One-pot two-step mechanochemical synthesis: ligand and complex preparation without isolating intermediates

Although the use of ball milling to induce reactions between solids (mechanochemical synthesis) can provide lower-waste routes to chemical products by avoiding solvent during the reaction, there are further potential advantages in using one-pot multistep syntheses to avoid the use of bulk solvents for the purification of intermediates. We report here two-step syntheses involving formation of salen-type ligands from diamines and hydroxyaldehydes followed directly by reactions with metal salts to provide the corresponding metal complexes. Five salen-type ligands 2,2'-[1,2-ethanediylbis[(E)-nitrilomethylidyne]] bisphenol, ` salenH2', 1; 2,2'-[(+/-)-1,2-cyclohexanediylbis-[(E)-nitrilomethylidyne]] bis-phenol, 2; 2,2'-[1,2-phenylenebis( nitrilomethylidyne)]-bis-phenol, ` salphenH2' 3; 2-[[(2-aminophenyl) imino] methyl]-phenol, 4; 2,2'-[(+/-)-1,2-cyclohexanediylbis[(E)-nitrilomethylidyne]]-bis[4,6-bis(1,1-dimethylethyl)]-phenol, ` Jacobsen ligand', 5) were found to form readily in a shaker-type ball mill at 0.5 to 3 g scale from their corresponding diamine and aldehyde precursors. Although in some cases both starting materials were liquids, ball milling was still necessary to drive those reactions to completion because precipitation of the product and or intermediates rapidly gave in thick pastes which could not be stirred conventionally. The only ligand which required the addition of solvent was the Jacobsen ligand 5 which required 1.75 mol equivalents of methanol to go to completion. Ligands 1-5 were thus obtained directly in 30-60 minutes in their hydrated forms, due to the presence of water by-product, as free-flowing yellow powders which could be dried by heating to give analytically pure products. The one-armed salphen ligand 4 could also be obtained selectively by changing the reaction stoichiometry to 1 : 1. SalenH(2) 1 was explored for the onepot two-step synthesis of metal complexes. In particular, after in situ formation of the ligand by ball milling, metal salts (ZnO, Ni(OAc)2 center dot 4H(2)O or Cu(OAc)(2)center dot H2O) were added directly to the jar and milling continued for a further 30 minutes. Small amounts of methanol (0.4-1.1 mol equivalents) were needed for these reactions to run to completion. The corresponding metal complexes [M(salen)] (M = Zn, 6; Ni, 7; or Cu, 8) were thus obtained quantitatively after 30 minutes in hydrated form, and could be heated briefly to give analytically pure dehydrated products. The all-at-once ` tandem' synthesis of [Zn(salen)] 6 was also explored by milling ZnO, ethylene diamine and salicylaldehyde together in the appropriate mole ratio for 60 minutes. This approach also gave the target complex selectively with no solvent needing to be added. Overall, these syntheses were found to be highly efficient in terms of time and the in avoidance of bulk solvent both during the reaction and for the isolation of intermediates. The work demonstrates the applicability of mechanochemical synthesis to one-pot multi-step strategies.

Serum amyloid A-related inflammation is lowered by increased fruit and vegetable intake, while high-sensitive C-reactive protein, IL-6 and E-selectin remain unresponsive

The present study assessed whether increased fruit and vegetable (F&V) intake reduced the concentrations of the inflammatory marker serum amyloid A (SAA) in serum, HDL2 and HDL3 and whether the latter reduction influenced any of the functional properties of these HDL subfractions. The present study utilised samples from two previous studies: (1) the FAVRIT (Fruit and Vegetable Randomised Intervention Trial) study - hypertensive subjects (systolic blood pressure (BP) range 140-190 mmHg; diastolic BP range 90-110 mmHg) were randomised to receive a 1-, 3- or 6-portion F&V/d intervention for 8 weeks, and (2) the ADIT (Ageing and Dietary Intervention Trial) study - older subjects (65-85 years) were randomised to receive a 2- or 5-portion F&V/d intervention for 16 weeks. HDL2 and HDL3 were isolated by rapid ultracentrifugation. Measurements included the following: serum high-sensitive C-reactive protein (hsCRP) by an immunoturbidimetric assay; serum IL-6 and E-selectin and serum-, HDL2- and HDL3-SAA by ELISA procedures; serum-, HDL2- and HDL3-cholesterol ester transfer protein (CETP) activity by a fluorometric assay. Although the concentrations of hsCRP, IL-6 and E-selectin were unaffected by increasing F&V intake in both studies (P>0·05 for all comparisons), those of SAA in HDL3 decreased in the FAVRIT cohort (P= 0·049) and those in HDL2 and HDL3 decreased in the ADIT cohort (P= 0·035 and 0·032), which was accompanied by a decrease in the activity of CETP in HDL3 in the FAVRIT cohort (P= 0·010) and in HDL2 in the ADIT cohort (P= 0·030). These results indicate that SAA responds to increased F&V intake, while other inflammatory markers remain unresponsive, and this leads to changes in HDL2 and HDL3, which may influence their antiatherogenic potential. Overall, the present study provides tangible evidence of the effectiveness of increased F&V intake, which may be of use to health policy makers and the general public.

Mineralogical Characteristics and Transformations during Long-Term Operation of a Zerovalent Iron Reactive Barrier

Design and operation of Fe⁰ permeable reactive barriers (PRBs) can be improved by understanding the long-term mineralogical transformations that occur within PRBs. Changes in mineral precipitates, cementation, and corrosion of Fe⁰ filings within an in situ pilot-scale PRB were examined after the first 30 months of operation and compared with results of a previous study of the PRB conducted 15 months earlier using X-ray diffraction and scanning electron microscopy employing energy dispersive X-ray and backscatter electron analyses. Iron (oxy)hydroxides, aragonite, and maghemite and/or magnetite occurred throughout the cores collected 30 mo after installation. Goethite, lepidocrocite, mackinawite, aragonite, calcite, and siderite were associated with oxidized and cemented areas, while green rusts were detected in more reduced zones. Basic differences from our last detailed investigation include (i) mackinawite crystallized from amorphous FeS, (ii) aragonite transformed into calcite, (iii) akaganeite transformed to goethite and lepidocrocite, (iv) iron (oxy)hydroxides and calcium and iron carbonate minerals increased, (v) cementation was greater in the more recent study, and (vi) oxidation, corrosion, and disintegration of Fe⁰ filings were greater, especially in cemented areas, in the more recent study. If the degree of corrosion and cementation that was observed from 15 to 30 mo after installation continues, certain portions of the PRB (i.e., up-gradient entrance of the ground water to the Fe⁰ section of the PRB) may last less than five more years, thus reducing the effectiveness of the PRB to mitigate contaminants.

Impact of sample preparation on mineralogical analysis of zero-valent iron reactive barrier materials

Permeable reactive barriers (PRBs) of zero-valent iron (Fe⁰) are increasingly being used to remediate contaminated ground water. Corrosion of Fe⁰ filings and tbe formation of precipitates can occur when the PRB material comes in contact with ground water and may reduce the lifespan and effectiveness of the barrier. At present, there are no routine procedures for preparing and analyzing the mineral precipitates from Fe⁰ PRB material. These procedures are needed because mineralogical composition of corrosion products used to interpret the barrier processes can change with iron oxidation and sample preparation. The objectives of this study were (i) to investigate a method of preparing Fe⁰ reactive barrier material for mineralogical analysis by X-ray diffraction (XRD), and (ii) to identify Fe mineral phases and rates of transformations induced by different mineralogical preparation techniques. Materials from an in situ Fe⁰ PRB were collected by undisturbed coring and processed for XRD analysis after different times since sampling for three size fractions and by various drying treatments. We found that whole-sample preparation for analysis was necessary because mineral precipitates occurred within the PRB material in different size fractions of the samples. Green rusts quickly disappeared from acetone-dried samples and were not present in air-dried and oven-dried samples Maghemite/magnetite content increased over time and in oven-dried samples, especially after heating to 105°C. We conclude that care must be taken during sample preparation of Fe⁰ PRB material, especially for detection of green rusts, to ensure accurate identification of minerals present within the barrier system.

Repeated measures of body mass index and C-reactive protein in relation to all-cause mortality and cardiovascular disease: Results from the consortium on health and ageing network of cohorts in Europe and the United States (CHANCES)

Obesity has been linked with elevated levels of C-reactive protein (CRP), and both have been associated with increased risk of mortality and cardiovascular disease (CVD). Previous studies have used a single ‘baseline’ measurement and such analyses cannot account for possible changes in these which may lead to a biased estimation of risk. Using four cohorts from CHANCES which had repeated measures in participants 50 years and older, multivariate time-dependent Cox proportional hazards was used to estimate hazard ratios (HR) and 95 % confidence intervals (CI) to examine the relationship between body mass index (BMI) and CRP with all-cause mortality and CVD. Being overweight (≥25–<30 kg/m2) or moderately obese (≥30–<35) tended to be associated with a lower risk of mortality compared to normal (≥18.5–<25): ESTHER, HR (95 % CI) 0.69 (0.58–0.82) and 0.78 (0.63–0.97); Rotterdam, 0.86 (0.79–0.94) and 0.80 (0.72–0.89). A similar relationship was found, but only for overweight in Glostrup, HR (95 % CI) 0.88 (0.76–1.02); and moderately obese in Tromsø, HR (95 % CI) 0.79 (0.62–1.01). Associations were not evident between repeated measures of BMI and CVD. Conversely, increasing CRP concentrations, measured on more than one occasion, were associated with an increasing risk of mortality and CVD. Being overweight or moderately obese is associated with a lower risk of mortality, while CRP, independent of BMI, is positively associated with mortality and CVD risk. If inflammation links CRP and BMI, they may participate in distinct/independent pathways. Accounting for independent changes in risk factors over time may be crucial for unveiling their effects on mortality and disease morbidity.

