1,512 research outputs found
Health consultation, W.R. Grace exfoliation facility : Phoenix, Maricopa County, Arizona
No full textabstract: The W.R. Grace facility in Phoenix, Arizona, received vermiculite concentrate from the Libby, Montana, vermiculite mine. W.R. Grace Company has owned and operated the Arizona site since 1964. In 1964 W.R. Grace purchased the company that had previously occupied the site and, following the relocation of its vermiculite exfoliation furnace from Glendale, Arizona, began processing vermiculite concentrate and marketing it under the Zonolite® brand. The objective of this health consultation is to evaluate exposure pathways and potential health effects in those persons who, between 1964 and 2002, may have been exposed to Libby asbestos as a result of vermiculite concentrate processing activities and waste materials from the W.R. Grace exfoliation facility in Phoenix.Under cooperative agreement with the Agency for Toxic Substances and Disease Registry.Includes bibliographical references (p. 22-24)
The individual and institutional experiences of the young apprenticeship ‘experiment’
No full textThe Young Apprenticeship (YA) programme is the latest in a long line of vocational qualifications to exist fleetingly within the English education system. Introduced in 2004, the YA programme offered Key Stage 4 students the opportunity to combine academic and vocational study within specific industrial sectors.Evaluative studies of a quantitative nature evidence a positive response, both in terms of perceived usefulness and actual success (90% completion rate of the Sport YA, (SkillsActive, 2009)) from students, providers and employers. Never attaining more than pilot status, the programme was closed to new entrants in 2011 following recommendations made by the Wolf Report, condemning it to the role of yet another vocational education ‘experiment’. Little is known about how the students experience a programme that occupies a significant proportion of their Key Stage 4 timetable. Outside of their immediate institutional context, the YA students are a hidden population.This study seeks to examine and give voice to the experiences of the individuals who have participated in the programme, within their institutional context. Through a qualitative research methodology, it is proposed that observed changes in individual disposition during participation in the YA programme allow it to be considered as a ‘lived experience’ for the participants. It is argued that Situated Learning theory and the Community of Practice concept are useful analytical tools through which to make sense of the learning processes in which the YA students engage
Description of the KNMI Operational Wave Forecast Model GONO
No full textThe purpose of this report is to give a description of the GONO computercode, which is operational at KNMI for many years now. The program was developed by J.W. Sanders, and its deep water version is based on a Norwegian wave prediction model. built by C. Haug in the sixties. Shallow water effects are however important in the southern part of the North Sea. giving a limitation of the wave growth and causing important swell dissipation. A discussion of the shallow water effects. as present in GONO, is given by Sanders in Ref. 3. The computer code GONO is written in ALGOL60 and operational on the Burroughs 6700 of KNMI. The program GONO calculates wind speed. wind direction and sea energy at every grid point of the GONO grid (cf. Appendix A) and swell energy at a limited number of points only (we call these points swell points). GONO runs every six hours and it gives a 12 and 24 hours forecast as well as results based on analyzed weather maps. In the winter of '79-'80 the output of GONO was compared with measurements and the operational wave model of Bracknell (United Kingdom). (The latter GONO version differs a little bit from the version here described). The preliminary results of this comparison are given in Ref. 4. A reasonable agreement of significant wave height and low-frequency energy, as given by GONO, with the observations was found. The present version of GONO, with small modifications compared to the previous version, is operational since the end of February 1980. Essentially, the GONO model is based on two steps. First. the sea energy at every grid point is determined. To this end advection of energy is treated by means of a finite difference scheme whereas the growth of the wave energy is calculated by means of an empirical growth curve (assuming that the wave spectrum has a fixed form). The second step is the calculation of the swell. Of course, in principle swell can be treated likewise, but then one has to store swell energy (and its direction) at every time, at every grid point for every frequency band. Also. this finite difference scheme is rather crude for swell propagation. whereas. because of stability reasons. there is an upper bound for the propagation speed (in the present case the upper bound is given by 13.87 m/s). If one is only interested in swell information at particular points (swell points), it is tempting to use a ray technique. The advantage of this technique is that it is very accurate. Swell is determined in this fashion in GONO.GON
Tungsten biochemistry of Pyrococcus furiosus
No full textTungsten is the heaviest element that exhibits biological activity (atomic number 74), when it is present in an enzyme. It is taken up by cells in the form of tungstate, and it is subsequently processed into an organic cofactor referred to as tungstopterin, which is found as active center in several enzymes. Pyrococcus furiosus is a hyperthermophilic archaeon that grows anaerobically at an optimal temperature of 100 ËšC, strictly dependent on the presence of tungstate. Over the last years, P. furiosus has become a model organism for hyperthermophiles, as many of its proteins have been the subject of research and its genome has been sequenced. Also regarding tungsten metabolism P. furiosus can be considered as a model: four tungsten containing aldehyde oxidoreductases were already characterized in some detail before the initiation of this study. In this thesis several aspects of tungsten metabolism in P. furiosus have been further explored: its tungstate transport system has been identified and characterized (WtpABC), studies on aspects of tungsten cofactor biosynthesis have been carried out and a new tungsten-containing aldehyde oxidoreductase (AOR), WOR5, has been purified and characterized.Applied Science
