Enzyme molecular choreography : studies on soluble inorganic pyrophosphatases

Inorganic pyrophosphatases (PPases, EC 3.6.1.1) hydrolyse pyrophosphate in a reaction that provides the thermodynamic 'push' for many reactions in the cell, including DNA and protein synthesis. Soluble PPases can be classified into two families that differ completely in both sequence and structure. While Family I PPases are found in all kingdoms, family II PPases occur only in certain prokaryotes. The enzyme from baker's yeast (Saccharomyces cerevisiae) is very well characterised both kinetically and structurally, but the exact mechanism has remained elusive. The enzyme uses divalent cations as cofactors; in vivo the metal is magnesium. Two metals are permanently bound to the enzyme, while two come with the substrate. The reaction cycle involves the activation of the nucleophilic oxygen and allows different pathways for product release. In this thesis I have solved the crystal structures of wild type yeast PPase and seven active site variants in the presence of the native cofactor magnesium. These structures explain the effects of the mutations and have allowed me to describe each intermediate along the catalytic pathway with a structure. Although establishing the ��choreography�� of the heavy atoms is an important step in understanding the mechanism, hydrogen atoms are crucial for the mechanism. The most unambiguous method to determine the positions of these hydrogen atoms is neutron crystallography. In order to determine the neutron structure of yeast PPase I perdeuterated the enzyme and grew large crystals of it. Since the crystals were not stable at ambient temperature, a cooling device was developed to allow neutron data collection. In order to investigate the structural changes during the reaction in real time by time-resolved crystallography a photolysable substrate precursor is needed. I synthesised a candidate molecule and characterised its photolysis kinetics, but unfortunately it is hydrolysed by both yeast and Thermotoga maritima PPases. The mechanism of Family II PPases is subtly different from Family I. The native metal cofactor is manganese instead of magnesium, but the metal activation is more complex because the metal ions that arrive with the substrate are magnesium different from those permanently bound to the enzyme. I determined the crystal structures of wild type Bacillus subtilis PPase with the inhibitor imidodiphosphate and an inactive H98Q variant with the substrate pyrophosphate. These structures revealed a new trimetal site that activates the nucleophile. I also determined that the metal ion sites were partially occupied by manganese and iron using anomalous X- ray scattering.

Schiff Base Complexes and their Assemblies on Surfaces

Schiff bases and their transition metal complexes are of significant current interest even though they have been prepared for decades. They have been used in various applications such as catalysis, corrosion protection, and molecular sensors. In this study, N-aryl Schiff base ketimine ligands as well as numerous new, differently substituted salen and salophen-type ligands and their cobalt(II), copper(II), iron(II), manganese(II), and nickel(II) complexes were synthesised. New solid state structures of the above compounds and the dioxygen coordination properties of cobalt(II) complexes and catalytic properties of three synthesised binuclear complexes were examined. The prepared complexes were applied in the formation of self-assembled layers on a polycrystalline gold surface and liquid-graphite interface. The effect of metal ion and ligand structure on the as-formed patterns was studied. When studying gold surfaces, a unique thiol-assisted dissolution of elemental gold was observed and a new thin gold foil preparation method was introduced. In the summary, synthesis, structures, and properties of Schiff base ligands and their transition metal complexes are described in detail and the applications of these reviewed. Assemblies of other complexes on a liquid-graphite interface and on a gold surface are also presented, and the surface characterisation methods and surfaces employed are described.

Studies on metal complex formation of environmentally friendly aminopolycarboxylate chelating agents

