Os mecanismos oxidativos pelos quais macrófagos exercem atividade microbicida permanecem em discussão, e estudos com hospedeiros animais serão essenciais para elucidar tal questão. Nesse trabalho, estudamos os mecanismos microbicidas de macrófagos in vivo comparando parâmetros de infecção nas lesões de camundongos resistentes (C57Bl/6) e suscetíveis (BALB/c) ao protozoário Leishmania amazonensis. A comparação mostrou que o controle da infecção pelos camundongos resistentes é dependente da ativação de macrófagos com expressão da enzima óxido nítrico sintase induzível, síntese de óxido nítrico e extensa nitração e hidroxilação das proteínas dos parasitas dentro dos fagolisossomos dos macrófagos. O principal agente tóxico aos parasitas parece ser derivado do peroxinitrito porque a nitração dos parasitas ocorreu na ausência virtual de células polimorfonucleares e foi acompanhada de hidroxilação. Além disso, tempol um inibidor de reações de nitração mediadas por peroxinitrito, inibiu a nitração de proteínas da lesão e aumentou o número de parasitas nelas presentes. Também, estudos com parasitas em cultura confirmaram que o peroxinitrito é citotóxico aos parasitas enquanto o óxido nítrico é citostático. O camundongo suscetível se mostrou capaz de sintetizar óxido nítrico mas o fez em estágios tardios da infecção e, provavelmente, em resposta a uma infecção secundária por bactérias. Tomados conjuntamente, os resultados indicam que o peroxinitrito e radicais dele derivados são os principais agentes leishmanicidas produzidos por macrófagos in vivo. / Macrophage oxidative microbicidal mechanisms remain debatable and their elucidation is likely to depend on studies with mammalian hosts. To examine macrophage microbicidal mechanisms in vivo, we compared infection parameters in the lesions of resistant (C57Bl/6) and susceptible (BALB/c) mice to the protozoan Leishmania amazonensis. This comparison demonstrated that infection control by resistant mice relied on macrophage activation with inducible nitric oxide synthase expression, nitric oxide synthesis and extensive nitration and hydroxylation of the proteins of the parasites inside macrophage phagolysosomes. The toxic agent to the parasite is likely to be peroxynitrite-derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells and was accompanied by parasite hydroxylation. In addition, tempol, an inhibitor of peroxynitrite-mediated nitrations, inhibited protein nitration of the lesions and increased the number of parasites in them. Also, studies with parasite cultures confmed that peroxynitrite is cytotoxic to the parasites whereas nitric oxide is cytostatic. The susceptible mice were also able to synthesize nitric oxide but only at late infection time and, most likely, in response to a secondary bacterial infection. Taken together, the results indicate that peroxynitrite and derived radicals are the main leishrnanicidal agents produced by macrophages in vivo.

Cieľom tejto diplomovej práce je stanoviť kvalitu a zloženie medu, taktiež stanoviť jeho botanický pôvod na základe profilu sacharidov v mede. Med je prírodný produkt vysokej kvality a vďaka značnej spotrebe a nemalej trhovej hodnote je tiež terčom na falšovanie. S cieľom identifikovať podvody a cudzorodé látky, pochádzajúce z cukrových sirupov, boli analyzované oligosacharidy a polysacharidy na báze maltodextrínov. Teoretická časť popisuje chemické zloženie medu a hlavné princípy aplikovanej inštrumentálnej techniky. Poskytuje literárny prehľad existujúcich analytických metód na stanovenie sacharidov prítomných v mede a na odhalenie jeho falšovania. Boli definované hlavné body falšovania a autenticity, vrátane legislatívnych aspektov a opisu bežných druhov falšovania. Experimentálna časť obsahuje postupy, ktoré vedú k vývoju a optimalizácii chromatografických podmienok a parametrov elektrochemického detektora na stanovenie sacharidov a maltodextrínov (oligosacharidov a polysacharidov) vo vzorkách medu. Použitá analytická technika bola vysoko účinná aniónovo-výmenná chromatografia (HPAEC) spojená s pulzným amperometrickým detektorom (PAD). Vyvinutá a optimalizovaná chromatografická metóda bola taktiež validovaná z hľadiska linearity, dynamického rozsahu, analytických limitov, presnosti a správnosti. Na záver bola táto vypracovaná metóda úspešne zhodnotená analýzou autentických a falšovaných vzoriek medu rôzneho botanického pôvodu pochádzajúcich z členských štátov EÚ.

