Shaping mechanisms of metal specificity in a family of metazoan Metallothioneins: evolutionary differentiation of Mollusc MTs.

Background: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order t o elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. Results: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. Conclusion: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Exploring the effect of aminoglycoside guanidinylation on ligands for Tau exon 10 splicing regulatory element RNA

We describe the effect of guanidinylation of the aminoglycoside moiety on acridine-neamine-containing ligands for the stem-loop structure located at the exon 10-5′-intron junction of Tau pre-mRNA, an important regulatory element of tau gene alternative splicing. On the basis of dynamic combinatorial chemistry experiments, ligands that combine guanidinoneamine and two different acridines were synthesized and their RNA-binding properties were compared with those of their amino precursors. Fluorescence titration experiments and UV-monitored melting curves revealed that guanidinylation has a positive effect both on the binding affinity and specificity of the ligands for the stemloop RNA, as well as on the stabilization of all RNA sequences evaluated, particularly some mutated sequences associated with the development of FTDP-17 tauopathy. However, this correlation between binding affinity and stabilization due to guanidinylation was only found in ligands containing a longer spacer between the acridine and guanidinoneamine moieties, since a shorter spacer produced the opposite effect (e.g. lower binding affinity and lower stabilization). Furthermore, spectroscopic studies suggest that ligand binding does not significantly change the overall RNA structure upon binding (circular dichroism) and that the acridine moiety might intercalate near the bulged region of the stem->loop structure (UV-Vis and NMR spectroscopy).

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Pitkäketjuisten keteenien valmistusreaktiot ja kinetiikka.

Tämän diplomityön tarkoituksena oli tutkia pitkäketjuisten rasvahappokloridien ja alkyyliketeenidimeerien (AKD) valmistusreaktioiden kinetiikkaa. Työn tavoitteena oli saada mittaustuloksia, joiden perusteella voitaisiin kehittää reaktioille kineettinen kineettinen malli ja suorittaa valmistusprosessien alustava optimointi. Teoreettisessa osassa on selvitetty rasvahappokloridien ja alkyyliketeenidimeerien (AKD) eri valmistustapoja. Lisäksi on perehdytty tarkemmin rasvahappokloridien dehydrohalogenointireaktioiden reaktiomekanismeihin ja valmistusprosessin problematiikkaan. Kokeellisessa osassa tutkittiin rasvahappokloridien valmistusta klooraamalla öljyhappoa 30 mol-% fosforitrikloridiylimäärällä lämpötiloissa 45, 50 ja 55OC. Alkyyliketeenidimeerien valmistusta tutkittiin dehydrohalogenoimalla palmitiini- ja öljyhappokloridia 0-30 mol-% trietyyliamiiniylimäärällä inertissä liuottimessa, lämpötiloissa 45 ja 50OC. Reaktiot toteutettiin puolipanosreaktiona, joissa reaktioastiassa olevaan reagenssin ja liuottimen seokseen lisättiin lähtöaine tasaisena massavirtana. Reaktioiden etenemistä seurattiin FT-IR- ja GC-analyysien avulla. Kalorimetrisilla kokeilla tutkittiin öljyhappokloridin dimeroitumisreaktion reaktiolämmön muodostumista ja UV-VIS-analyyseillä seurattiin öljyhappokloridin vanhenemista. Öljyhappokloridin valmistusreaktiolle saatiin hyvä kineettinen malli. Kineettisen mallin puutteena voidaan pitää sitä, ettei kokeissa saatu tietoa mahdollisten sivutuotteiden muodostumisesta. Sovitusohjelmalla saatiin sovitettua estimaatit reaktionopeusvakioille lämpötiloissa 45, 50 ja 55OC. Happokloridien dimeroitumisreaktioiden kineettisen mallin sopivuudelle saatiin suhteellisen hyvä kuva Kuten öljyhappokloridin tapauksessa, myöskään AKD:n valmistusreaktioissa syntynyt sivutuotteita, joten niiden osuutta oletettuun kineettiseen malliin ei tunneta. Sovitusohjelmalla saatiin sovitettua estimaatit reaktionopeusvakioille lämpötiloissa 45 ja 50OC.

