The role of the adenovirus DNA binding protein in DNA replication and recombination

Replication of adenovirus DNA in infected cells is an efficient process that, compared to cellular replication, has the use of a protein primer as a hallmark. The mechanism of this DNA replication process and especially the role of one of the replication proteins, the DNA binding protein DBP, is the main subject of this thesis. Adenovirus DNA replication can be reconstituted in vitro, using three viral proteins, adenovirus DNA polymerase (pol), precursor terminal protein (pTP), and DBP. Optimal replication efficiency is obtained when two cellular transcription factors are added, nuclear factor I (NFI) and Oct-1. The adenoviral dsDNA genome contains two terminal proteins (TP) covalently linked to the 5. ends. The inverted terminal repeats contain the origins of replication. pTP and pol are tightly associated in solution. During initiation of replication pTP functions as a primer to which the first nucleotide, dCTP, is covalently coupled. Both NFI and Oct-1 stimulate the initiation by recruiting the pTP-pol complex to the origin of replication. Initiation starts opposite position 4 of the template strand. After formation of a pTP-trinucleotide (pTP-CAT), the complex jumps back and CAT becomes paired with template residues 1.3. Shortly after jumping back, the polymerase dissociates from pTP and elongation proceeds via strand displacement. The adenovirus DNA binding protein is an important player in adenovirus DNA replication, where it serves multiple functions. In the first step of adenovirus DNA replication DBP stimulates the coupling of the first nucleotide to pTP. Also binding of NFI to the origin is stimulated by DBP. Subsequently, during elongation DBP unwinds the dsDNA ahead of pol and removes secondary structures. Adenovirus DBP binds with high affinity and cooperativity to ssDNA, whereas binding to dsDNA is non- cooperative and with lower affinity. These differences in binding affinity are the driving force for dsDNA unwinding which is required for processive DNA chain elongation by pol. DBP is a 529 amino acids long protein with a molecular weight of 59 kD. The structure of adenovirus DBP possesses a remarkable feature; the C-terminal arm (aa. 512-529), which is important for the cooperative binding of DBP to another DBP molecule. In addition, the C-terminal arm is flexible and can rotate around a fixed point, called the hinge-region (aa. 512-515). In Chapter 1, an introduction to the adenovirus DNA replication machinery and the role of several single stranded DNA binding proteins originating from different organisms is presented. In Chapter 2 we present data on the function of the flexibility of the C-terminal arm of DBP and we discuss the implications of an altered flexibility on adenovirus DNA replication by mutating several aa in the hinge-region. In Chapter 3, we present data on the function of DBP in the stimulation of initiation. In contrast to a direct protein-protein interaction of DBP with pol we demonstrate that DBP stimulates the binding of pol to the dsDNA origin. We assume that the structure of the dsDNA template is changed by DBP in such a way that polymerase binds more efficiently to the dsDNA. In Chapter 4 we demonstrate that DBP functions in unwinding of the dsDNA donor and that it has an additional role in renaturation of the unwound donor with the complementary acceptor ssDNA. For the first time we were able to demonstrate a direct role for an adenovirus replication protein in homologous recombination. Finally, in Chapter 5 new questions, that have surfaced as a result of the research presented in this thesis, are discussed

Association of cardiovascular risk factors with carotid intima media thickness in patients with rheumatoid arthritis with low disease activity compared to controls: A cross-sectional study

Objectives Rheumatoid arthritis (RA) has been identified as an independent cardiovascular risk factor. The importance of risk factors such as hypertension and hyperlipidemia in the generation of atherosclerosis in RA patients is unclear. This study analyzed clinical parameters associated with carotid intima media thickness (cIMT) in patients with RA. Methods Subjects with RA and healthy controls without RA, both without known cardiovascular disease, were included. Participants underwent a standard physical examination and laboratory measurements including a lipid profile. cIMT was measured semi-automatically by ultrasound. Results In total 243 RA patients and 117 controls were included. The median RA disease duration was 7 years (IQ

Bayesian Cluster Finder: Clusters in the CFHTLS Archive Research Survey

The detection of galaxy clusters in present and future surveys enables measuring mass-to-light ratios, clustering properties, galaxy cluster abundances and therefore, constraining cosmological parameters. We present a new technique for detecting galaxy clusters, which is based on the Matched Filter Algorithm from a Bayesian point of view. The method is able to determine the position, redshift and richness of the cluster through the maximization of a filter depending on galaxy luminosity, density and photometric redshift combined with a galaxy cluster prior that accounts for color-magnitude relations and BCG-redshift relation. We tested the algorithm through realistic mock galaxy catalogs, revealing that the detections are 100% complete and 80% pure for clusters up to z $$20 (Abell Richness $\sim$0, M$\sim4\times10^{14} M_{\odot}$). The completeness and purity remains approximately the same if we do not include the prior information, implying that this method is able to detect galaxy cluster with and without a well defined red sequence. We applied the algorithm to the CFHTLS Archive Research Survey (CARS) data, recovering similar detections as previously published using the same or deeper data plus additional clusters which appear to be real.Comment: Accepted for publication in MNRAS; 17 pages, 38 figure