Aminopolykarboksyylaatteja, kuten etyleenidiamiinitetraetikkahappoa (EDTA), on k��ytetty useiden vuosikymmenien ajan erinomaisen metalli-ionien sitomiskyvyn vuoksi kelatointiaineena lukuisissa sovelluksissa sek�� analytiikassa ett�� monilla teollisisuuden aloilla. N��iden yhdisteiden biohajoamattomuus on kuitenkin her��tt��nyt huolta viime aikoina, sill�� niiden on havaittu olevan hyvin pysyvi�� luonnossa. T��m�� ty�� on osa laajempaa tutkimushanketta, jossa on tavoitteena l��yt���� korvaavia kelatointiaineita EDTA:lle. Tutkimuksen aiheena on kuuden kelatointiaineen metalli-ionien sitomiskyvyn kartoitus. EDTA:a paremmin luonnossa hajoavina n��m�� ovat ymp��rist��yst��v��llisi�� ehdokkaita korvaaviksi kelatointiaineiksi useisiin sovelluksiin. Ty��ss�� tutkittiin niiden kompleksinmuodostusta useiden metalli-ionien kanssa potentiometrisella titrauksella. Metalli-ionivalikoima vaihteli hieman kelatointiaineesta riippuen sis��lt��en magnesium-, kalsium-, mangaani-, rauta-, kupari-, sinkki-, kadmium-, elohopea-, lyijy- ja lantaani-ionit. Tutkittavat metallit oli valittu t��ht��imess�� olevien sovellusten, synteesiss�� ilmenneiden ongelmien tai ymp��rist��n��k��kohtien perusteella. Tulokset osoittavat n��iden yhdisteiden metallinsitomiskyvyn olevan jonkin verran heikompi kuin EDTA:lla, mutta kuitenkin riitt��v��n useisiin sovelluksiin kuten sellunvalkaisuprosessiin. Myrkyllisten raskasmetallien, kadmiumin, elohopen ja lyijyn kohdalla EDTA:a heikompi sitoutuminen on eduksikin, koska se yhdistettyn�� parempaan biohajoavuuteen saattaa alentaa tutkittujen yhdisteiden kyky�� mobilisoida kyseisi�� metalleja sedimenteist��. Useimmilla tutkituista yhdisteist�� on ymp��rist��n��k��kulmasta etuna my��s EDTA:a pienempi typpipitoisuus.

Multiscale study on hydrogen mobility in metallic fusion divertor material

For achieving efficient fusion energy production, the plasma-facing wall materials of the fusion reactor should ensure long time operation. In the next step fusion device, ITER, the first wall region facing the highest heat and particle load, i.e. the divertor area, will mainly consist of tiles based on tungsten. During the reactor operation, the tungsten material is slowly but inevitably saturated with tritium. Tritium is the relatively short-lived hydrogen isotope used in the fusion reaction. The amount of tritium retained in the wall materials should be minimized and its recycling back to the plasma must be unrestrained, otherwise it cannot be used for fueling the plasma. A very expensive and thus economically not viable solution is to replace the first walls quite often. A better solution is to heat the walls to temperatures where tritium is released. Unfortunately, the exact mechanisms of hydrogen release in tungsten are not known. In this thesis both experimental and computational methods have been used for studying the release and retention of hydrogen in tungsten. The experimental work consists of hydrogen implantations into pure polycrystalline tungsten, the determination of the hydrogen concentrations using ion beam analyses (IBA) and monitoring the out-diffused hydrogen gas with thermodesorption spectrometry (TDS) as the tungsten samples are heated at elevated temperatures. Combining IBA methods with TDS, the retained amount of hydrogen is obtained as well as the temperatures needed for the hydrogen release. With computational methods the hydrogen-defect interactions and implantation-induced irradiation damage can be examined at the atomic level. The method of multiscale modelling combines the results obtained from computational methodologies applicable at different length and time scales. Electron density functional theory calculations were used for determining the energetics of the elementary processes of hydrogen in tungsten, such as diffusivity and trapping to vacancies and surfaces. Results from the energetics of pure tungsten defects were used in the development of an classical bond-order potential for describing the tungsten defects to be used in molecular dynamics simulations. The developed potential was utilized in determination of the defect clustering and annihilation properties. These results were further employed in binary collision and rate theory calculations to determine the evolution of large defect clusters that trap hydrogen in the course of implantation. The computational results for the defect and trapped hydrogen concentrations were successfully compared with the experimental results. With the aforedescribed multiscale analysis the experimental results within this thesis and found in the literature were explained both quantitatively and qualitatively.