Cytokine and C-reactive protein profiles induced by porcine circovirus type 2 experimental infection in 3-week-old piglets

The purpose of this study was to determine serum profiles of cytokines at a protein level and Creactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a 35-day period in 10 piglets experimentally infected with PCV2 at 3 weeks of age. Four of the infected piglets developed severe PMWS at 14 to 21 days post-infection (d.p.i.) and died prior to termination of the experiment. The remaining six PCV2-infected piglets experienced transient fever, but did not display overt clinical signs of PMWS and were considered as subclinically infected. A bioassay was used to detect IL-6 and ELISAs were used to detect IFN-alpha, IL-10, and CRP. There were no significant differences in cytokine or CRP expression from 0 to 7 d.p.i. between the PMWS-affected and the subclinically infected piglets. Levels of IL-10 and CRP were elevated from 10 and 14 d.p.i. respectively in the PMWS-affected piglets compared to the subclinically infected piglets. There were no significant differences in IFN-alpha and IL-6 expression between the PMWS-affected piglets and the subclinically infected piglets. The present study shows that elevated levels of serum CRP and IL-10 were associated with PCV2-infected piglets that subsequently developed severe PMWS. This may help to provide further insight into the immunoaetiogenesis of this syndrome.

A field and reactive transport model study of arsenic in a basaltic rock aquifer

The use of geothermal energy as a source for electricity and district heating has increased over recent decades. Dissolved As can be an important constituent of the geothermal fluids brought to the Earth's surface. Here the field application of laboratory measured adsorption coefficients of aqueous As species on basaltic glass surfaces is discussed. The mobility of As species in the basaltic aquifer in the Nesjavellir geothermal system, Iceland was modelled by the one-dimensional (1D) reactive transport model PHREEQC ver. 2, constrained by a long time series of field measurements with the chemical composition of geothermal effluent fluids, pH, Eh and, occasionally, Fe- and As-dissolved species measurements. Di-, tri- and tetrathioarsenic species (As(OH)S22-, AsS₃H^2-, AsS33- and As(SH)4-) were the dominant form of dissolved As in geothermal waters exiting the power plant (2.556μM total As) but converted to some extent to arsenite (H₃AsO₃) and arsenate HAsO42- oxyanions coinciding with rapid oxidation of S2- to S2O32- and finally to SO42- during surface runoff before feeding into a basaltic lava field with a total As concentration of 0.882μM following dilution with other surface waters. A continuous 25-a data set monitoring groundwater chemistry along a cross section of warm springs on the Lake Thingvallavatn shoreline allowed calibration of the 1D model. Furthermore, a series of ground water wells located in the basaltic lava field, provided access along the line of flow of the geothermal effluent waters towards the lake. The conservative ion Cl^- moved through the basaltic lava field (4100m) in less than10a but As was retarded considerably due to surface reactions and has entered a groundwater well 850m down the flow path as arsenate in accordance to the prediction of the 1D model. The 1D model predicted a complete breakthrough of arsenate in the year 2100. In a reduced system arsenite should be retained for about 1ka. © 2011 Elsevier Ltd.

Developmental Antecedents of Proactive and Reactive Rebelliousness: The Role of Parenting Style, Childhood Adversity, and Attachment

Adolescence is a time of developmental transition that for one in five young people is characterised by feelings of oppositionality, rebellion, and negativism. Despite the prevalent experience of teen turbulence and despite its significance within the phenomenological framework provided by reversal theory (RT), the childhood antecedents of rebelliousness in adolescence and adulthood have not been given extensive empirical attention within RT, although such work has been carried out using other constructs and theories. We examined recalled parenting style, childhood adversity, and attachment style in adulthood as correlates of proactive and reactive rebelliousness in a sample of 80 participants, aged 18 to 50 years. Each participant responded to a questionnaire package containing the revised Adult Attachment Scale, the Parental Bonding Instrument, the Childhood Experience of Care and Abuse Questionnaire, and the Negativism Dominance Scale. We found that paternal abusive parenting, followed by paternal parenting style, paternal neglect, and paternal antipathy were independently predictive of scores on proactive rebelliousness, the sensation-seeking form. Maternal and paternal indifferent parenting styles each were found to equivalently and independently predict scores on reactive rebelliousness, the interpersonal disaffection form. The results of this study suggest these two forms of rebelliousness may have distinctly different antecedents. A longitudinal study is needed to examine the potentially causal pathways that are suggested by the results of this cross-sectional research. We consider reversal theory explanations of these results and contrast them with complementary theoretical frameworks.

