Characterization of the nifA regulatory gene of Rhizonium leguminosarum PRE

This thesis describes the characterization of the nif A regulatory gene of the pea endosymbiont Rhizobiumleguminosarum PRE.Chapter I gives a general overview on the regulation of nitrogen fixation in diazotrophs, with special focus on the regulatory NifA protein. The regulation of genes involved in nitrogen fixation in two bacteria is discussed in detail: the free living Klebsiellapneumoniae and the endosymbiont of alfalfa R . meliloti . Major differences exist between these organisms where the onset of nitrogen fixation is concerned. K . pneumoniae has a general nitrogen regulatory circuitry which senses an internal biochemical signal i.e. the level of available ammonia as defined by the glutamine to 2-ketoglutarate ratio, a high ratio indicating a surplus, a low ratio a deficit. Sensing of a N-deficit results is translated, through a chain reaction of protein modifications, into activation of the regulatory NtrC product by phosphorylation. The resulting NtrC-P activates transcription of the regulatory nif LA operon, which encodes the inhibitor NifL and the activator NifA. The Klebsiella NifA thereupon activates transcription of the genes involved in nitrogen fixation. In a recently published paper David et al. (1988) suggest that the onset of nitrogen fixation in R . meliloti starts with the sensing of the external oxygen level. The FixL protein is hypothesized to sense a decrease in oxygen level. This protein is thus activated and in turn activates the FixJ protein, which directly or indirectly activates transcription of the nif A gene. The Rhizobium NifA protein activates transcription of the nitrogen fixation genes. In this overview we hypothesize that the oxygen sensing protein FNR instead of FixL senses the internal oxygen level. FNR then activates transcription of the fix LJ operon. The FixL protein may be a moderator of the activity of FixJ, comparable to the role of NtrB in activating NtrC. To date all rhizobial NifA proteins, in contrast to Klebsiella NifA, were shown to be oxygen sensitive. The structural analysis of the NifA protein is described and possible functions ascribed to domains identified in this protein are discussed. A model for NifA activity emerging from data presented for K . pneumoniae isdiscussed. At present a complete model cannot be presented for Rhizobiaceae . The similarities and differences between the models for K . pneumoniae and Rhizobium species are discussed.In Chapter 2 the DNA sequence and deduced amino acid sequence of R . leguminosarum PRE are presented. The amino acid sequence differs in 30 amino acids from that published for R . leguminosarum 3855 (Grönger et al., 1987). A possible explanation for this difference is discussed. The NifA Open Reading Frame (ORF) reveals two potential translation start sites, which in a heterologous E.coli background appear to be used both. The second translation start, which leads to a 488 amino acids, 53 kD protein, is preferred over the first, which leads to a 519 amino acids, 56.1 kD protein. The R . meliloti (Weber et al., 1985, Buikema et al., 1985) and B . japonicumnif A genes (Thöny et al., 1987) also have two translation start sites. It was shown for R . meliloti NifA (Beynon et al., 1988) the full length protein is the active form in an E . coli background. It is discussed that a translational preference for the second translational start site, leading to the inactive protein, as was found in pulse labeling experiments in E . coli may also exist in Rhizobium . We therefore suggest that the experiments presented by Beynon et al. (1988) are not conclusive as to the size of the functional protein in a Rhizobium background. Primer extension experiments and S 1 -nuclease protection were used to identify the putative nif A promoter. A transcription terminator was identified by S1-nuclease protection.Chapter 3 deals with a phenomenon reported by Hawkins and Johnston (1988) and Roelvink et al. (1988). A nif A::Tn 5 mutant can not be complemented by a plasmid having only the nif A coding DNA fragment. A detailed analysis of the nif A- nif B intergenic region is presented. The nif A gene has a transcriptional terminator typical of bacterial genes (Brendel et al., 1986) consisting of a four GC basepairs stem and a nine base loop followed by a thymidine rich DNA stretch. This terminator was sapped by S1-nuclease protection. The nif B gene has a RpoN dependent promoter, having all nucleotides thought to be crucial to its activity. The nif A terminator was fused to the Tet - promoter and this fusion was cloned in a low copytranscriptional lac Z vector. The results show that the nif A terminator allows 85% readthrough. RNA::DNA hybridisation studies show that the nif A gene is transcribed at a level twice of that of nif B. By using a plasmid, which has a DNA region encompassing nif A, nif B and a ferredoxin like gene downstream of nifB (Grönger et al., 1988, Klipp et al., 1988) it was shown that nif A::Tn 5 mutants can be fully complemented. Taken together these findings suggest that the nif A and the nifB gene are in one operon. The failure of plasmids having the nifA encoding DNA fragment alone to complement a nif A::Tn 5 mutant results from a polar effect of the Tn 5 transposon on nif B transcription.Chapter 4 deals with the nif H promoter region of R . leguminosarum PRE as one of the target sites of the NifA protein. We determined the nucleotide sequence of this region and identified a pseudo upstream activator sequence (UAS), a pseudo promoter, a consensus UAS and a consensus promoter. The promoter, mapped by primer extension experiments, differs from the consensus in one of the nucleotides thought to be invariant (see Gussin et al., 1986). The function of the nif H promoter elements was tested in a heterolo gous E . coli and a homologous Rhizobium background. Fusions of the nif H promoter region to lac Z, and fusions of deleted nif H promoter regions to lac Z, were used in activation studies byE .pneumoniae NifA in E . coli . Both high and low copy (deletion) nif H:: lac Z fusions were conjugated to Rhizobium . The activation study in an E . coli background showed that the pseudo UAS and the pseudo promoter are not involved in the function of the promoter. A different result was obtained with low copy nif H:: lac Z constructs in a Rhizobium background. The construct having both pseudo and consensus UAS, when compared with a construct having the consensus UAS only, seems to delay the onset of nitrogen fixation by three days. We suggest that this indicates that the presence of one or more UAS's modulates the expression of nif and fix genes, as was suggested for UAS's of B . Japonicumnif and fix genes (Gubler and Hennecke, 1988). A nif promoter region holding a UAS, when cloned in a multi copy vector, can inhibit nitrogen fixation by capturing the NifA activator needed for expression of nif and fix genes. A multicopy Inhibition study with (deleted) nif H:: lac Zfusions led to a surprising finding: deletion of part of the consensus UAS on the multicopy plasmid did not result in inhibition of nitrogen fixation. The relevance of this finding is discussed. We conclude that R . leguminosarumnif H can function without an UAS as was found forR .meliloti nifH inplanta (Better et al., 1985). We suggest that NifA way form a complex withRpoN-RNAPthat can bind directly at the promoter to activate transcription.</TT