Radiation effects in supported nanoparticles

Nanotechnology applications are entering the market in increasing numbers, nanoparticles being among the main classes of materials used. Particles can be used, e.g., for catalysing chemical reactions, such as is done in car exhaust catalysts today. They can also modify the optical and electronic properties of materials or be used as building blocks for thin ���lm coatings on a variety of surfaces. To develop materials for speci���c applications, an intricate control of the particle properties, structure, size and shape is required. All these depend on a multitude of factors from methods of synthesis and deposition to post-processing. This thesis addresses the control of nanoparticle structure by low-energy cluster beam deposition and post-synthesis ion irradiation. Cluster deposition in high vacuum offers a method for obtaining precisely controlled cluster-assembled materials with minimal contamination. Due to the clusters small size, however, the cluster-surface interaction may drastically change the cluster properties on deposition. In this thesis, the deposition process of metal and alloy clusters on metallic surfaces is modelled using molecular dynamics simulations, and the mechanisms in���uencing cluster structure are identi���ed. Two mechanisms, mechanical melting upon deposition and thermally activated dislocation motion, are shown to determine whether a deposited cluster will align epitaxially with its support. The semiconductor industry has used ion irradiation as a tool to modify material properties for decades. Irradiation can be used for doping, patterning surfaces, and inducing chemical ordering in alloys, just to give a few examples. The irradiation response of nanoparticles has, however, remained an almost uncharted territory. Although irradiation effects in nanoparticles embedded inside solid matrices have been studied, almost no work has been done on supported particles. In this thesis, the response of supported nanoparticles is studied systematically for heavy and light ion irradiation. The processes leading to damage production are identi���ed and models are developed for both types of irradiation. In recent experiments, helium irradiation has been shown to induce a phase transformation from multiply twinned to single-crystalline nanoparticles in bimetallic alloys, but the nature of the transition has remained unknown. The alloys for which the effect has been observed are CuAu and FePt. It is shown in this thesis that transient amorphization leads to the observed transition and that while CuAu and FePt do not amorphize upon irradiation in bulk or as thin ���lms, they readily do so as nanoparticles. This is the ���rst time such an effect is demonstrated with supported particles, not embedded in a matrix where mixing is always an issue. An understanding of the above physical processes is essential, if nanoparticles are to be used in applications in an optimal way. This thesis clari���es the mechanisms which control particle morphology, and paves way for the synthesis of nanostructured materials tailored for speci���c applications.

Characterisation of cellulose- and lignin-based materials using x-ray scattering methods

Wood is an important material for the construction and pulping industries. Using x-ray diffraction the microfibril angle of Sitka spruce wood was studied in the first part of this thesis. Sitka spruce (Picea sitchensis [Bong.] Carr.) is native to the west coast of North America, but due to its fast growth rate, it has also been imported to Europe. So far, its nanometre scale properties have not been systematically characterised. In this thesis the microfibril angle of Sitka spruce was shown to depend significantly on the origin of the tree in the first annual rings near the pith. Wood can be further processed to separate lignin from cellulose and hemicelluloses. Solid cellulose can act as a reducer for metal ions and it is also a porous support for nanoparticles. By chemically reducing nickel or copper in the solid cellulose support it is possible to get small nanoparticles on the surfaces of the cellulose fibres. Cellulose supported metal nanoparticles can potentially be used as environmentally friendly catalysts in organic chemistry reactions. In this thesis the size of the nickel and copper containing nanoparticles were studied using anomalous small-angle x-ray scattering and wide-angle x-ray scattering. The anomalous small-angle x-ray scattering experiments showed that the crystallite size of the copper oxide nanoparticles was the same as the size of the nanoparticles, so the nanoparticles were single crystals. The nickel containing nanoparticles were amorphous, but crystallised upon heating. The size of the nanoparticles was observed to be smaller when the reduction of nickel was done in aqueous ammonium hydrate medium compared to reduction made in aqueous solution. Lignin is typically seen as the side-product of wood industries. Lignin is the second most abundant natural polymer on Earth, and it possesses potential to be a useful material for many purposes in addition to being an energy source for the pulp mills. In this thesis, the morphology of several lignins, which were produced by different separation methods from wood, was studied using small-angle and ultra small-angle x-ray scattering. It was shown that the fractal model previously proposed for the lignin structure does not apply to most of the extracted lignin types. The only lignin to which the fractal model could be applied was kraft lignin. In aqueous solutions the average shape of the low molar mass kraft lignin particles was observed to be elongated and flat. The average shape does not necessarily correspond to the shape of the individual particles because of the polydispersity of the fraction and due to selfassociation of the particles. Lignins, and especially lignosulfonate, have many uses as dispersants, binders and emulsion stabilisers. In this thesis work the selfassociation of low molar mass lignosulfonate macromolecules was observed using small-angle x-ray scattering. By taking into account the polydispersity of the studied lignosulfonate fraction, the shape of the lignosulfonate particles was determined to be flat by fitting an oblate ellipsoidal model to the scattering intensity.

