Revisão sobre fármacos quirais e a sua importância na medicina atual

Aspects are discussed on the chirality of drugs and their action in the human body, the benefits of using a drug in enantiomerically pure form and why, even today, despite the risks, many drugs are marketed as racemic mixture. Among the methods of separation there is the High Performance Liquid Chromatography (HPLC) which can be given in different ways using chiral additives in the mobile phase as in the system of ligand exchange in the ion pair system and the system of cavity or inclusion. Note also the wide variety of chiral stationary phases available in the market that allow high specificity using them according to the need and purpose of the method. The review of the topic is extremely important since it is a matter of public interest world that brings into play issues and financial policies

Study of photorespiration in marine microalgae through the determination of glycolic acid using hydrophilic interaction liquid chromatography

Determination of organic acids in intracellular extracts and in the cultivation media of marine microalgae aid investigations about metabolic routes related to assimilation of atmospheric carbon by these organisms, which are known by their role in the carbon dioxide sink. The separation of these acids was investigated by hydrophilic interaction liquid chromatography (HILIC) using isocratic elution with a mobile phase composed of 70: 30 v/v acetonitrile/20 mmol/L ammonium acetate buffer (pH 6.8) and detection at 220 nm. HILIC allowed the determinations of glycolic acid, the most important metabolite for the evaluation of the photorespiration process in algae, to be made with better selectivity than that achieved by reversed phase liquid chromatography, but with less detectability. The concentration of glycolic acid was determined in the cultivation media and in intracellular extracts of the algae Tetraselmis gracilis and Phaeodactylum tricornutum submitted to different conditions of aeration: (i) without forced aeration, (ii) aeration with atmospheric air, and (iii) bubbling with N(2). The concentration of glycolic acid had a higher increase as the cultures were aerated with nitrogen, showing higher photorespiratory flux than that occurring in the cultures aerated with atmospheric air.

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r a parts per thousand yenaEuro parts per thousand 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r a parts per thousand yenaEuro parts per thousand 0.9977) and 6-ODD (r a parts per thousand yenaEuro parts per thousand 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

Studying Electrophoretic Separations Using Cholate Micelles: Effects Of Ph, Temperature, And Composition

Micelle-forming bile salts have previously been shown to be effective pseudo-stationary phases for separating the chiral isomers of binaphthyl compounds with micellar electrokinetic capillary chromatography (MEKC). Here, cholate micelles are systematically investigated via electrophoretic separations and NMR using R, S-1, 1¿- binaphthyl- 2, 2¿-diylhydrogenphosphate (BNDHP) as a model chiral analyte. The pH, temperature, and concentration of BNDHP were systematically varied while monitoring the chiral resolution obtained with MEKC and the chemical shift of various protons in NMR. NMR data for each proton on BNDHP is monitored as a function of cholate concentration: as cholate monomers begin to aggregate and the analyte molecules begin to sample the micelle aggregate we observe changes in the cholate methyl and S-BNDHP proton chemical shifts. From such NMR data, the apparent CMC of cholate at pH 12 is found to be about 13-14 mM, but this value decreases at higher pH, suggesting that more extreme pHs may give rise to more effective separations. In general, CMCs increase with temperature indicating that one may be able to obtain better separations at lower temperatures. S-BNDHP concentrations ranging from 50 ¿M to 400 ¿M (pH 12.8) gave rise to apparent cholate CMC values from 10 mM to 8 mM, respectively, indicating that S-BNDHP, the chiral analyte molecule, may play an active role in stabilizing cholate aggregates. In all, these data show that NMR can be used to systematically investigate a complex multi-variable landscape of potential optimizations of chiral separations.