Photocontrolled DNA Binding of a Receptor-Targeted Organometallic Ruthenium(II) Complex

A photoactivated ruthenium(II) arene complex has been conjugated to two receptor-binding peptides, a dicarba analogue of octreotide and the Arg-Gly-Asp (RGD) tripeptide. These peptides can act as"tumor-targeting devices" since their receptors are overexpressed on the membranes of tumor cells. Both ruthenium-peptide conjugates are stable in aqueous solution in the dark, but upon irradiation with visible light, the pyridyl-derivatized peptides were selectively photodissociated from the ruthenium complex, as inferred by UV-vis and NMR spectroscopy. Importantly, the reactive aqua species generated from the conjugates, [(η6-p-cym)Ru(bpm)(H2O)]2+, reacted with the model DNA nucleobase 9-ethylguanine as well as with guanines of two DNA sequences, 5′dCATGGCT and 5′dAGCCATG. Interestingly, when irradiation was performed in the presence of the oligonucleotides, a new ruthenium adduct involving both guanines was formed as a consequence of the photodriven loss of p-cymene from the two monofunctional adducts. The release of the arene ligand and the formation of a ruthenated product with a multidentate binding mode might have important implications for the biological activity of such photoactivated ruthenium(II) arene complexes. Finally, photoreactions with the peptide-oligonucleotide hybrid, Phac-His-Gly-Met-linker-p5′dCATGGCT, also led to arene release and to guanine adducts, including a GG chelate. The lack of interaction with the peptide fragment confirms the preference of such organometallic ruthenium(II) complexes for guanine over other potential biological ligands, such as histidine or methionine amino acids.

Marjojen flavonoidien uuttaminen

Flavonoidit ovat kasvien polyfenolisia sekundäärimetaboliitteja, jotka koostuvat 15-hiilisestä C6-C3-C6-perusrungosta. Niillä on havaittu useita terveyden kannalta edullisia ominaisuuksia kuten antioksidatiivisia, antikarsinogeenisia, antiallergeenisia, anti-inflammatorisia, estrogeenisia sekä antimikrobiaalisia vaikutuksia. Kasvimateriaalia käyttävän elintarviketeollisuuden sivuvirrat ovat flavonoidi-pitoisten jakeiden arvokkaita raaka-aineita. Tässä työssä tarkoituksena oli tutkia marjojen puristusjäännöksen sisältämien flavonoidien uuttamista ja uuttautumiseen vaikuttavia tekijöitä kuten liuottimen koostumusta ja määrää, uuttolämpötilaa ja -aikaa sekä liuottimen lisäaineita. Uutteet analysointiin HPLC:lla käyttäen ODS-2-kolonnia ja gradienttieluointia. Komponentit detektoitiin niille tyypillisillä UV-VIS-aallonpituuksilla ja identifioitiin vertaamalla flavonoidiprofiililtaan tunnettujen marjojen kromatogrammeihin. Antosyaanien kvantitatiiviseen analysointiin käytettiin spektrofotometrista pH-eromenetelmää ja tulokset ilmoitettiin syanidiini-3-glukosidina. Uutteiden kestävyyttä tutkittiin säilyttämällä näytteitä eri olosuhteissa sekä lisäämällä niihin uuton saantoon ja komponenttien säilyvyyteen vaikuttavia aineita. Useimmat tutkituista hapoista paransivat flavonoidien saantoa ja uutteiden säilyvyyttä, mutta esim. askorbiinihapon lisääminen nopeutti tiettyjen uutteen sisältämien flavonoidien tuhoutumista.

Estudo da Determinação dos Grupos Silanóis ( º Si-OH) em Sílica: Aplicação à Materiais de Recheio Utilizados em Cromatografia Gasosa

A simple chemical method has been developed to quantify the silanol groups (º Si-OH) in silica as well as in coated chromatographic supports for use in packed - column Gas Chromatography. After adsorption of 10 mg/mL methylene blue, centrifuging action and filtration, the absorbance of the solution was inversely proportional to the silanol quantity. The difference between the absorbance of the pure solution and that of the solid - free filtrate was related to the silica weight, yielding a quantitative analysis of these groups.

Vidros recobertos com camadas delgadas transparentes de SnO2

SnO2 thin layers, prepared from aqueous colloidal suspensions by the sol-gel process, have been dip-coated on commercial borosilicate glasses. The effect of the conditions of deposition on the optical and structural characteristics of the thin layers was analysed by UV-Vis spectroscopy, x-ray reflectometry and electron scanning microscopy. Layers prepared with withdrawal speed in between 0.1 and 10cm/min show thickness smaller than 90nm, roughness of the order of 2nm and transmittance higher than 80%, resulting in good optical quality samples. The roughness increases from 2 to 11nm as the withdrawal speed increases from 10 to 80cm/min, what seems to be associated to the enlargement of the layers thickness (> 90nm). The measurements of mass loss, done after etching with fluoridric acid show that the coated samples are more corrosion resistant than the uncoated borosilicate glass.

Caracterização de ácidos húmicos de vermicomposto de esterco bovino compostado durante 3 e 6 meses

The main objective of this research was the characterization of the humic fractions isolated from vermicomposting, originating from cattle manure and treated with Eisenea foetida or Lumbricus rubellus, during 3 and 6 months. Elemental analysis and Infrared and UV-vis spectroscopy were used for their characterizations. The results obtained shown that both humic acids are very similar, but six-month humic acid shown lower percentage of organic material than three month humic acid. The spectroscopy analysis shown that the humic acid studied can be compared with other humic acids reported in the literature. By comparing both vermicomposts, the one produced in three months presents a great potential as fertilizer and it is more economical than the vermicompost produced during a six month period.