The WiggleZ Dark Energy Survey: Survey Design and First Data Release

The WiggleZ Dark Energy Survey is a survey of 240,000 emission line galaxies in the distant universe, measured with the AAOmega spectrograph on the 3.9-m Anglo-Australian Telescope (AAT). The target galaxies are selected using ultraviolet photometry from the GALEX satellite, with a flux limit of NUV<22.8 mag. The redshift range containing 90% of the galaxies is 0.2<z<1.0. The primary aim of the survey is to precisely measure the scale of baryon acoustic oscillations (BAO) imprinted on the spatial distribution of these galaxies at look-back times of 4-8 Gyrs. Detailed forecasts indicate the survey will measure the BAO scale to better than 2% and the tangential and radial acoustic wave scales to approximately 3% and 5%, respectively. This paper provides a detailed description of the survey and its design, as well as the spectroscopic observations, data reduction, and redshift measurement techniques employed. It also presents an analysis of the properties of the target galaxies, including emission line diagnostics which show that they are mostly extreme starburst galaxies, and Hubble Space Telescope images, which show they contain a high fraction of interacting or distorted systems. In conjunction with this paper, we make a public data release of data for the first 100,000 galaxies measured for the project.Comment: Accepted by MNRAS; this has some figures in low resolution format. Full resolution PDF version (7MB) available at http://www.physics.uq.edu.au/people/mjd/pub/wigglez1.pdf The WiggleZ home page is at http://wigglez.swin.edu.au

Modelling high redshift Lyman-alpha Emitters

We present a new model for high redshift Lyman-Alpha Emitters (LAEs) in the cosmological context which takes into account the resonant scattering of Ly-a photons through expanding gas. The GALICS semi-analytic model provides us with the physical properties of a large sample of high redshift galaxies. We implement a gas outflow model for each galaxy based on simple scaling arguments. The coupling with a library of numerical experiments of Ly-a transfer through expanding or static dusty shells of gas allows us to derive the Ly-a escape fractions and profiles. The predicted distribution of Ly-a photons escape fraction shows that galaxies with a low star formation rate have a f_esc of the order of unity, suggesting that, for those objects, Ly-a may be used to trace the star formation rate assuming a given conversion law. In galaxies forming stars intensely, the escape fraction spans the whole range from 0 to 1. The model is able to get a good match to the UV and Ly-a luminosity function (LF) data at 3 < z < 5. We find that we are in good agreement with both the bright Ly-a data and the faint population observed by Rauch et al. (2008) at z=3. Most of the Ly-a profiles of our LAEs are redshifted by the diffusion in the outflow which suppresses IGM absorption. The bulk of the observed Ly-a equivalent width (EW) distribution is recovered by our model, but we fail to obtain the very large values sometimes detected. Predictions for stellar masses and UV LFs of LAEs show a satisfactory agreement with observational estimates. The UV-brightest galaxies are found to show only low Ly-a EWs in our model, as it is reported by many observations of high redshift LAEs. We interpret this effect as the joint consequence of old stellar populations hosted by UV-bright galaxies, and high HI column densities that we predict for these objects, which quench preferentially resonant Ly-a photons via dust extinction.Comment: 17 pages, 12 figures, 3 tables, accepted for publication in MNRA

The Contribution of Social Networks to the Health and Self-Management of Patients with Long-Term Conditions: A Longitudinal Study

Evidence for the effectiveness of patient education programmes in changing individual self-management behaviour is equivocal. More distal elements of personal social relationships and the availability of social capital at the community level may be key to the mobilisation of resources needed for long-term condition self-management to be effective. Aim: To determine how the social networks of people with long-term conditions (diabetes and heart disease) are associated with health-related outcomes and changes in outcomes over time. Methods: Patients with chronic heart disease (CHD) or diabetes (n = 300) randomly selected from the disease registers of 19 GP practices in the North West of England. Data on personal social networks collected using a postal questionnaire, alongside face-to-face interviewing. Follow-up at 12 months via postal questionnaire using a self-report grid for network members identified at baseline. Analysis: Multiple regression analysis of relationships between health status, self-management and health economics outcomes, and characteristics of patients’ social networks. Results: Findings indicated that: (1) social involvement with a wider variety of people and groups supports personal self-management and physical and mental well-being; (2) support work undertaken by personal networks expands in accordance with health needs helping people to cope with their condition; (3) network support substitutes for formal care and can produce substantial saving in traditional health service utilisation costs. Health service costs were significantly (p0.01) reduced for patients receiving greater levels of illness work through their networks. Conclusions: Support for self-management which achieves desirable policy outcomes should be construed less as an individualised set of actions and behaviour and more as a social network phenomenon. This study shows the need for a greater focus on harnessing and sustaining the capacity of networks and the importance of social involvement with community groups and resources for producing a more desirable and cost-effective way of supporting long term illness management

