiate the mitotic entry network in G2 phase, ensuring welldelineated progression to mitosis. Identifying the function of RPRM in G2 phase adds to our limited understanding of cell cycle regulation in relation to CHH. Moreover, this thesis reveals that DNA damage-induced cell cycle termination in G2 phase can lead to cell fates other than senescence, an implication that could have relevance in tumourigenesis

A snapshot of Australian nurse practitioners' extended practice activities

Introduction The Australian Nurse Practitioner Project (AUSPRAC) was initiated to examine the introduction of nurse practitioners into the Australian health service environment. The nurse practitioner concept was introduced to Australia over two decades ago and has been evolving since. Today, however, the scope of practice, role and educational preparation of nurse practitioners is well defined (Gardner et al, 2006). Amendments to specific pre-existing legislation at a State level have permitted nurse practitioners to perform additional activities including some once in the domain of the medical profession. In the Australian Capital Territory, for example 13 diverse Acts and Regulations required amendments and three new Acts were established (ACT Health, 2006). Nurse practitioners are now legally authorized to diagnose, treat, refer and prescribe medications in all Australian states and territories. These extended practices differentiate nurse practitioners from other advanced practice roles in nursing (Gardner, Chang & Duffield, 2007). There are, however, obstacles for nurse practitioners wishing to use these extended practices. Restrictive access to Medicare funding via the Medicare Benefit Scheme (MBS) and the Pharmaceutical Benefit Scheme (PBS) limit the scope of nurse practitioner service in the private health sector and community settings. A recent survey of Australian nurse practitioners (n=202) found that two-thirds of respondents (66%) stated that lack of legislative support limited their practice. Specifically, 78% stated that lack of a Medicare provider number was ‘extremely limiting’ to their practice and 71% stated that no access to the PBS was ‘extremely limiting’ to their practice (Gardner et al, in press). Changes to Commonwealth legislation is needed to enable nurse practitioners to prescribe medication so that patients have access to PBS subsidies where they exist; currently patients with scripts which originated from nurse practitioners must pay in full for these prescriptions filled outside public hospitals. This report presents findings from a sub-study of Phase Two of AUSPRAC. Phase Two was designed to enable investigation of the process and activities of nurse practitioner service. Process measurements of nurse practitioner services are valuable to healthcare organisations and service providers (Middleton, 2007). Processes of practice can be evaluated through clinical audit, however as Middleton cautions, no direct relationship between these processes and patient outcomes can be assumed

Public trust and genomic medicine in Canada and the UK.

Background: Genomic medicine could improve precise risk stratification, early prevention, and personalised treatment across a broad spectrum of disease. As this reality approaches, questions on the importance of public trust arise. The success of genomic medicine initiatives is influenced by the public's trust and willingness to engage. Specific social actors influential in the public's trust have been identified by the "Your DNA, Your Say" study, including doctors, researchers, and governments. This paper aims to identify and examine which specific social actors, if any, in Canada and the United Kingdom (UK) are the most trustworthy and influential to engage the public in genomic medicine research. Methods: Using data from the 'Your DNA, Your Say' study, logistic regression models and Pearson's chi-square tests were conducted to explore trust in social actors across Canada and the UK. Results: The results demonstrate Canada and the UK significantly differ in public trust and willingness to donate. Non-profit researchers, domestic doctors, and personal doctors were identified to be the most influential and trustworthy social actors in Canada and the UK. Conclusions: The comparative results indicate that both countries would benefit from engaging the public through doctors and non-profit researchers. The UK could additionally support public trust by engaging with the public through the National Health Service. However, the results suggest that whilst public trust is significant, it may be neither necessary nor sufficient in influencing willingness to donate. Future research could do well to investigate how the importance of public trust compares in countries with lower public trust

Medical discourse and avant-garde art in France, 1905-1925

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Whole body vibration training in chronic disease: muscle, bone, function.

Muscle pull from regular physical activity is crucial for optimal development of the skeleton during growth and maintenance of bone mineral density (BMD) throughout life. Mitochondrial Respiratory Chain Disorders (MRCD) and Cystic Fibrosis (CF) are two chronic diseases that exhibit reduced lean tissue mass and impaired exercise capacity, which negatively impacts bone health in these populations. Whole body vibration training (WBVT) is an emerging therapeutic modality that has been successful in improving BMD and muscle mass and function in heath and disease. Aim: To evaluate whether 6 months of home-based WBVT improves BMD, muscle function, exercise capacity and quality of life (QoL) in people with MRCD or CF. Methods: Participants were enrolled for 15-18 months: 3-6 months observation; 6 months home-based WBVT (3 x 3mins daily at 20Hz on a Galileo® Home vibration platform); 6 months follow-up. Participants attended four study visits and completed: dual energy x-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) to assess BMD; muscle function testing on the Leonardo Jumping Platform (LJP); 6-minute walk test (6MWT) and/or formal exercise testing to assess exercise capacity; and disease specific QoL questionnaires. Linear mixed models analysis was used to assess changes between visits. Results: The MRCD cohort had 23 participants (13 male) mean (SD) age 31.0 (19.8) years and the CF cohort, 16 participants (8 male) mean (SD) age 12.8 (3.5) years. Statistically significant improvements in BMD of the legs were seen in the MRCD and CF cohorts for both DXA and pQCT. Muscle force during hopping and co-ordination during the chair rise test on the LJP improved significantly post WBVT in the CF. Exercise capacity did not change in the MRCD or CF cohorts after WBVT. QoL showed improvements in both cohorts. Conclusions: WBVT was well tolerated. WBVT improved BMD, aspects of muscle function, and QoL in people with MRCD or CF and may be a useful adjunct to physiotherapy exercise programs

Why women say hybrid working enhances productivity, while men don't

There is a complex interplay between gender, remote work and productivity. Nikita, Anna Lane, Grace Lordan and Paul Middleton conducted a survey of more than 1000 employees in financial and professional services in the UK and found that men and women rank remote and hybrid working arrangements differently. The authors say that understanding these differences is key for an inclusive and productive work environment

Towards equitable and trustworthy genomics research.

Funder: National Institutes of HealthThe representation of traditionally scientifically underserved groups in genomic research continues to be low despite concerns about equity and social justice and the scientific and clinical need. Among the factors that account for this are a lack of trust in the research community and limited diversity in this community. The success of the multiple initiatives that aim to improve representation relies on the willingness of underrepresented populations to make data and samples available for research and clinical use. In this narrative review, we propose that this requires building trust, and set out four approaches to demonstrating trustworthiness, including increasing diversity in the research workforce, and meaningful engagement with underrepresented communities in a culturally and linguistically appropriate manner. Capacity building globally will ensure that actual and perceived exploitation and 'helicopter' research could be eliminated