Biochemistry of Tungstoenzymes from Pyrococcus furiosus
No full textThe cell uses a variety of transition metals to provide greater catalytic diversity than could be achieved using only the functional groups of amino acids. The biochemistry of molybdenum and tungsten is unusual: they are the only 4d and 5d metal ions with established biological role(s). This thesis reports studies on the identification of tungstoenzymes using functional proteomics and on the physiological substrate, function and catalytic mechanism of one of the abundantly present tungstoenzymes of Pyrococcus furiosus; formaldehyde ferredoxin oxidoreductase (FOR). A new method of native-native two dimensional gel electrophoresis (2DGE) was developed in which no denaturants such as urea or SDS were applied (Chapter 2). Radioactively labeled tungsten (187WO42-) was added to the growth medium of P. furiosus to detect tungsten proteins for subsequent identification with mass spectrometry. The radiograms of gels containing radioactively labeled tungsten revealed six spots with tungsten-associated proteins. These spots were excised and analyzed by mass spectrometry. Two tungstoenzymes were identified as aldehyde ferredoxin oxidoreductase and formaldehyde ferredoxin oxidoreductase. No tungsten containing proteins could be identified from the other spots. A new tungsten containing oxidoreductase (WOR5) was discovered. It is the fifth and last member of the family of tungsten containing oxidoreductases from the hyperthermophilic archaeon P. furiosus. WOR5 was purified and characterized with EPR spectroscopy and electrochemistry (Chapter 3). It was found to be a homo-dimeric protein (subunit: 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. The enzyme has a broad substrate specificity with a high affinity for several substituted and non-substituted aliphatic and aromatic aldehydes with variable chain lengths, and it can use ferredoxin as electron acceptor in catalysis. The redox chemistry of the tungsten and iron-sulfur prosthetic groups in P. furiosus formaldehyde ferredoxin oxidoreductase (Chapter 4) and steady and pre-steady state kinetics (Chapter 5) for this enzyme were studied using formaldehyde as substrate and ferredoxin as electron acceptor. The intermediate, paramagnetic W(V) state could be trapped only by reduction of FOR with substrate, with consecutive one-electron intraprotein electron transfer to the single [4Fe-4S](2+;1+) cluster and partial comproportionation of the tungsten over W(IV, V, VI); this is a stable state in the absence of an external electron acceptor. Due to the very unfavorable hydratation equilibrium of the formaldehyde/methylene glycol couple no W(V) was found in dye mediated equilibrium redox titrations. The development of this intermediate was slow even at elevated temperatures and with a excess of substrate. The free formaldehyde, and not the methylene glycol, is the enzyme's substrate, implying that the KM for formaldehyde is three orders-of-magnitude less than the value previously reported in literature. The steady state kinetics of FOR is consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The determined KM of ferredoxin (14 μM) was an order of magnitude less than previously reported values, due to the fact that at high concentrations of substrate the enzyme is inhibited and denatured. Pre-steady state difference spectra revealed peak shifts and a lack of isosbestic points, an indication that several out-of-phase processes happen simultaneously in the first seconds of the reaction. The binding and the oxidation of the substrate are both fast processes. The release of the product and the electron shuffling over the tungsten and iron-sulfur center in the absence of an external electron acceptor are slower. Based on these results, in combination with results from previous EPR studies two alternatives for a catalytic redox cycle are proposed.Applied Science
Structural and functional studies of the iron storage protein ferritin from Pyrococcus furiosus
No full textThis research focuses on the iron storage protein ferritin. Ferritin is a protein involved in iron homeostasis by storing Fe(II) excess in the form of an Fe(III) mineral core in the presence of oxygen and by releasing iron during iron deficiency. Ferritins are vital for human health. Their malfunction may lead among other diseases to anemia, iron overload, Parkinson or Alzheimer. In addition to its medical significance, there are industrial applications of ferritin such as in nanotechnology, catalysis and environmental cleaning. The research of this study focused on ferritin from hyperthermophile, archaeon and anaerobe Pyrococcus furiosus and involved both the fundamental biochemistry of ferritin and ferritin's possible application. The results include the development of a highly efficient overproduction system of P. furiosus ferritin, the determination of ferritin's redox chemistry that gave insight into ferritin's possible physiological redox partners, and determination of P. furiosus ferritin's crystal structure. Additionally, ferritin has been used for the production of carbon nanotubes and a method to increase the yield of carbon nanotubes was developed and presented.Applied Science
Redox biochemistry of Pyrococcus furiosus: Fundamental and applied aspects
