p53 and responses to DNA damage in small airway epithelium

Acute lung injury is implicated in many respiratory diseases, including lung adenocarcinoma and emphysema. In this thesis, the hypothesis that the stress protein p53 is important in acute lung injury was investigated. p53 is a short-lived, latent transcription factor which is activated and stabilised in response to a wide range of cellular stresses, including DNA damage. In certain cell types, wild type p53 protein mediates a variety of DNA damage responses and transcriptionally modulates a battery of downstream genes involved in DNA repair, growth control, and apoptosis.The effects of p53 were investigated in a mouse model of acute lung injury and in short-term primary cultures of isolated Clara cells. Gene targeted mice, germline deficient in p53, were exposed to y-irradiation and compared to wild type controls. The in vivo response to DNA damage was characterised in terms of growth arrest, apoptosis, morphology, and gene expression. An acute stress response was observed in vivo, and localised to a subpopulation of the lung epithelium, the bronchiolar cells. p53 was stabilised in this population and was associated with transcriptional induction of Bax, but not other bcl-2 family members. p53 deficient mice did not display this rapid accumulation of Bax transcripts, as assessed by RNase Protection Assay. Within wild type and p53 null mice, y-irradiation did not induce apoptosis in lung epithelial cells at any timepoints studied, as assessed by morphology, but did induce strand breaks that were detectable by TUNEL. Cell cycle activity, as assessed by BrdU incorporation, was infrequent in the lung at all timepoints, regardless of p53 status, and hence an effect of p53 on cell cycle progression was not detected in vivo.The effects of p53-deficiency were additionally investigated in short-term primary cultures of murine bronchiolar Clara cells. Culturing of Clara cells allowed an assessment of the functional consequences of p53 deficiency in proliferating cells. Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells derived from wild type littermates. Baseline proliferation rates, as determined by BrdU incorporation, were similar irrespective of p53 status. p53 null cultures displayed abnormal morphology; specifically, a high incidence of multinucleation, which increased with time in culture. Multinucleated cells maintained expression of the Clara cell marker CC10, and were proficient in S phase DNA synthesis, as determined by BrdU incorporation. Nucleation defects in p53 -/- Clara cells associated with abnormalities in mitosis and cytokinesis, as documented by timelapse videomicroscopy, and with increased centrosome number, determined by confocal microscopy. Defects in centrosome homeostasis, mitotic fidelity, and cytokinesis in p53-null Clara cells may reflect a novel role of p53 in preserving genomic integrity in lung epithelium.Effects of p53-deficiency were also studied following exposure to DNA damage. A p53-dependent reduction in the BrdU index was observed in Clara cells following ionizing radiation. The reduction in BrdU index in wild type cells displayed serumdependency, and occurred only in the absence of serum. Apoptosis was infrequent in both genotypes, as determined by time-lapse videomicroscopy. Taken together, these findings demonstrate that in murine primary Clara cell culture, growth arrest but not apoptosis is a p53-mediated response to DNA damage, and that extracellular factors, such as serum, influence this response.In addition, a transgenic model employing lung-specific Cre/lox technology was evaluated. The Cre/lox recombinase system evolved within bacteriophage PI as a mechanism to maintain correct unit copy segregation of the prophage within host cells. This thesis reports application of this system to regulate gene expression in murine lung epithelial cells in vivo, with the eventual goal of generating improved mouse models of acute lung injury. Transgenic mice expressing Cre from the lungspecific promoter human SP-C were crossed to a Floxed DNA Ligase I line, and transgene stability and function assessed by PCR and Southern blot methodologies. The SP-C Cre transgene was demonstrated as stable, but of low copy-number. Excision of the floxed allele, as determined by PCR, was specific to the lung, and was not observed in other tissues. However, the level of excision was poor as assessed by Southern analysis of the excision event. Furthermore, Cre expression was undetectable by RT-PCR. Low expression levels of Cre may reflect the low copy-number of the SP-C/Cre transgene.In summary, this thesis reports on the role of p53 in lung epithelial cells, and on the feasibility of using Cre/lox technologies to regulate gene expression in the lung epithelium of transgenic mice. Bax mRNA induction, but not apoptosis, is a DNA damage response of small airway epithelial cells. Transactivation of Bax was p53- dependent in irradiated lung, as determined by RNase protection assay, and did not occur in p53-/- mice. To further investigate the role of p53 in the lung, a method for extracting, isolating, and culturing Clara cells, a progenitor cell of the lung, was established and incorporated into this analysis. Absence of p53 favours multinucleation and loss of cell cycle arrest in primary Clara cell culture, and highlight additional roles of p53 in cell division and growth control. In addition, this thesis explored the feasibility of using Cre/lox technologies to regulate gene expression in murine lung epithelium. SP-C/Cre mice were assessed in their ability to excise a Floxed DNA Ligase I cassette in the tissues of double transgenic mice. Cre-mediated excision was specific to lung epithelium, but infrequent