Investigação espectroscópica dos carbonilos heterobimetálicos cis-[Fe(CO)4(HgX)2], X = Cl, Br,I

The series of compounds cis-[Fe(CO)4(HgX)2], X=Cl,Br,I shows an octahedral geometry around the iron atom with the two HgX groups cis to each other. In this paper the assignment for the carbonyl stretching modes and the calculation of their force constants were performed on the basis of the Cotton-Krainhanzel model. Taking into account all the data from the IR, 199Hg NMR and UV-vis spectra it is possible to verify the influence of X on the electronic densities at the metallic centers.

Estudo comparativo sobre filtragem de sinais instrumentais usando transformadas de Fourier e Wavelet

A comparative study of the Fourier (FT) and the wavelet transforms (WT) for instrumental signal denoising is presented. The basic principles of wavelet theory are described in a succinct and simplified manner. For illustration, FT and WT are used to filter UV-VIS and plasma emission spectra using MATLAB software for computation. Results show that FT and WT filters are comparable when the signal does not display sharp peaks (UV-VIS spectra), but the WT yields a better filtering when the filling factor of the signal is small (plasma spectra), since it causes low peak distortion.

Distribuição de metais e caracterização das constantes de troca entre espécies metálicas e frações húmicas aquáticas de diferentes tamanhos moleculares

In this work the metal distribution and exchange constants between metal species and aquatic humic fractions with different molecular sizes were studied. The aquatic humic substances (AHS) were extracted by XAD-8 resin from water sample collected from Itapitanguí river, São Paulo State, Brazil. The AHS were fractionated in six fractions with different molecular sizes (>100 - <5 kDa) and characterized by several techniques. Molar ratios H/C suggested higher aromaticity for fractions F1 and F6 whereas molar ratios C/N didn´t show any differences regarding the humification degree between the fractions. The UV-Vis absorbance a254/a436 ratio showed higher results for F4 and F5, probably by less condensed features. FTIR studies showed high similarity in the functional groups in the fractions. The highest percentage of traces of Co, Al, Fe, Mn, Cu, Zn and Ni (determined by ICP-AES) was preferably complexed by fractions F3 and F4 with a greater amount of dissolved organic carbon (DOC). In addition, the exchange constants, determined by ultrafiltration method, showed complexes AHS-Fe and AHS-Al with higher stability than complexes AHS-Co in all fractions.

Dimensão fractal de ácidos húmicos em diferentes condições experimentais

The determination of fractal dimension (D) of humic particles was achieved by the turbidimetric technique where diluted suspensions of humic acids, in different experimental conditions, were analyzed by spectrophotometry UV-Vis. The slope of the lines (beta) was taken from the graphics (logtauvs loglambda) to obtain D. The results show that the values of D changed according to pH (3.0, 5.0 and 7.0), temperature (25 and 5 ºC) and shaking (magnetic and horizontal). In general, the value of D decreased with the increment of pH, increase of shaking and decrease of temperature.

Aplicação da CLAE para determinação do ácido 10-Hidróxi-2-decenóico (10-HDA) em geléia real pura e adicionada a mel brasileiro

The aim of this paper was to determine the 10-HDA in pure royal jelly and products containing royal jelly, using HPLC methodology. 10-HDA is the natural indicator of the presence of royal jelly in products and also gives the authenticity of pure royal jelly. The chromatographic conditions used were: isocratic system, C18-H column, auto sampler, diode array UV-VIS detector (225 nm), mobile phase with methanol/water (45:55), pH= 2.5 and a-naphtol as internal standard. The results obtained using laboratory samples for pure royal jelly were 2.37%, varying from 0.15% for honey with 10% of royal jelly to 2.10% for honey with 90% of royal jelly respectivelly. For commercial products, the 10-HDA content varied from no detectable to 0.026%. The recovery test presented a minumum of 100.44% The detection limit was 45.92 ng/mL and the quantification limit was 76.53 ng/mL.

Preparação e caracterização do complexo cobaloxima e sua utilização na construção de um eletrodo modificado: um experimento eletroquímico no curso de graduação

The present experiment describes the preparation, characterization of n-butyl(pyridil)cobaloxime complex and its electrochemical property. The infrared and uv-visible absorption spectra were used to characterize the complex obtained. The infrared spectrum of the compound showed characteristics bands that indicated the formation of the Co-C chemical bond formation. The electronic absorption spectrum in acetonitrile showed transition bands attributed to p-p*, metal-to-ligand charge transfer, d-d transitions and charge transfer Co-C. The electrochemical property was investigated by the pulse differential voltammetry technique. Two oxidation processes: Co(I)/Co(II) at -423 mV and Co(II)/Co(III) at 752 mV were observed.