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

Absence of p53 in Clara cells favours multinucleation and loss of cell cycle arrest

BACKGROUND: The p53 oncosuppressor protein is a critical mediator of the response to injury in mammalian cells and is mutationally inactivated in the majority of lung malignancies. In this analysis, the effects of p53-deficiency were investigated in short-term primary cultures of murine bronchiolar Clara cells. Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells derived from wild type littermates. RESULTS: p53 null cultures displayed abnormal morphology; specifically, a high incidence of multinucleation, which increased with time in culture. Multinucleated cells were proficient in S phase DNA synthesis, as determined by BrdU incorporation. However, multinucleation did not reflect altered rates of S phase synthesis, which were similar between wild type and p53-/- cultures. Nucleation defects in p53-/- Clara cells associated with increased centrosome number, as determined by confocal microscopy of pericentrin-stained cultures, and may highlight a novel role of p53 in preserving genomic integrity in lung epithelial cells. 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 serum-dependency, and occurred only in the absence of serum. Taken together, these findings demonstrate that in murine primary Clara cell culture, cell cycle arrest is a p53-mediated response to DNA damage, and that extracellular factors, such as serum, influence this response. CONCLUSION: These findings highlight functions of wild type p53 protein in bipolar spindle formation, centrosome regulation, and growth control in bronchiolar Clara cells

The accuracy of intramedullary femoral alignment in total knee replacement in the prescence of ipsilateral hip replacement

Objectives: During total knee replacement (TKR) surgery, the most commonly used method for aligning the distal femur appropriately is via an intramedullary (IM) distal femoral alignment rod. The alignment of the rod itself is reliant on the isthmus which is used to most accurately place the rod in the correct anatomical axis. In the instance of something preventing the rod from entering the isthmus correctly, such as a hip replacement, then the degree of accuracy could be assumed to be even less. Mechanical-anatomical malalignment has been shown to decrease the implant (TKR) survival and so methods of increasing accuracy of alignment relative to the mechanical axis have been developed. At present the most accurate method intraoperatively is computer navigation and several studies have demonstrated improved alignment. An increasing number of patients year on year are having both knee and hip replacements and as the population ages the likelihood of having both a knee and hip replacement will also increase. We propose that the presence of a hip replacement within the isthmus of the femur may further decrease the accuracy of the IM alignment of the femur leading to incorrect implant positioning. Methods: The study was conducted on 10 cadaveric specimens (20 femurs). Computational navigation instrumentation was attached in turn to each femur and the ideal alignment data recorded in a standard fashion by a single operator (principal investigator). A standard entry port was then be made in the femur for the introduction of the IM rod. An IM rod was then inserted with the distal femoral cutting block in the accepted position recorded blindly on the computer navigation (both in terms of varus/valgus alignment to the mechanical axis and the degree of flexion). The process was then repeated at 3 levels to represent primary and revision hip lengths from the greater trochanter (replicating the changes that would occur in the presence of a hip replacement) The process was recorded three times at each level. Results: The resection angles between the cutting surface and the mechanical axis were measured and collected by means of computer navigation system. The results show that the IM alignment had mean Valgus of 0 degrees +/- 0.8 but with a hip replacement in situ this increased to 0.46 degrees +/- 1.49 (range 2.5 varus to 4.5 valgus), with a revision stem 0.825 +/- 1.68 (range 2.5 varus to 4.5 valgus)and long stemmed revision 1.325 +/- 2.09 (range 5 varus to 6.5 valgus). In terms of Flexion IM alignment had a mean flexion of 0.92 +/- 1.7 (range 3 extension to 4 flexion) but with a hip replacement in situ this increased to 1.88 degrees +/- 2.03 (range 2.5 extension to 8.5 flexion), with a revision stem 2.35 +/- 2.2 (range 2.5 extension to 8 flexion) and long stemmed revision 2.75 +/- 2.16(range 3.5 extension to 7 flexion). Conclusion: This Study concludes that the prescence of a hip replacement, in particular long stemmed prosthesis, further reduces the accuracy of IM alignment in the Femur for Total Knee Replacement. Consideration of an alternative method, such as navigation, should be considered insuch situations