Strong Down-Valley Low-Level Jets over the Atacama Desert: Observational Characterization

The near-surface wind and temperature regime at three points in the Atacama Desert of northern Chile is described using two-year multi-level measurements from 80-m towers located in an altitude range between 2100 and 2700 m ASL. The data reveal the frequent development of strong nocturnal drainage flows at all sites. Down-valley nose-shaped wind speed profiles are observed with maximum values occurring at heights between 20 m and 60 m AGL. The flow intensity shows considerable inter-daily variability and a seasonal modulation of maximum speeds, which in the cold season can attain hourly average values larger than 20 m s−1. Turbulent mixing appears significant over the full tower layer, affecting the curvature of the nighttime temperature profile and possibly explaining the observed increase of surface temperatures in the down-valley direction. Nocturnal valley winds and temperatures are weakly controlled by upper-air conditions observed at the nearest aerological station. Estimates of terms in the momentum budget for the development and the quasi-stationary phases of the down-valley flows suggest that the pressure gradient force due to the near-surface cooling along the sloping valley axes plays an important role in these drainage flows. A scale for the jet nose height of equilibrium turbulent down-slope jets is proposed, based on surface friction velocity and surface inversion intensity. At one of the sites this scale explains about 70% of the case-to-case observed variance of jet nose heights. Further modeling and observational work is needed, however, in order to better define the dynamics, extent and turbulence structure of this flow system, which has significant wind-energy, climatic and environmental implications.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

The mechanical properties of Saccharomyces cerevisiae

Cell-wall mechanical properties play an integral part in the growth and form of Saccharomyces cerevisiae. In contrast to the tremendous knowledge on the genetics of S. cerevisiae, almost nothing is known about its mechanical properties. We have developed a micromanipulation technique to measure the force required to burst single cells and have recently established a mathematical model to extract the mechanical properties of the cell wall from such data. Here we determine the average surface modulus of the S. cerevisiae cell wall to be 11.1 ± 0.6 N/m and 12.9 ± 0.7 N/m in exponential and stationary phases, respectively, giving corresponding Young's moduli of 112 ± 6 MPa and 107 ± 6 MPa. This result demonstrates that yeast cell populations strengthen as they enter stationary phase by increasing wall thickness and hence the surface modulus, without altering the average elastic properties of the cell-wall material. We also determined the average breaking strain of the cell wall to be 82% ± 3% in exponential phase and 80% ± 3% in stationary phase. This finding provides a failure criterion that can be used to predict when applied stresses (e.g., because of fluid flow) will lead to wall rupture. This work analyzes yeast compression experiments in different growth phases by using engineering methodology.

Temperature effects in hydrophobic interaction chromatography.

The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.

Studies on survival of Pseudomonas aeruginosa 6750

The growth of Pseudomonas aeruginosa 6750 as a biofilm was investigated using a novel system based on that of Gilbert et al (1989). The aim was to test the effect of controlled growth of the organism on antibiotic susceptibility and examine the survival of the organism as a biofilm. During the investigations it became clear that, because of the increasing growth of P.aeruginosa and production of exopolysaccharide, a growth rate controlled monolayer could not be achieved and so the method was not used further. The data, however, showed that there was an increase in the smooth colony type of the organism during growth. Investigations were focused on the survival of P.aeruginosa in batch and chemostat studies. Survival or percentage culturability, as measured by total and colony count ratio, was found to decrease both in extended batch culture and for chemostat cells with decreasing growth rate. Extended batch culture, however, did not exhibit further increases in resistance to ciprofloxacin and polymyxin B. Survival was also measured using other parameters namely the direct viable count, vital staining, effect of temperature downshift and measurement of lag. In batch culture, the most notable change was a decrease in cell size along the growth curve. This was accompanied by an increase in the cellular protein content. Protein per volume was calculated from the data which showed a marked increase in batch culture, which was not demonstrated for chemostat cells with decreasing growth rate. Outer membrane protein profiles were obtained for batch and chemostat cells. An LPS profile of batch culture cells was also demonstrated. In general, there was little difference in the outer membrane protein profiles of cells from early and late stationary phases.The result of the LPS profile showed that there appeared to be an increase in the B-band of the region of the LPS in the older stationary phase cultures.

Improved production of industrially relevant recombinant proteins: application of multi-factorial design of experiments and scalable process modelling

The work described in this thesis focuses on the use of a design-of-experiments approach in a multi-well mini-bioreactor to enable the rapid establishments of high yielding production phase conditions in yeast, which is an increasingly popular host system in both academic and industrial laboratories. Using green fluorescent protein secreted from the yeast, Pichia pastoris, a scalable predictive model of protein yield per cell was derived from 13 sets of conditions each with three factors (temperature, pH and dissolved oxygen) at 3 levels and was directly transferable to a 7 L bioreactor. This was in clear contrast to the situation in shake flasks, where the process parameters cannot be tightly controlled. By further optimisating both the accumulation of cell density in batch and improving the fed-batch induction regime, additional yield improvement was found to be additive to the per cell yield of the model. A separate study also demonstrated that improving biomass improved product yield in a second yeast species, Saccharomyces cerevisiae. Investigations of cell wall hydrophobicity in high cell density P. pastoris cultures indicated that cell wall hydrophobin (protein) compositional changes with growth phase becoming more hydrophobic in log growth than in lag or stationary phases. This is possibly due to an increased occurrence of proteins associated with cell division. Finally, the modelling approach was validated in mammalian cells, showing its flexibility and robustness. In summary, the strategy presented in this thesis has the benefit of reducing process development time in recombinant protein production, directly from bench to bioreactor.

Synthesis and characterization of silica-supported L-Lysine-based dendritic branches

Two series of novel modified silicas have been prepared in which individual dendritic branches have been attached to aminopropylsilica using standard peptide coupling methodology. The dendritic branches are composed of enantiomerically pure l-lysine building blocks, and hence, the modified silicas have the potential to act as chiral stationary phases in chromatography. In one series of modified silicas, the surface of the dendritic branch consists of Boc carbamate groups, whereas the other has benzoyl amide surface groups. Different coupling reagents have been investigated in order to maximize the loading onto the solid phase. The new supported dendritic materials have been fully characterized with properties of the bulk material determined by elemental analysis, 13C NMR, and IR spectroscopy, whereas XPS provides important information about the surface of the modified silica exposed to the incident X-rays, the key region in which potential chromatographic performance of these materials will take place. Although the bulk analyses indicate that loading of the dendritic branch onto silica decreases with increasing dendritic generation (and consequently steric bulk), XPS indicates that the optimum surface coverage is actually obtained at the second generation of dendritic growth.

Characterization of water-soluble organic compounds in bioaerosols by liquid chromatography and fluorescence spectroscopy

