Upravljanje ljudskim potencijalima u funkciji učinkovitosti policijske organizacije – percepcija policijskih rukovoditelja

Upravljanje ljudskim potencijalima u funkciji učinkovitosti policijske organizacije – percepcija policijskih rukovoditelj

Koncentracije ostataka veterinarskih lijekova u mlijeku s individualnih farmi u Hrvatskoj

A total of 119 raw milk samples collected at individual small milk-producing facilities and collection tanks of milk routes from five counties of east and north continental Croatia were examined for chloramphenicol, sulfonamides, tetracyclines, gentamicin, streptomycin, dihydrostreptomycin, flumequine and enrofloxacin from January to March of 2011. Immunoassay methods used for drug determination were validated according to the guidelines laid down by European Commission Decision 2002/657/EC. Data indicated that the methods are appropriate for the detection of antibiotics measured. Measured mean values (μg L-1) of antibiotics were: 0.005 for chloramphenicol, 3.67 for sulfonamides, 2.83 for tetracyclines, 1.10 for gentamicin, 2.64 for streptomycin, 7.67 for dihydrostreptomycin, 10.4 for flumequine and 4.11 for enrofloxacin. None of samples analyzed showed the presence of veterinary drug residues above the maximum residues levels (MRLs) established by European Union and Croatian legislation. The calculated estimated daily intakes (EDIs) for the average daily milk consumption of 300 mL for an adult in Croatia for examined antibiotics showed levels 20 to 1640 times lower than the values of acceptable daily intakes (ADIs) fixed by European Medicines Agency and World Health Organization. This suggested that toxicological risk associated with the consumption of analysed milk could not be considered a public health issue with regards to these veterinary drugs.Tijekom 3 mjeseca analizirano je ukupno 119 uzoraka mlijeka na veterinarske lijekove: kloramfenikol, sulfonamide, tetraciklin, gentamicin, streptomicin, dihidrostreptomicin, flumekin i enrofloksacin. Imunoenzimske metode korištene za određivanje veterinarskih lijekova validirane su prema odredbama propisanim Odlukom Europske komisije 2002/657/ EC. Rezultati validacije metoda pokazuju da su primijenjene metode prikladne za tu namjenu. Ni u jednom uzorku mlijeka nije utvrđena koncentracija ostataka veterinarskih lijekova iznad najviših dopuštenih količina (NDK) utvrđenih Europskom legislativom. Izračunate procjene unosa određivanih lijekova mlijekom su za 20 do 1640 puta niže od zadanih prihvatljivih dnevnih količina unosa. Prema dobivenim rezultatima može se zaključiti da određivani veterinarski lijekovi ne predstavljaju toksikološki rizik za potrošače s obzirom na rezultate analize mlijeka na ostatke antibiotika s malih farmi

Ectoine can enhance structural changes in DNA in vitro

Strand breaks and conformational changes of DNA have consequences for the physiological role of DNA. The natural protecting molecule ectoine is beneficial to entire bacterial cells and biomolecules such as proteins by mitigating detrimental effects of environmental stresses. It was postulated that ectoine-like molecules bind to negatively charged spheres that mimic DNA surfaces. We investigated the effect of ectoine on DNA and whether ectoine is able to protect DNA from damages caused by ultraviolet radiation (UV-A). In order to determine different isoforms of DNA, agarose gel electrophoresis and atomic force microscopy experiments were carried out with plasmid pUC19 DNA. Our quantitative results revealed that a prolonged incubation of DNA with ectoine leads to an increase in transitions from supercoiled (undamaged) to open circular (single-strand break) conformation at pH 6.6. The effect is pH dependent and no significant changes were observed at physiological pH of 7.5. After UV-A irradiation in ectoine solution, changes in DNA conformation were even more pronounced and this effect was pH dependent. We hypothesize that ectoine is attracted to the negatively charge surface of DNA at lower pH and therefore fails to act as a stabilizing agent for DNA in our in vitro experiments

Kontrola ostataka levamisola u mlijeku primjenom validirane metode tekućinske kromatografije-tandemske-spektrometrije masa

Concentrations of the anthelmintic agent levamisole were measured in a total of 85 raw cow milk samples collected during 2014 from dairy farms across Croatia. The liquid chromatographytandem mass spectrometry (LC-MS/MS) method used for levamisole quantification was validated according to the criteria set by Commission Decision 2002/657/EC. The following validation parameters were determined: limit of decision (CCα) 0.55 μg kg-1, detection of capability (CCβ) 0.59 μg kg-1, limit of detection (LOD) 0.06 μg kg-1, limit of quantification (LOQ) 0.22 μg kg-1, precision and accuracy (expressed as recovery) 97.3-100 %, intra-laboratory reproducibility (RSD) 4.2-5.6 %. Levamisole levels for 45 of the total of 85 samples were below the LOD value (0.06 µg kg-1). In the remaining 40 milk samples, levamisole was measured in the range from 0.061 to 0.142 µg kg-1 and the mean value was 0.092 µg kg-1. Accordingly, all concentrations analysed were below the LOQ value (0.22 µg kg-1) and limit of decision (CCα) of the method used.Koncentracije anthelmintika levamisola određene su u ukupno 85 uzoraka sirovog kravljeg mlijeka prikupljenih tijekom 2014. na mliječnim farmama iz cijele Hrvatske. Za kvantifikaciju levamisola korištena je metoda tekućinske kromatografije-tandemske- spektrometrije masa (LC-MS/MS) koja je validirana u skladu s kriterijima utvrđenim Odlukom Komisije 2002/657/EZ. Utvrđeni su sljedeći parametri validacije: granična koncentracija određivanja (CCα) 0,55 ug kg-1, sposobnost dokazivanja (CCβ) 0,59 ug kg-1, granica određivanja (LOD) 0,06 ug kg-1, granica kvantifikacije (LOQ) 0,22 ug kg-1, preciznost i točnost (izraženi kao iskorištenje) 97,3- 100,0 %, unutarlaboratorijska obnovljivost (RSD) 4,2- 5,6 %. Koncentracije levamisola za 45 od ukupno 85 uzoraka bile su ispod vrijednosti LOD (0,06 ug kg-1). U preostalih 40 uzoraka mlijeka, levamisol je izmjeren u rasponu od 0,061 do 0,142 ug kg-1, a srednja vrijednost je bila 0,092 ug kg-1. Zaključno, sve koncentracije analizirane su ispod LOQ vrijednosti (0,22 ug kg-1) i CCα, granične koncentracije metode

Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods

Combined influence of ectoine and salt: spectroscopic and numerical evidence for compensating effects on aqueous solutions

Ectoine is an important osmolyte, which allows microorganisms to survive in extreme environmental salinity. The hygroscopic effects of ectoine in pure water can be explained by a strong water binding behavior whereas a study on the effects of ectoine in salty solution is yet missing. We provide Raman spectroscopic evidence that the influence of ectoine and NaCl are opposing and completely independent of each other. The effect can be explained by the formation of strongly hydrogen-bonded water molecules around ectoine which compensate the influence of the salt on the water dynamics. The mechanism is corroborated by first principles calculations and broadens our understanding of zwitterionic osmolytes in aqueous solution. Our findings allow us to provide a possible explanation for the relatively high osmolyte concentrations in halotolerant bacteria

Structure and Bonding in Some Platinum Complexes

In this thesis the technique of X-ray crystal structure analysis is applied to the study of molecular structure, conformation and bonding in some platinum(ll) and platinum(I) complexes. The contents are divided into three parts. In Part I some of the theoretical and practical aspects of the X-ray diffraction methods, pertinent to the work described in Parts II and III, are surveyed. Part II, which is presented in six Chapters, is concerned with the structures of eight square-planar platinum(ll) complexes. Current views on trans- and cis-influence of ligands and some aspects of metal-phosphorus bonding are first reviewed (Chapter 1). This is followed by a description of the crystal structure analyses of three platinum(II) complexes containing the novel ligand PMe2C6F5 (Chapter 2). The interest is centred on the effect of electron-withdrawal of phosphine substituents on the metal-ligand bonding. Chapter 3 is devoted to the structure analyses of the complexes cis-PtCl2(PEt3)L, where L=PEt3 and CO. This work completes a systematic study of such complexes and the results are discussed in terms of the cis and trans-influence of the ligands. The constancy of the observed triethyl-phosphine conformation in square-planar platinum complexes has led to molecular mechanics calculations on the triethylphosphine molecule, which are also described. In Chapter 4 the structure of a platinum(II) complex which provides the first known example of a metallated phosphine-carborane is described. The interest in the effect of strongly electron-withdrawing substituents on sulphur- donor atom on metal-ligand bonding has led to determination of the crystal structure of cis-PtCl2(CF3SCH2CHMeSCF3), presented in Chapter 5. Chapter 6 is devoted to the structure analysis of trans-[PtCl(COEt)(PMe2Ph)2 . This complex displays an unusually large 1J(Pt-P) coupling constant. The X-ray study was carried out in order to examine the correlation between Pt-P bond lengths and coupling constants in bridged binuclear platinum(II) species. Part III is concerned with the structure analyses of two closely similar and novel platinum(l) complexes, which contain direct metal-ligand bonds

Molecular Mechanisms

Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free- vibrational density of states of water due to ectoine, as well as the use of ectoine as an hydroxyl-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR)

Design and optimization of cationic hydrophobic polymer-based delivery systems for nucleic acids

There is high interest in the therapeutic delivery of nucleic acids, as it offers considerable potential for the treatment of genetic diseases, cancer therapy and vaccination. Due to their degradable and highly charged nature, suitable carriers are required for delivery of nucleic acids into targeted cells. The carrier should protect the nucleic acids from degradation, possess high stability, facilitate crossing of cell membranes, release the nucleic acids inside the cell and show high biocompatibility. There is a growing interest in polymeric carriers, due to their versatile synthesis, offering numerous possibilities of fine-tuning and their high stability. Typically, cationic polymers are used, electrostatically complexing the negatively charged genetic material. However, the cationic charge is often associated with cytotoxicity, low serum stability, changed biodistribution and thus reduced transfection efficiency. A promising approach to address these challenges of cationic polymers is their hydrophobic modification to increase stability and interaction with cell membranes. This thesis deals with different hydrophobically modified cationic polymers and strategies to optimize their performance in vitro and in vivo. Overall, hydrophobic modification results in highly efficient carriers for various kinds of genetic material (e.g., pDNA, siRNA and mRNA). Thereby, a novel pH-dependent formulation method enables to exploit poorly water-soluble but highly efficient cationic/hydrophobic polymers for gene delivery. By pH-responsive shielding concepts, biocompatibility and in vivo applicability of these polymers was enhanced, without substantially diminishing transfection efficiency. These polymeric gene delivery systems can find a wide range of applications in future gene therapy, for example for delivery to tumor and inflamed tissue due to their pH- responsive nature, immune therapies or vaccination and form the basis for further optimization for cell specific delivery

Implications for the Binding of the Protein G5P to DNA

Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration- dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms