Molecular architecture and function of adenovirus DNA polymerase

Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and needs to accomodate both DNA and protein as a primer, it is not surprising that a large number of protein-protein and protein-DNA interactions are involved in efficient replication. Indeed, Ad pol is known to interact with pTP, NFI and DNA, although our understanding of these interactions is limited. In this thesis, these interactions have been studied in greater detail. After an introductory chapter on DNA dependent DNA polymerases and Ad replication, the jumping back mechanism that characterizes the change from initiation to elongation is extensively reviewed in chapter 2. In chapter 3, the highly conserved (I/Y)XGG motif of Ad pol is studied. In chapter 4, the interaction between Ad pol and DNA is further studied by the use of biotinylated oligo-nucleotides with a bulky streptavidin block. Chapter 5 examines the termination of Ad pol on the native TP-containing viral DNA. Finally, in chapter 6 the recruitment of the pTP-pol complex via a direct interaction between Ad pol and NFI is studied in detail

Observations of deep coral and sponge assemblages in Olympic Coast National Marine Sanctuary, Washington. Cruise Report: NOAA Ship McArthur II Cruise AR06-07/07

From May 22 to June 4, 2006, NOAA scientists led a research cruise using the ROPOS Remotely Operated Vehicle (ROV) to conduct a series of dives at targeted sites in the Olympic Coast National Marine Sanctuary (OCNMS) with the goal of documenting deep coral and sponge communities. Dive sites were selected from areas for which OCNMS had side scan sonar data indicating the presence of hard or complex substrate. The team completed 11 dives in sanctuary waters ranging from six to 52 hours in length, at depths ranging from 100 to 650 meters. Transect surveys were completed at 15 pre-selected sites, with additional observations made at five other sites. The survey locations included sites both inside and outside the Essential Fish Habitat (EFH) Conservation Area, known as Olympic 2, established by the Pacific Fishery Management Council, enacted on June 12, 2006. Bottom trawling is prohibited in the Olympic 2 Conservation Area for nontribal fishermen. The Conservation Area covers 159.4 square nautical miles or about 15 percent of the sanctuary. Several species of corals and sponges were documented at 14 of the 15 sites surveyed, at sites both inside and outside the Conservation Area, including numerous gorgonians and the stony corals Lophelia pertusa and Desmophyllum dianthus, as well as small patches of the reef building sponge Farrea occa. The team also documented Lophelia sp. and Desmophyllum sp. coral rubble, dead gorgonians, lost fishing gear, and other anthropogenic debris, supporting concerns over potential risks of environmental disturbances to coral health. (PDF contains 60 pages.

The use of a SQUID magnetometer for middle ear research

A new technique is described for the measurement of vibrations in the temporal bones of an isolated middle ear. The precise recording of vibrations in the middle ear is of importance for the construction and improvement of a middle ear prosthesis.1 The method of measurement is based on a transformation of mechanical vibrations into magnetic flux variations. This is performed by attaching a small piece of permanent magnetic material to the eardrum or middle ear ossicles. The magnetic flux variations caused by vibrations of the eardrum or ossicles during application of sound can be measured by means of a SQUID magnetometer.\ud \ud Measurements showed that it is possible to measure vibratory displacement amplitudes of the eardrum down to about 10−10 m in a frequency range between 200 Hz and 10 kHz, although the acoustical and magnetometer conditions were not optimal. The method offers several advantages compared to already existing methods.2–5,

A utopia concreta da poesia: "Uma árvore de veneno" de Blake

The essay examines some broad perspectives on the art that comes from the tradition of “critical Marxism”, by analyzing a poem of Songs of Experience, written by William Blake. The reading is related to hermeneutics and post-structuralism, as the aesthetic writings of the Frankfurt School.O ensaio examina algumas perspectivas amplas sobre a arte que vêm da tradição do “marxismo crítico”, por meio da análise de um poema de Canções da experiência, de William Blake. A leitura deve tanto à hermenêutica e ao pós-estruturalismo quanto aos escritos estéticos da Escola de Frankfurt

Mdm2 Induces Mono-Ubiquitination of FOXO4

Background: The Forkhead box O (FOXO) class of transcription factors are involved in the regulation of several cellular responses including cell cycle progression and apoptosis. Furthermore, in model organisms FOXOs act as tumor suppressors and affect aging. Previously, we noted that FOXOs and p53 are remarkably similar within their spectrum of regulatory proteins [1]. For example, the de-ubiquitinating enzyme USP7 removes ubiquitin from both FOXO and p53. However, Skp2 has been identified as E3 ligase for FOXO1, whereas Mdm2 is the prime E3 ligase for p53. Principal Findings/Methodology: Here we provide evidence that Mdm2 acts as an E3 ligase for FOXO as well. In vitro incubation of Mdm2 and FOXO results in ATP-dependent (multi)mono-ubiquitination of FOXO similar to p53. Furthermore, in vivo co-expression of Mdm2 and FOXO induces FOXO mono-ubiquitination and consistent with this result, siRNAmediated depletion of Mdm2 inhibits mono-ubiquitination of FOXO induced by hydrogen peroxide. Regulation of FOXO ubiquitination by Mdm2 is likely to be direct since Mdm2 and FOXO co-immunoprecipitate. In addition, Mdm2-mediated ubiquitination regulates FOXO transcriptional activity. Conclusions/Significance: These data identify Mdm2 as a novel E3 ligase for FOXOs and extend the analogous mode o

Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex

Efficient transcription is linked to modification of chromatin. For instance, tri-methylation of lysine 4 on histone H3 (H3K4) strongly correlates with transcriptional activity and is regulated by the Bur1/2 kinase complex. We found that the evolutionarily conserved Ccr4-Not complex is involved in establishing H3K4 tri-methylation in Saccharomyces cerevisiae. We observed synthetic lethal interactions of Ccr4-Not components with BUR1 and BUR2. Further analysis indicated that the genes encoding the Not-proteins are essential for efficient regulation of H3K4me3, but not H3K4me1/2, H3K36me2 or H3K79me2/3 levels. Moreover, regulation of H3K4me3 levels by NOT4 is independent of defects in RNA polymerase II loading. We found NOT4 to be important for ubiquitylation of histone H2B via recruitment of the PAF complex, but not for recruitment or activation of the Bur1/2 complex. These results suggest a mechanism in which the Ccr4-Not complex functions parallel to or downstream of the Bur1/2 kinase to facilitate H3K4me3 via PAF complex recruitment

SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner

The Forkhead transcription factor FOXA2 plays a fundamental role in controlling metabolic homeostasis in the liver during fasting. The precise molecular regulation of FOXA2 in response to nutrients is not fully understood. Here, we studied whether FOXA2 could be controlled at a post-translational level by acetylation. By means of LC-MS/MS analyses, we identified five acetylated residues in FOXA2. Sirtuin family member SIRT1 was found to interact with and deacetylate FOXA2, the latter process being dependent on the NAD +-binding catalytic site of SIRT1. Deacetylation by SIRT1 reduced protein stability of FOXA2 by targeting it towards proteasomal degradation, and inhibited transcription from the FOXA2-driven G6pase and CPT1a promoters. While mutation of the five identified acetylated residues weakly affected protein acetylation and stability, mutation of at least seven additional lysine residues was required to abolish acetylation and reduce protein levels of FOXA2. The importance of acetylation of FOXA2 became apparent upon changes in nutrient levels. The interaction of FOXA2 and SIRT1 was strongly reduced upon nutrient withdrawal in cell culture, while enhanced Foxa2 acetylation levels were observed in murine liver in vivo after starvation for 36 hours. Collectively, this study demonstrates that SIRT1 controls the acetylation level of FOXA2 in a nutrient-dependent manner and in times of nutrient shortage the interaction between SIRT1 and FOXA2 is reduced. As a result, FOXA2 is protected from degradation by enhanced acetylation, hence enabling the FOXA2 transcriptional program to be executed to maintain metabolic homeostasis

Movement of transplanted adult salmonids in previously inaccessible habitat in the Elwha River

The removal of the Elwha and Glines Canyon Dams on the Elwha River will renew access for anadromous salmonids to 70 miles of high quality habitat located primarily within Olympic National Park. Concurrent dam removals began in 2011, with complete fish passage projected in 2014. While the long-term benefits to anadromous populations are undisputed, release of stored sediment behind the dams is temporarily elevating suspended solids and degrading existing spawning habitat downstream of the Elwha dam. To minimize deleterious effects in the lower river, give populations an early opportunity to spawn and imprint on upstream habitats, and examine the response of anadromous fish to the newly available areas, Chinook and coho salmon and steelhead were moved upstream of Elwha and Glines Canyon dams in 2011 and 2012. We radiotagged and tracked 20 adult Chinook salmon, 47 adult coho salmon, and 37 steelhead to determine spatial and temporal movements and spawning in tributaries and the main stem river. An additional 10 coho and 1 chum salmon were tagged in the lower river and released to continue their migration. Fish movements were monitored using fixed sites and mobile tracking. We also observed substantial volitional fallback and subsequent spawning or migrational movement in the lower river by all both Chinook and coho salmon. Kelting behavior was common in tagged steelhead. We observed coho salmon and steelhead redds in Little River, Indian Creek, the mainstem Elwha River, and side channels of the river. Two Chinook redds were seen in the area upstream of Glines Canyon Dam. Tributaries seeded with adults retained spawers while mainstem releases provided for more exploratory migrational movements. The offspring from these relocated adults will have direct outmigration access to the ocean and the river will be open for upstream migration when they return as adults. Transplanting adult salmonids is labor intensive and requires source populations from downstream for seeding, but can result in spawning activity in new areas and can be managed to include hatchery and wild fish to supplement naturally occurring colonizations

Phosphorylation of Not4p Functions Parallel to BUR2 to Regulate Resistance to Cellular Stresses in Saccharomyces cerevisiae

Background The evolutionarily conserved Ccr4-Not and Bur1/2 kinase complexes are functionally related in Saccharomyces cerevisiae. In this study, we further explore the relationship between the subunits Not4p and Bur2p. Methodology/Principal Findings First, we investigated the presence of post-translational modifications on the Ccr4-Not complex. Using mass spectrometry analyses we identified several SP/TP phosphorylation sites on its Not4p, Not1p and Caf1p subunits. Secondly, the influence of Not4p phosphorylation on global H3K4 tri-methylation status was examined by immunoblotting. This histone mark is severely diminished in the absence of Not4p or of Bur2p, but did not require the five identified Not4p phosphorylation sites. Thirdly, we found that Not4p phosphorylation is not affected by the kinase-defective bur1-23 mutant. Finally, phenotypic analyses of the Not4p phosphomutant (not4S/T5A) and bur2Δ strains showed overlapping sensitivities to drugs that abolish cellular stress responses. The double-mutant not4S/T5A and bur2Δ strain even revealed enhanced phenotypes, indicating that phosphorylation of Not4p and BUR2 are active in parallel pathways for drug tolerance. Conclusions Not4p is a phospho-protein with five identified phosphorylation sites that are likely targets of a cyclin-dependent kinase(s) other than the Bur1/2p complex. Not4p phosphorylation on the five Not4 S/T sites is not required for global H3K4 tri-methylation. In contrast, Not4p phosphorylation is involved in tolerance to cellular stresses and acts in pathways parallel to BUR2 to affect stress responses in Saccharomyces cerevisiae