In the last decades, the effects of the air pollution have been increasing, especially in the case of the human health diseases. In order to overcome this problem, scientists have been studying the components of the air. As a part of water-soluble organic compounds, amino acids are present in the atmospheric environment as components of diverse living organisms which can be responsible for spreading diseases through the air. Liquid chromatography is one technique capable of distinguish the different amino acids from each other. In this work, aiming at separating the amino acids found in the aerosols samples collected in Aveiro, the ability of four columns (Mixed-Mode WAX-1, Mixed-Mode HILIC-1, Luna HILIC and Luna C18) to separate four amino acids (aspartic acid, lysine, glycine and tryptophan) and the way the interaction of the stationary phases of the columns with the analytes is influenced by organic solvent concentration and presence/concentration of the buffer, are being assessed. In the Mixed-Mode WAX-1 column, the chromatograms of the distinct amino acids revealed the separation was not efficient, since the retention times were very similar. In the case of lysine, in the elution with 80% (V/V) MeOH, the peaks appeared during the volume void. In the Mixed-Mode HILIC-1 column, the variation of the organic solvent concentration did not affect the elution of the four studied amino acids. Considering the Luna HILIC column, the retention times of the amino acids were too close to each other to ensure a separation among each other. Lastly, the Luna C18 column revealed to be useful to separate amino acids in a gradient mode, being the variation of the mobile phase composition in the organic solvent concentration (ACN). Luna C18 was the column used to separate the amino acids in the real samples and the mobile phase had acidified water and ACN. The gradient consisted in the following program: 0 – 2 min: 5% (V/V) ACN, 2 – 8 min: 5 – 2 % (V/V) ACN, 8 – 16 min: 2% (V/V) ACN, 16 – 20 min: 2 – 20 % (V/V) ACN, 20 – 35 min: 20 – 35 % (V/V) ACN. The aerosols samples were collected by using three passive samplers placed in two different locations in Aveiro and each sampler had two filters - one faced up and the other faced down. After the sampling, the water-soluble organic compounds was extracted by dissolution in ultra-pure water, sonication bath and filtration. The resulting filtered solutions were diluted in acidified water for the chromatographic separation. The results from liquid chromatography revealed the presence of the amino acids, although it was not possible to identify each one of them individually. The chromatograms and the fluorescence spectra showed the existence of some patterns: the samples that correspond to the up filters had more intense peaks and signals, revealing that the up filters collected more organic matter.

In vitro activity of gallium maltolate against Staphylococci in logarithmic, stationary, and biofilm growth phases: comparison of conventional and calorimetric susceptibility testing methods.

Ga(3+) is a semimetal element that competes for the iron-binding sites of transporters and enzymes. We investigated the activity of gallium maltolate (GaM), an organic gallium salt with high solubility, against laboratory and clinical strains of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), methicillin-susceptible Staphylococcus epidermidis (MSSE), and methicillin-resistant S. epidermidis (MRSE) in logarithmic or stationary phase and in biofilms. The MICs of GaM were higher for S. aureus (375 to 2000 microg/ml) than S. epidermidis (94 to 200 microg/ml). Minimal biofilm inhibitory concentrations were 3,000 to >or=6,000 microg/ml (S. aureus) and 94 to 3,000 microg/ml (S. epidermidis). In time-kill studies, GaM exhibited a slow and dose-dependent killing, with maximal action at 24 h against S. aureus of 1.9 log(10) CFU/ml (MSSA) and 3.3 log(10) CFU/ml (MRSA) at 3x MIC and 2.9 log(10) CFU/ml (MSSE) and 4.0 log(10) CFU/ml (MRSE) against S. epidermidis at 10x MIC. In calorimetric studies, growth-related heat production was inhibited by GaM at subinhibitory concentrations; and the minimal heat inhibition concentrations were 188 to 4,500 microg/ml (MSSA), 94 to 1,500 microg/ml (MRSA), and 94 to 375 microg/ml (MSSE and MRSE), which correlated well with the MICs. Thus, calorimetry was a fast, accurate, and simple method useful for investigation of antimicrobial activity at subinhibitory concentrations. In conclusion, GaM exhibited activity against staphylococci in different growth phases, including in stationary phase and biofilms, but high concentrations were required. These data support the potential topical use of GaM, including its use for the treatment of wound infections, MRSA decolonization, and coating of implants.

Shared information in stationary states of stochastic processes

We present four estimators of the shared information (or interdepency) in ground states given that the coefficients appearing in the wave function are all real non-negative numbers and therefore can be interpreted as probabilities of configurations. Such ground states of Hermitian and non-Hermitian Hamiltonians can be given, for example, by superpositions of valence bond states which can describe equilibrium but also stationary states of stochastic models. We consider in detail the last case, the system being a classical not a quantum one. Using analytical and numerical methods we compare the values of the estimators in the directed polymer and the raise and peel models which have massive, conformal invariant and nonconformal invariant massless phases. We show that like in the case of the quantum problem, the estimators verify the area law with logarithmic corrections when phase transitions take place.