The Beaker phenomenon and the genomic transformation of northwest Europe

From around 2750 to 2500 bc, Bell Beaker pottery became widespread across western and central Europe, before it disappeared between 2200 and 1800 bc. The forces that propelled its expansion are a matter of long-standing debate, and there is support for both cultural diffusion and migration having a role in this process. Here we present genome-wide data from 400 Neolithic, Copper Age and Bronze Age Europeans, including 226 individuals associated with Beaker-complex artefacts. We detected limited genetic affinity between Beaker-complex-associated individuals from Iberia and central Europe, and thus exclude migration as an important mechanism of spread between these two regions. However, migration had a key role in the further dissemination of the Beaker complex. We document this phenomenon most clearly in Britain, where the spread of the Beaker complex introduced high levels of steppe-related ancestry and was associated with the replacement of approximately 90% of Britain’s gene pool within a few hundred years, continuing the east-to-west expansion that had brought steppe-related ancestry into central and northern Europe over the previous centuries

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

The benefits and harms of intravenous thrombolysis with recombinant tissue plasminogen activator within 6 h of acute ischaemic stroke (the third international stroke trial [IST-3]): a randomised controlled trial

BACKGROUND: Thrombolysis is of net benefit in patients with acute ischaemic stroke, who are younger than 80 years of age and are treated within 4·5 h of onset. The third International Stroke Trial (IST-3) sought to determine whether a wider range of patients might benefit up to 6 h from stroke onset. METHODS: In this international, multicentre, randomised, open-treatment trial, patients were allocated to 0·9 mg/kg intravenous recombinant tissue plasminogen activator (rt-PA) or to control. The primary analysis was of the proportion of patients alive and independent, as defined by an Oxford Handicap Score (OHS) of 0-2 at 6 months. The study is registered, ISRCTN25765518. FINDINGS: 3035 patients were enrolled by 156 hospitals in 12 countries. All of these patients were included in the analyses (1515 in the rt-PA group vs 1520 in the control group), of whom 1617 (53%) were older than 80 years of age. At 6 months, 554 (37%) patients in the rt-PA group versus 534 (35%) in the control group were alive and independent (OHS 0-2; adjusted odds ratio [OR] 1·13, 95% CI 0·95-1·35, p=0·181; a non-significant absolute increase of 14/1000, 95% CI -20 to 48). An ordinal analysis showed a significant shift in OHS scores; common OR 1·27 (95% CI 1·10-1·47, p=0·001). Fatal or non-fatal symptomatic intracranial haemorrhage within 7 days occurred in 104 (7%) patients in the rt-PA group versus 16 (1%) in the control group (adjusted OR 6·94, 95% CI 4·07-11·8; absolute excess 58/1000, 95% CI 44-72). More deaths occurred within 7 days in the rt-PA group (163 [11%]) than in the control group (107 [7%], adjusted OR 1·60, 95% CI 1·22-2·08, p=0·001; absolute increase 37/1000, 95% CI 17-57), but between 7 days and 6 months there were fewer deaths in the rt-PA group than in the control group, so that by 6 months, similar numbers, in total, had died (408 [27%] in the rt-PA group vs 407 [27%] in the control group). INTERPRETATION: For the types of patient recruited in IST-3, despite the early hazards, thrombolysis within 6 h improved functional outcome. Benefit did not seem to be diminished in elderly patients. FUNDING: UK Medical Research Council, Health Foundation UK, Stroke Association UK, Research Council of Norway, Arbetsmarknadens Partners Forsakringsbolag (AFA) Insurances Sweden, Swedish Heart Lung Fund, The Foundation of Marianne and Marcus Wallenberg, Polish Ministry of Science and Education, the Australian Heart Foundation, Australian National Health and Medical Research Council (NHMRC), Swiss National Research Foundation, Swiss Heart Foundation, Assessorato alla Sanita, Regione dell'Umbria, Italy, and Danube University