Morphological modelling of the Western Scheldt: validation of DELFT3D

Morphological changes of the Scheldt estuary are extensively monitored by the Dutch and Belgian authorities. To predict effects of long-term natural changes (sea level rise) and the consequences of various human interventions, such as dredging/dumping/sand mining and deepening of the navigation channel, there is an urgent need for adequate and reliable tools in these matters. The present report describes the results of the calibration and verification of the DEFLT3D morphological model for the Western Scheldt on the short term (years) as well as on the mid term (decades)

Change Is in the Air: The Hypoxic Induction of Phenotype Switching in Melanoma

Melanoma cells can switch from a highly proliferative, less invasive state to a highly invasive, less proliferative state, a phenomenon termed phenotype switching. This results in a highly heterogenous tumor, where a slow-growing, aggressive population of cells may resist tumor therapy, and it predicts tumor recurrence. Here we discuss the observation made by Widmer et al. that hypoxia may drive phenotype switching

Observations of velocities, sand concentrations, and fluxes under velocity-asymmetric oscillatory flows

Peer reviewedPublisher PD

A Climate Index Optimized for Longshore Sediment Transport Reveals Interannual and Multidecadal Littoral Cell Rotations

A recent 35-year endpoint shoreline change analysis revealed signi&#64257;cant counterclockwiserotations occurring in north-central Oregon, USA, littoral cells that extend 10s of kilometers in length.While the potential for severe El Niños to contribute to littoral cell rotations at seasonal to interannual scalewas previously recognized, the dynamics resulting in persistent (multidecadal) rotation were unknown,largely due to a lack of historical wave conditions extending back multiple decades and the dif&#64257;culty ofseparating the timescales of shoreline variability in a high energy region. This study addresses this questionby (1) developing a statistical downscaling framework to characterize wave conditions relevant for longshoresediment transport during data-poor decades and (2) applying a one-line shoreline change model toquantitatively assess the potential for such large embayed beaches to rotate. A climateINdex was optimizedto capture variability in longshore wave power as a proxy for potentialLOngshore Sediment Transport(LOST_IN), and a procedure was developed to simulate many realizations of potential wave conditions fromthe index. Waves were transformed dynamically with Simulating Waves Nearshore to the nearshore asinputs to a one-line model that revealed shoreline rotations of embayed beaches at multiple time and spatialscales not previously discernible from infrequent observations. Model results indicate that littoral cellsrespond to both interannual and multidecadal oscillations, producing comparable shoreline excursions toextreme El Niño winters. The technique quantitatively relates morphodynamic forcing to speci&#64257;c climatepatterns and has the potential to better identify and quantify coastal variability on timescales relevant to achanging climate.This work would not have been possible without funding from the NSF Graduate Research Fellowship Program (GRFP) through NSF grant DGE-1314109, the Coastal and Ocean Climate Applications (COCA) program through NOAA grant NA15OAR4310243, NOAA’s Regional Integrated Sciences and Assessments Program (RISA), under NOAA grant NA15OAR4310145, and the Spanish Ministerio de Educación Cultura y Deporte FPU (Formación del Profesorado Universitario) studentship BOE-A-2013-12235. Beach survey data collection undertaken on the Oregon coast was made possible by the Northwest Association of Networked Ocean Observing Systems (NANOOS) through NOAA grant NA16NOS0120019