Occupational chronic solvent encephalopathy in Finland 1995 - 2007 : incidence and diagnostic methods

The occurrence of occupational chronic solvent encephalopathy (CSE) seems to decrease, but still every year reveals new cases. To prevent CSE and early retirement of solvent-exposed workers, actions should focus on early CSE detection and diagnosis. Identifying the work tasks and solvent exposure associated with high risk for CSE is crucial. Clinical and exposure data of all the 128 cases diagnosed with CSE as an occupational disease in Finland during 1995-2007 was collected from the patient records at the Finnish Institute of Occupational Health (FIOH) in Helsinki. The data on the number of exposed workers in Finland were gathered from the Finnish Job-exposure Matrix (FINJEM) and the number of employed from the national workforce survey. We analyzed the work tasks and solvent exposure of CSE patients and the findings in brain magnetic resonance imaging (MRI), quantitative electroencephalography (QEEG), and event-related potentials (ERP). The annual number of new cases diminished from 18 to 3, and the incidence of CSE decreased from 8.6 to 1.2 / million employed per year. The highest incidence of CSE was in workers with their main exposure to aromatic hydrocarbons; during 1995-2006 the incidence decreased from 1.2 to 0.3 / 1 000 exposed workers per year. The work tasks with the highest incidence of CSE were floor layers and lacquerers, wooden surface finishers, and industrial, metal, or car painters. Among 71 CSE patients, brain MRI revealed atrophy or white matter hyperintensities or both in 38% of the cases. Atrophy which was associated with duration of exposure was most frequently located in the cerebellum and in the frontal or parietal brain areas. QEEG in a group of 47 patients revealed increased power of the theta band in the frontal brain area. In a group of 86 patients, the P300 amplitude of auditory ERP was decreased, but at individual level, all the amplitude values were classified as normal. In 11 CSE patients and 13 age-matched controls, ERP elicited by a multimodal paradigm including an auditory, a visual detection, and a recognition memory task under single and dual-task conditions corroborated the decrease of auditory P300 amplitude in CSE patients in single-task condition. In dual-task conditions, the auditory P300 component was, more often in patients than in controls, unrecognizable. Due to the paucity and non-specificity of the findings, brain MRI serves mainly for differential diagnostics in CSE. QEEG and auditory P300 are insensitive at individual level and not useful in the clinical diagnostics of CSE. A multimodal ERP paradigm may, however, provide a more sensitive method to diagnose slight cognitive disturbances such as CSE.

Sob outro olhar: a narrativa jesu��tica sobre o hindu��smo e sua rela����o com a pr��tica mission��ria no sul da ��ndia (S��culo XVII)

No contexto da expans��o ultramarina portuguesa no Oriente, a regi��o do Madur��, localizada ao sul da ��ndia, tamb��m vivenciou a experi��ncia pol��tica e, principalmente, religiosa a partir da presen��a da Companhia de Jesus nesse espa��o. Em 1596, o jesu��ta portugu��s Gon��alo Fernandes Trancoso tornou-se respons��vel por cuidar da comunidade crist�� existente no Madur��, ganhando como companheiro de miss��o, em 1606, o jesu��ta italiano Roberto de Nobili. A presente disserta����o tem por objetivo central promover uma an��lise acerca das atua����es mission��rias dos jesu��tas Gon��alo Fernandes Trancoso e Roberto de Nobili na miss��o do Madur��, no in��cio do s��culo XVII. Seus trabalhos mission��rios podem ser compreendidos a partir do estudo de suas narrativas sobre os costumes e pr��ticas bram��nicas, sendo poss��vel identificar, para al��m das diferentes estrat��gias de convers��o utilizadas por ambos inacianos, o modo como estes articularam seus conhecimentos sobre a sociedade indiana e o hindu��smo com o desenvolvimento de um projeto de convers��o. Assim, destaca-se a falta homogeneidade no interior da Companhia de Jesus no que toca a pr��ticas e m��todos mission��rios.

A study of the male reproductive organs of nine species of penaeid prawns (Crustacea: Penaeinae)

This paper deals with the male reproductive organs of nine species of penaeid prawns; Penaeus penicillatus Alcock, P. merguiensis De Man, P. semisulcatus De Haan, Metapenaeus affinis (H.Milne Edwards), M. monoceros (Fabricius), M. stebbingi Nobili, Parapenaeopsis hardwickii (Miers), P. sculptilis (Heller) and P. stylifera (H. Milne Edwards). The male reproductive organs exhibited structural variations, which were more pronounced at generic level. These variations are mainly due to the type of spermatophore they possess. One species of each genus, that is, P. merguiensis, M. affinis and P. sculptilis were also studied histologically to examine the internal structure of the male reproductive organs. Spermatophores of the six species belonging to the genera Penaeus and Parapenaeopsis are also described and illustrated.