No full textIn Pyrococcus furiosus (Pfu) ferredoxin (Fd) replaces NAD+ for carrying reducing equivalent to the oxidative phosphorylation machinery, and it also takes part in the regeneration of NADPH, which is necessary for biosynthetic pathways. Therefore, it plays a central role in various metabolic pathways of the organism. PfuFd is one of the most extensively studied ferredoxins, however, some fundamental aspects remain elusive. This thesis work focuses on some of the unresolved features of the protein. Initially PfuFd was isolated as a dimer of 12-13 kDa, however, all the subsequent studies described the protein as a monomer without any experimental evidence. All (putative) natural electron-transfer partners of PfuFd are redox enzymes catalyzing two-electron reactions while ferredoxin is a one-electron carrier. It is possible that ferredoxin may interact with the redox partner enzymes in the dimeric form. Therefore, it is important to resolve the dilemma regarding the oligomeric state of the protein (chapter 2). The intra-cellular ionic strength of P. furiosus was determined to be ca. 350 mM, at which ferredoxin occurs predominantly in the dimeric form. Transition from the dimeric to monomeric form is observed at a salt concentration higher than 350 mM. We hypothesize that ferrdoxin is a dimer in vivo. PfuFd demonstrates conservation of the minimal domain containing the cluster coordinating consensus sequence. However, the cluster is coordinated by three cysteines and one aspartate residue instead of by four cysteines. In addition, two additional cysteines are present which raises the possibility of a disulfide bond formation between them. In some earlier studies the formation of a disulfide bond was demonstrated under aerobic condition and a possible contribution of the disulfide bond to the redox chemistry of the protein has been described. However, formation of a disulfide bond under the strongly reducing intracellular condition of an anaerobic organism like P. furiosus is not understandable. We demonstrate (in chapter 3) that these additional cysteines do not form a disulfide bond under intra-cellular mimicking conditions. Observing the effects of site directed mutagenesis, we attribute to the free cysteines an important structural role in the hyperthermostability of the protein. Ferredoxin, cytochrome c, rubredoxin, etc. are small electron-transfer protein and they possess well characterised metal cofactors. Electrochemistry has been successfully applied in studying the intricate properties of these electron-transfer proteins mainly in solution. However, solution voltammetry suffers from various problems like mass transport / diffusion limitation, requirement of promoters, which can be avoided by immobilizing the protein on the electrode surface. A stable and functional PfuFd immobilized gold electrode has been developed, which shows a similar electrochemical response as in the solution voltammetry. The transition between the dimeric and monomeric state on the electrode is observed in an atomic force microscopic (AFM) setup. X-ray photoelectron spectrocopy (XPS) confirms the formation of gold-thiol bonds. We find the ferredoxin electrode to be a useful tool for studying the catalytic mechanisms of the associated redox enzymes from P. furiosus. We have thus studied two redox enzymes, glyceraldehyde-3-phosphate oxidoreductase (GAPOR) and aldehyde oxidoreductase (AOR), which are involved in ferredoxin-mediated redox processes in the organism. Addition of these enzymes at room temperature results in complex formation between the electrode-bound ferredoxin and the enzyme. At 60 °C a catalytic wave appears upon addition of the substrate, glyceraldehyde-3-phosphate to the Fd-GAPOR complex. In the case of AOR at 80 °C reversible oxidation of crotonadehyde to crotonic acid and vice versa was observed. This work opens the way for the application of Fd electrodes in achieving controlled reduction of carboxylic acids on a preparative scale.Applied Science
Metalloproteins containing iron and tungsten: Biocatalytic links between organic and inorganic redox chemistry
No full textApplied Science
"Leishmania infantum" : molecular analysis for identification of potential virulence factors and genes of diagnostic use
No full textLeishmania infantum is the causative agents of zoonotic visceral leishmaniasis, a fatal disease if left untreated. In Southern Europe, the number of HIV/Leishmania infantum co-infections is increasing and becomes an emerging concern for the public health. Dogs represent the main reservoir of this parasite. In order to control canine leishmaniasis, an early diagnosis system is needed because dogs are capable to transmit the parasite, before showing any symptoms of the disease. Here fore, cDNA expression libraries were screened with sera of infected dogs. We selected several antigens reacting with the sera. These were different histones, ribosomal and mitochondrial proteins. Four of them were subcloned into expression plasmids, and recombinantly expressed in E. coli. The purified proteins were sent to a collaborating laboratory for further examination. Although several diagnostic polymerase chain reactions (PCRs) for Leishmania species causing human leishmaniasis are published, none of them is able to detect all different species and distinguish among them at the same time. Therefore, a diagnostic PCR was developed using the mini-exon gene (encoding the spliced leader) as template. A restriction fragment length polymorphism (RFLP) scheme was elaborated in order to identify different Leishmania species causing human leishmaniasis. The PCR and RFLP scheme is now in routine use at the STI. In order to identify potential virulence factors, vaccine candidates and drug targets of Leishmania infantum, two cDNA subtractions were performed: (1) A none-infective L. infantum strain was subtracted from a highly infective strain. Several genes which were higher or exclusively expressed in the infective strain were identified. However, association with virulence could not be shown, since the two strains differed not only in virulence but also in growth rate. (2) Non-infective early promastigote stages were subtracted from late promastigote stages (metacyclic transmission stage) of the same strain. Several genes showing differential expression were selected including genes encoding nutrient transporters, cytochrome b5 and unknown proteins. One of them was the previously described metacyclic specific gene MAT-1. Three of the genes were recombinantly expressed in E. coli in order to perform immunization and preliminary localization studies
- …