New energy geographies : a case study of yoga, meditation and healthfulness

Beginning with a routine day in the life of a practitioner of yoga and meditation and emphasising the importance of nurturing, maintaining and preventing the dissipation of diverse ‘energies’, this paper explores the possibilities for geographical health studies which take seriously ‘new energy geographies’. It is explained how this account is derived from in-depth fieldwork tracing how practitioners of yoga and meditation find times and spaces for these practices, often in the face of busy urban lifestyles. Attention is paid to the ‘energy talk’ featuring heavily in how practitioners describe the benefits that they perceive themselves to derive from these practices, and to claims made about ‘energies’ generated during the time-spaces of these practices which seemingly flow, usually with positive effects, into other domains of their lives. The paper then discusses the implications of this energy talk in the context of: (a) critically reviewing conventional approaches to studying ‘energy geographies’; (b) identifying an alertness to the likes of ‘affective energies’ surfacing in recent theoretically-attuned works of human geography (and cognate disciplines); and (c) exploring differing understandings of energy/energies extant in geographical studies of health and in step with the empirical research materials presented about yoga, meditation and healthfulness. While orientated towards explicitly geographical inquiries, the paper is intended as a statement of interest to the wider medical humanities

Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Expression of coxsackie and adenovirus receptor distinguishes transitional cancer states in therapy-induced cellular senescence

Therapy-induced cellular senescence describes the phenomenon of cell cycle arrest that can be invoked in cancer cells in response to chemotherapy. Sustained proliferative arrest is often overcome as a contingent of senescent tumor cells can bypass this cell cycle restriction. The mechanism regulating cell cycle re-entry of senescent cancer cells remains poorly understood. This is the first report of the isolation and characterization of two distinct transitional states in chemotherapy-induced senescent cells that share indistinguishable morphological senescence phenotypes and are functionally classified by their ability to escape cell cycle arrest. It has been observed that cell surface expression of coxsackie and adenovirus receptor (CAR) is downregulated in cancer cells treated with chemotherapy. We show the novel use of surface CAR expression and adenoviral transduction to differentiate senescent states and also show in vivo evidence of CAR downregulation in colorectal cancer patients treated with neoadjuvant chemoradiation. This study suggests that CAR is a candidate biomarker for senescence response to antitumor therapy, and CAR expression can be used to distinguish transitional states in early senescence to study fundamental regulatory events in therapy-induced senescence

A perspective on radical transformations to sustainability: resistances, movements and alternatives

A transformation to sustainability calls for radical and systemic societal shifts. Yet what this entails in practice and who the agents of this radical transformation are require further elaboration. This article recenters the role of environmental justice movements in transformations, arguing that the systemic, multi-dimensional and intersectional approach inherent in EJ activism is uniquely placed to contribute to the realization of equitable sustainable futures. Based on a perspective of conflict as productive, and a “conflict transformation” approach that can address the root issues of ecological conflicts and promote the emergence of alternatives, we lay out a conceptual framework for understanding transformations through a power analysis that aims to confront and subvert hegemonic power relations; that is, multi-dimensional and intersectional; balancing ecological concerns with social, economic, cultural and democratic spheres; and is multi-scalar, and mindful of impacts across place and space. Such a framework can help analyze and recognize the contribution of grassroots EJ movements to societal transformations to sustainability and support and aid radical transformation processes. While transitions literature tends to focus on artifacts and technologies, we suggest that a resistance-centred perspective focuses on the creation of new subjectivities, power relations, values and institutions. This recenters the agency of those who are engaged in the creation and recuperation of ecological and new ways of being in the world in the needed transformation

Sandy coastlines under threat of erosion

Sandy beaches occupy more than one-third of the global coastline(1) and have high socioeconomic value related to recreation, tourism and ecosystem services(2). Beaches are the interface between land and ocean, providing coastal protection from marine storms and cyclones(3). However the presence of sandy beaches cannot be taken for granted, as they are under constant change, driven by meteorological(4,5), geological(6) and anthropogenic factors(1,7). A substantial proportion of the world's sandy coastline is already eroding(1,7), a situation that could be exacerbated by climate change(8,9). Here, we show that ambient trends in shoreline dynamics, combined with coastal recession driven by sea level rise, could result in the near extinction of almost half of the world's sandy beaches by the end of the century. Moderate GHG emission mitigation could prevent 40% of shoreline retreat. Projected shoreline dynamics are dominated by sea level rise for the majority of sandy beaches, but in certain regions the erosive trend is counteracted by accretive ambient shoreline changes; for example, in the Amazon, East and Southeast Asia and the north tropical Pacific. A substantial proportion of the threatened sandy shorelines are in densely populated areas, underlining the need for the design and implementation of effective adaptive measures. Erosion is a major problem facing sandy beaches that will probably worsen with climate change and sea-level rise. Half the world's beaches, many of which are in densely populated areas, could disappear by the end of the century under current trends; mitigation could lessen retreat by 40%.info:eu-repo/semantics/publishedVersio