Preparing for the future: A reappraisal of archaeo-geophysical surveying on Irish National Road Schemes 2001-2010

yesThis document reviews Legacy Data generated from 10 years’ worth of road scheme activity in Ireland to determine how archaeological geophysical surveys could be carried out on national roads in the future. The geophysical surveys were carried out by several different contractors across a range of challenging field conditions, geologies, weather and seasons. The research is based upon the results of linear schemes but also has validity for wider approaches. The findings of this research are based upon the compilation of all terrestrial archaeological geophysical surveys carried out on behalf of the National Roads Authority (NRA), a review of the success or otherwise of those surveys in comparison with ground-observed excavations and in combination with experimental surveys that tested previously held assumptions or knowledge to determine best practice methods for the future. The use and success of geophysical surveys in Ireland differ quite significantly from those in the UK, from where many of the methods of assessment were derived or adapted. Many of these differences can be attributed to geology. Ireland has a very high percentage of Carboniferous limestone geology, overlain mostly by tills and frequent occurrences of peat. These soils can reduce, to some extent, the effectiveness of magnetometer surveys; the most frequently used geophysical technique in Ireland. However, magnetometer data can be maximised in these cases by increasing the spatial resolution to produce effective results. An increase in spatial resolution is also effective generally, for enhancing the chances of identifying archaeological features by discriminating between archaeological and geological anomalies as well as increasing anomaly definition and visualisation of small and subtle archaeological features. Seasonal tests have determined that Irish soils are generally suitable for year round earth resistance assessments although some counties in the southeast of the country may experience very dry soils at the surface during some periods of the year. A variety of sampling strategies were used in the past, however it is now apparent that detailed assessments across the full length and width of a proposed road corridor are the most appropriate form of geophysical investigation. Magnetometer surveys are generally suitable for most Irish soils and geologies, although exceptions apply in areas of near-surface igneous deposits, deep peat and alluvial soils; however magnetometer surveys are not capable of identifying all types of archaeological features and other methods will be required for a full evaluation. Analysis of the Legacy Data has determined that in general the NRA archaeological geophysical surveys were historically used in a very positive way on road schemes. The range of features assessed or identified account for most types of archaeological sites in Ireland. These have provided a significant archive of case studies that will be of benefit to future archaeological geophysical research and will help to protect the globally dwindling archaeological resource that is threatened by development-led or commercially driven projects

Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: A systematic analysis for the Global Burden of Disease Study 2017