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

Anaerobic oxidation of methane associated with sulfate reduction in a natural freshwater gas source

The occurrence of anaerobic oxidation of methane (AOM) and trace methane oxidation (TMO) was investigated in a freshwater natural gas source. Sediment samples were taken and analyzed for potential electron acceptors coupled to AOM. Long-term incubations with 13C-labeled CH4 (13CH4) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, 13C-labeled CO2 (13CO2) simultaneously increased with methane formation, which is typical for TMO. In the presence of nitrate, neither methane formation nor methane oxidation occurred. Net AOM was measured only with sulfate as electron acceptor. Here, sulfide production occurred simultaneously with 13CO2 production and no methanogenesis occurred, excluding TMO as a possible source for 13CO2 production from 13CH4. Archaeal 16S rRNA gene analysis showed the highest presence of ANME-2a/b (ANaerobic MEthane oxidizing archaea) and AAA (AOM Associated Archaea) sequences in the incubations with methane and sulfate as compared with only methane addition. Higher abundance of ANME-2a/b in incubations with methane and sulfate as compared with only sulfate addition was shown by qPCR analysis. Bacterial 16S rRNA gene analysis showed the presence of sulfate-reducing bacteria belonging to SEEP-SRB1. This is the first report that explicitly shows that AOM is associated with sulfate reduction in an enrichment culture of ANME-2a/b and AAA methanotrophs and SEEP-SRB1 sulfate reducers from a low-saline environment.We thank Douwe Bartstra (Vereniging tot Behoud van de Gasbronnen in Noord-Holland, The Netherlands), Carla Frijters (Paques BV, The Netherlands) and Teun Veuskens (Laboratory of Microbiology, WUR, The Netherlands) for sampling; Martin Meirink (Hoogheemraadschap Hollands Noorderkwartier, The Netherlands) for physicochemical data; Freek van Sambeek for providing Figure 1; Lennart Kleinjans (Laboratory of Microbiology, WUR, The Netherlands) for help with pyrosequencing analysis, Irene Sánchez-Andrea (Laboratory of Microbiology, WUR, The Netherlands) for proof-reading and Katharina Ettwig (Department of Microbiology, Radboud University Nijmegen, The Netherlands) for providing M. oxyfera DNA. We want to thank all anonymous reviewers for valuable contributions. This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)

A high-resolution stable isotope record from a Peruvian stalagmite

Speleothems are known as carbonate formations in caves. The last few years there is a growing scientific interest in speleothems for climate reconstruction (McDermott, in press; Sancho et al., in press; Jirnénez de Cisneros et al., 2003; Genty et al., 2003; Baker et al., 2002; Proctor et al., 2002; Linge et al., 2001; Wang et al., 2001). Speleothems have proven their potential to create a terrestrial high-resolution paleo-temperature record (Schwarcz, 1986). The advantage of caves for paleoclimatic studies is the yearly stability of climatic conditions in the cave (Jirnénez de Cisneros et al., 2003). Observations have shown that the temperature in any deep cave is clase to the mean annual temperature of the surrounding (Schwarcz et al., 1976; Yonge et al., 1985). This makes speleothems a successful too! paleo-temperature reconstruction (Hendy and Wilson, 1968). The carbonate, ofwhich the speleothem is build, can be used for stable isotope measurements (o13C and o180). Oxygen isotopes are world-wide used to reconstruct paleo-temperatures, especially for biogenic carbonate in marine cores. Precise age-dating on speleothems can be done by U-Th measurements on TIMS, which makes it possible to calculate absolute ages for the isotope record

Modelling mammalian energetics: the heterothermy problem

Global climate change is expected to have strong effects on the world’s flora and fauna. As a result, there has been a recent increase in the number of meta-analyses and mechanistic models that attempt to predict potential responses of mammals to changing climates. Many models that seek to explain the effects of environmental temperatures on mammalian energetics and survival assume a constant body temperature. However, despite generally being regarded as strict homeotherms, mammals demonstrate a large degree of daily variability in body temperature, as well as the ability to reduce metabolic costs either by entering torpor, or by increasing body temperatures at high ambient temperatures. Often, changes in body temperature variability are unpredictable, and happen in response to immediate changes in resource abundance or temperature. In this review we provide an overview of variability and unpredictability found in body temperatures of extant mammals, identify potential blind spots in the current literature, and discuss options for incorporating variability into predictive mechanistic models