Enhancement of fish production in a reservoir after partitioning by dikes through community participation

A reservoir of 70 acres was portioned by dikes into four manageable big ponds to get more production of fishes at Basurhat, Noakhali, Bangladesh under the supervision of local community through a society of 40 people ownership. Pangus (Pangasius hypophthalmus) @ 20,000/acre, and then fry and fingerlings of different types of fishes such as catla (Catla catla), rohu (Labeo rohita), mrigal (Cirrhina mrigala), grass carp (Ctenophmyngodon idella), bighead (Aristichthys nobili), silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus cmpio) and rajpunti (Puntius gonionatus) @ 500/acre were stocked. Feed containing 25% protein was used two times daily and feed was adjusted fortnightly. After 8 months, all the fishes were weighed 0.80-2.10 kg except rajpunti (150-200 g) and tilapia (150-220 g), and a total of 25 ton of fish was harvested which was five times higher than the previous production under signal ownership. The production of fishes were increased after partitioning the lake with dikes due to proper management and control.

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������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������5������12��� 40������������4���������������9���������������������������1���������Pinnotheres sp.������������������������������������������������������������������������������������������������������ ������������������������������������������������������������������Tritodynamia intermedia Shen, 1935������������������������ Tritodynamia horvathi Nobili, 1905������������������ ���������������������������������������������������������Sakai���1976���������T. intermedia Shen���T. horvathi Nobili������������������������������������������������������������ ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������Pinnotheres������8������������������Pinnotheres excussus Dai et al., 1980������������Pinnotheres pilulus Dai et al., 1980���������������Pinnotheres serrignathus Shen, 1932������������Pinnotheres dilatatus Shen, 1932���������������Pinnotheres tsingtaoensis Shen, 1932���������������Pinnotheres haiyangensis Shen, 1932���������������Pinnotheres obtusidentata���Dai et al., 1980���������������Pinnotheres luminatus Dai et al., 1980���������������������������������������������Tritodynamia���4���������������������Tritodynamia fujianensis Chen, 1979������������������Tritodynamia longipropodum Dai et al., 1980������������������ Tritodynamia dilatatum Yang et Sun,1996������������������ Tritodynamia hainanensis Dai et al.g, 1980������������������������������������������������������13��������������������������������������������������������������������������������������������������������������� ���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������

Report on some species of Palaemonidae (Crustacea, Decapoda) from French Polynesia

Based on material collected from French Polynesia and deposited in the Museum national d'Histoire naturelle, Paris, the present paper reports 31 palaemonid shrimp species, which belong to the Palaemoninae (two genera, three species) and to the Pontoniinae (12 genera, 28 species), including six new species. The new species are: Izucaris crosnieri n. sp., Periclimenes alexanderi n. sp., P. josephi n. sp., P platydactylus n. sp., P polynesiensis n. sp. and P vicinus n. sp. Detailed descriptions and illustrations of the new species are provided. Besides the six new species, ten other species are recorded for the first time from French Polynesia: Exoclimenella maldivensis Duris & Bruce, 1995, Kemponia rapanui (Fransen, 1987) n. comb., Palaemonella crosnieri Bruce, 1978, P spinulata Yokoya, 1936, Periclimenaeus hecate (Nobili, 1904), P orbitocarinatus Fransen, 2006, Periclimenes aleator Bruce, 199 1, P paralcocki Li & Bruce, 2006, P uniunguiculatus Bruce, 1990, Pontonides loloata Bruce, 2005.

Neurology and psychiatry: waking up to opportunities of sleep. State of the art and clinical/research priorities for the next decade.

In recent years, evidence has emerged for a bidirectional relationship between sleep and neurological and psychiatric disorders. First, sleep-wake disorders (SWDs) are very common and may be the first/main manifestation of underlying neurological and psychiatric disorders. Secondly, SWDs may represent an independent risk factor for neuropsychiatric morbidities. Thirdly, sleep-wake function (SWF) may influence the course and outcome of neurological and psychiatric disorders. This review summarizes the most important research and clinical findings in the fields of neuropsychiatric sleep and circadian research and medicine, and discusses the promise they bear for the next decade. The findings herein summarize discussions conducted in a workshop with 26 European experts in these fields, and formulate specific future priorities for clinical practice and translational research. More generally, the conclusion emerging from this workshop is the recognition of a tremendous opportunity offered by our knowledge of SWF and SWDs that has unfortunately not yet entered as an important key factor in clinical practice, particularly in Europe. Strengthening pre-graduate and postgraduate teaching, creating academic multidisciplinary sleep-wake centres and simplifying diagnostic approaches of SWDs coupled with targeted treatment strategies yield enormous clinical benefits for these diseases.