Background: The Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD 2017) includes a comprehensive assessment of incidence, prevalence, and years lived with disability (YLDs) for 354 causes in 195 countries and territories from 1990 to 2017. Previous GBD studies have shown how the decline of mortality rates from 1990 to 2016 has led to an increase in life expectancy, an ageing global population, and an expansion of the non-fatal burden of disease and injury. These studies have also shown how a substantial portion of the world's population experiences non-fatal health loss with considerable heterogeneity among different causes, locations, ages, and sexes. Ongoing objectives of the GBD study include increasing the level of estimation detail, improving analytical strategies, and increasing the amount of high-quality data. Methods: We estimated incidence and prevalence for 354 diseases and injuries and 3484 sequelae. We used an updated and extensive body of literature studies, survey data, surveillance data, inpatient admission records, outpatient visit records, and health insurance claims, and additionally used results from cause of death models to inform estimates using a total of 68 781 data sources. Newly available clinical data from India, Iran, Japan, Jordan, Nepal, China, Brazil, Norway, and Italy were incorporated, as well as updated claims data from the USA and new claims data from Taiwan (province of China) and Singapore. We used DisMod-MR 2.1, a Bayesian meta-regression tool, as the main method of estimation, ensuring consistency between rates of incidence, prevalence, remission, and cause of death for each condition. YLDs were estimated as the product of a prevalence estimate and a disability weight for health states of each mutually exclusive sequela, adjusted for comorbidity. We updated the Socio-demographic Index (SDI), a summary development indicator of income per capita, years of schooling, and total fertility rate. Additionally, we calculated differences between male and female YLDs to identify divergent trends across sexes. GBD 2017 complies with the Guidelines for Accurate and Transparent Health Estimates Reporting. Findings: Globally, for females, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and haemoglobinopathies and haemolytic anaemias in both 1990 and 2017. For males, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and tuberculosis including latent tuberculosis infection in both 1990 and 2017. In terms of YLDs, low back pain, headache disorders, and dietary iron deficiency were the leading Level 3 causes of YLD counts in 1990, whereas low back pain, headache disorders, and depressive disorders were the leading causes in 2017 for both sexes combined. All-cause age-standardised YLD rates decreased by 3·9% (95% uncertainty interval [UI] 3·1-4·6) from 1990 to 2017; however, the all-age YLD rate increased by 7·2% (6·0-8·4) while the total sum of global YLDs increased from 562 million (421-723) to 853 million (642-1100). The increases for males and females were similar, with increases in all-age YLD rates of 7·9% (6·6-9·2) for males and 6·5% (5·4-7·7) for females. We found significant differences between males and females in terms of age-standardised prevalence estimates for multiple causes. The causes with the greatest relative differences between sexes in 2017 included substance use disorders (3018 cases [95% UI 2782-3252] per 100 000 in males vs 1400 [1279-1524] per 100 000 in females), transport injuries (3322 [3082-3583] vs 2336 [2154-2535]), and self-harm and interpersonal violence (3265 [2943-3630] vs 5643 [5057-6302]). Interpretation: Global all-cause age-standardised YLD rates have improved only slightly over a period spanning nearly three decades. However, the magnitude of the non-fatal disease burden has expanded globally, with increasing numbers of people who have a wide spectrum of conditions. A subset of conditions has remained globally pervasive since 1990, whereas other conditions have displayed more dynamic trends, with different ages, sexes, and geographies across the globe experiencing varying burdens and trends of health loss. This study emphasises how global improvements in premature mortality for select conditions have led to older populations with complex and potentially expensive diseases, yet also highlights global achievements in certain domains of disease and injury

Global, regional, and national age-sex-specific mortality and life expectancy, 1950-2017: a systematic analysis for the Global Burden of Disease Study 2017