Optimisation of an aptamer-biosensor to detect C-reactive protein

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009

Study of transcripts and reactive oxygen species involved in the defence responses of Quercus suber against Phytophthora cinnamomi

The present work has the merit of exploring an insight into the activation of defence genes of Quercus suber during response to infection by Phytophthora cinnamomi. Thus, cDNA-AFLP methodology was used to identify gene fragments differentially present in the mRNA profiles of host cells of micropropagated Q. suber plantlets roots infected with zoospores of P. cinnamomi at different post challenge time points. Six candidate genes were selected based on their interesting cDNA-AFLP expression patterns and homology to genes known to play a role in defence. These six genes encode a cinnamyl alcohol dehydrogenase 2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), thaumatin-like protein (QsTLP), chitinase (QsCHI) and a 1,3-beta glucanase (QsGLU). The current work has been successful in evaluation of the expression of these genes by qRT-PCR. Data analysis revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the early hours of inoculation, while transcript profiles of thaumatin-like protein showed decreasing. No expression was detected for 1,3-beta-glucanase (QsGLU). Furthermore, the choice of suitable reference genes in any new experimental system is absolutely crucial in qRT-PCR; for this reason in this study and for the first time a set of potential reference genes were analyzed and validated for qRT-PCR normalization in the patho-system Phytophthora-Q. suber. Four candidate reference genes polimerase II (QsRPII), eukaryotic translation initiation factor 5A(QsEIF-5A), b-tubulin (QsTUB) and a medium subunit family protein of Clathrin adaptor complexes (QsCACs) were evaluated to determine the most stable internal references in Q. suber. Analysis of stability of genes was carried out using Genex software. Results indicated all these four potential reference genes assumed stable expression. Data analysis revealed that QsRPII and QsCACs were the two most stable genes, while genes QsTUB and QsEIF-5A were the third and the fourth most stable gene, respectively. In this study, a plasmid-based quantitative PCR method was developed to measure P. cinnamomi colonization during infection process of Q. suber. Plasmid-based detection of P. cinnamomi showed a gradual accumulation of the pathogen DNA in cork oak root tips up to 24 h post infection. The higher increase in P. cinnamomi/plasmid DNA ratio occurred between 18 and 24 h. One of the primary objectives of this research was to study the effect of cinnamomins (elicitins secreted by P. cinnamomin) on inducing defence mechanism against the pathogen, as recent histological and ultra-structural studies showed that P. cinnamomi was restricted to the outer cortex root fragments pre-treated with capsicien and cryptogein, suggesting that elicitins can stimulate plant defence reactions against P. cinnamomi. To complement these studies and to have a clear view of the nature of the interaction, the role of cinnamomins in the production of the oxidative burst [ROS and ROS scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)] and in the defence responses was evaluated. Cork oak seedlings were pretreated with alpha-cinnamomin and then inoculated with P. cinnamomi mycelia. Results showed a significant higher production of reactive oxygen species (ROS) (H2O2 and O2•-) in elicitin and non-elicitin treated roots in interaction with P. cinnamomi in comparison to the corresponding control. The plant group inoculated with the pathogen after cinnamomin treatment showed an earlier increase in H2O2 production but this was lower as compared with that group inoculated with P. cinnamomi alone. Also, in elicitin pre-treated group generally, a lower level of O2•− production during infection was observed as compared with inoculated roots with P. cinnamomi alone without elicitin treatment. Furthermore, in this study, we evaluated activities of antioxidant enzymes upon challenge with P. cinnamomi, with and without pretreatment with alpha cinnamomin. Results indicated that the activities of defense enzymes POD, SOD and CAT increased after P. cinnamomi inoculation when compared with those in the control group. Also, in the group treated with alpha-cinnamomin followed by P. cinnamomi inoculation, a higher level of enzymatic activities was detected as compared with elicitin non-treated group, which suggest the protective effect of alpha-cinnamomin against the pathogen due to higher elevated levels of defense enzymes POD, SOD and CAT during the infection period. Furthermore, a sensitive qPCR method was applied to measure the pathogen biomass in elicited and non-elicited Q. suber roots challenged with P. cinnamomi to elucidate the effect of cinnamomins on the colonization of P. cinnamomi. Plasmid-based quantification of P. cinnamomi showed a significant decrease in accumulation of the pathogen DNA in cork oak roots after treatment with alpha and beta-cinnamomins which attest the role of cinnamomins in promoting defense responses in cork oak against P. cinnamomi invasion.

New synthetic methodologies for the transformation of biomass derived intermediates to valuable molecules

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014