Background: Assessments of age-specific mortality and life expectancy have been done by the UN Population Division, Department of Economics and Social Affairs (UNPOP), the United States Census Bureau, WHO, and as part of previous iterations of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD). Previous iterations of the GBD used population estimates from UNPOP, which were not derived in a way that was internally consistent with the estimates of the numbers of deaths in the GBD. The present iteration of the GBD, GBD 2017, improves on previous assessments and provides timely estimates of the mortality experience of populations globally. Methods: The GBD uses all available data to produce estimates of mortality rates between 1950 and 2017 for 23 age groups, both sexes, and 918 locations, including 195 countries and territories and subnational locations for 16 countries. Data used include vital registration systems, sample registration systems, household surveys (complete birth histories, summary birth histories, sibling histories), censuses (summary birth histories, household deaths), and Demographic Surveillance Sites. In total, this analysis used 8259 data sources. Estimates of the probability of death between birth and the age of 5 years and between ages 15 and 60 years are generated and then input into a model life table system to produce complete life tables for all locations and years. Fatal discontinuities and mortality due to HIV/AIDS are analysed separately and then incorporated into the estimation. We analyse the relationship between age-specific mortality and development status using the Socio-demographic Index, a composite measure based on fertility under the age of 25 years, education, and income. There are four main methodological improvements in GBD 2017 compared with GBD 2016: 622 additional data sources have been incorporated; new estimates of population, generated by the GBD study, are used; statistical methods used in different components of the analysis have been further standardised and improved; and the analysis has been extended backwards in time by two decades to start in 1950. Findings: Globally, 18·7% (95% uncertainty interval 18·4–19·0) of deaths were registered in 1950 and that proportion has been steadily increasing since, with 58·8% (58·2–59·3) of all deaths being registered in 2015. At the global level, between 1950 and 2017, life expectancy increased from 48·1 years (46·5–49·6) to 70·5 years (70·1–70·8) for men and from 52·9 years (51·7–54·0) to 75·6 years (75·3–75·9) for women. Despite this overall progress, there remains substantial variation in life expectancy at birth in 2017, which ranges from 49·1 years (46·5–51·7) for men in the Central African Republic to 87·6 years (86·9–88·1) among women in Singapore. The greatest progress across age groups was for children younger than 5 years; under-5 mortality dropped from 216·0 deaths (196·3–238·1) per 1000 livebirths in 1950 to 38·9 deaths (35·6–42·83) per 1000 livebirths in 2017, with huge reductions across countries. Nevertheless, there were still 5·4 million (5·2–5·6) deaths among children younger than 5 years in the world in 2017. Progress has been less pronounced and more variable for adults, especially for adult males, who had stagnant or increasing mortality rates in several countries. The gap between male and female life expectancy between 1950 and 2017, while relatively stable at the global level, shows distinctive patterns across super-regions and has consistently been the largest in central Europe, eastern Europe, and central Asia, and smallest in south Asia. Performance was also variable across countries and time in observed mortality rates compared with those expected on the basis of development. Interpretation: This analysis of age-sex-specific mortality shows that there are remarkably complex patterns in population mortality across countries. The findings of this study highlight global successes, such as the large decline in under-5 mortality, which reflects significant local, national, and global commitment and investment over several decades. However, they also bring attention to mortality patterns that are a cause for concern, particularly among adult men and, to a lesser extent, women, whose mortality rates have stagnated in many countries over the time period of this study, and in some cases are increasing

You know its summer in Ireland when the rain gets warmer: Analysing repetitive time-lapse earth resistance data to determine ‘optimal’ survey climate conditions

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A decade of ground-truthing: reappraising magnetometer prospection surveys on linear corridors in light of excavation evidence

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Large-scale migration into Britain during the Middle to Late Bronze Age

Present-day people from England and Wales harbour more ancestry derived from Early European Farmers (EEF) than people of the Early Bronze Age . To understand this, we generated genome-wide data from 793 individuals, increasing data from the Middle to Late Bronze and Iron Age in Britain by 12-fold, and Western and Central Europe by 3.5-fold. Between 1000 and 875 BC, EEF ancestry increased in southern Britain (England and Wales) but not northern Britain (Scotland) due to incorporation of migrants who arrived at this time and over previous centuries, and who were genetically most similar to ancient individuals from France. These migrants contributed about half the ancestry of Iron Age people of England and Wales, thereby creating a plausible vector for the spread of early Celtic languages into Britain. These patterns are part of a broader trend of EEF ancestry becoming more similar across central and western Europe in the Middle to Late Bronze Age, coincident with archaeological evidence of intensified cultural exchange . There was comparatively less gene flow from continental Europe during the Iron Age, and Britain's independent genetic trajectory is also reflected in the rise of the allele conferring lactase persistence to ~50% by this time compared to ~7% in central Europe where it rose rapidly in frequency only a millennium later. This suggests that dairy products were used in qualitatively different ways in Britain and in central Europe over this period. [Abstract copyright: © 2021. The Author(s), under exclusive licence to Springer Nature Limited.