Ziel dieser Arbeit ist die Abtrennung von monovalenten Anionen wie Chlorid, Fluorid, Bromid und Nitrat aus hochkonzentrierten alkalischen wässrigen Lösungen, die als Matrix zweiwertigen Anionen (Carbonat und Sulfat) besitzen. Nach Auswertung der Literatur eignen sich vor allem die Verfahren Ionenaustausch und Nanofiltration für diesen Zweck. Die untersuchten Ionenaustauscher weisen eine geringe Selektivität für die einwertigen Anionen auf, so dass die für einwertige Anionen nutzbare Kapazität mit steigendem Gehalt an zweiwertigen Anionen sinkt. Dabei steigt die Kapazität in der Reihenfolge Fluorid→Chlorid→Bromid→Nitrat. Die beobachteten Selektivitäten bei der Nanofiltration steigen in der gleichen Reihenfolge, wobei die Trennrate zwischen ein- und zweiwertigen Anionen vom Membrantyp abhängt. Es konnte ein Zusammenhang zwischen den Kapazitäten der untersuchten stark basischen Anionenaustauscher für monovalente Anionen und den Rückhalten für diese Anionen bei den Nanofiltrationsmembranen mit den Ionenhydratationsparametern festgestellt werden.

No description available.

Läkemedel och per- och polyfluorerade alkylsubstanser (PFAS) har identifierats i utgående vatten från avloppsvattenverk där förekomsten av dessa mikroföroreningar har fått mycket uppmärksamhet då dessa ämnen har visats kunna leda till negativa effekter på den akvatiska miljön. De traditionella reningsteknikerna som idag används på avloppsreningsverk (ARV) har begränsad reningseffekt på mikroföroreningar där ett särskilt reningssteg för avancerad rening av mikroföroreningar krävs för att minska utsläppen av dessa till miljön.                                                              Denna studie syftade till att undersöka reduktionen av mikroföroreningar vid kombination av granulärt aktivt kol (GAK) och anjonbytare (AIX). Samt att undersöka vilken utformning, uppströmsflöde med fluidiserad bädd eller nedströmsflöde med fixerade bäddar, som fungerade mest effektivt med avseende på driftstabiliteten. Vidare syftade arbetet till att utifrån tidigare insamlade data från liknande kolonnförsök på Uppsala Vatten utreda eventuell korrelation mellan totalt organiskt kol (TOC), löst organiskt kol (DOC) och UVA med läkemedel och PFAS som skulle kunna användas som uppföljningsmetod i det senare fullskaliga reningssteget på Kungsängsverket. Kolonnförsöken bestod av sju kolonner vilka delades in i tre driftlinjer. Driftlinje ett bestod av ett GAK-filter med Cyclecarb 401 följt av ett AIX-filter med Purolite 694E, liknande utformning användes för driftlinje två men där GAK-filter med Filtrasorb 400 användes i stället för Cyclecarb 401. Samtliga filter i driftlinje ett och två drevs med nedströmsflöde. Den sista driftlinjen som undersöktes bestod av Cyclecarb 401 följt av två AIX-filter med uppströmsflöde och fluidiserade bäddar. Till varje driftlinje pumpades utgående vatten från Kungsängsverket ARV genom en förbehandling bestående av två patronfilter. I GAK- och AIX-filtren med nedströmsflöde användes en kontakttid på 15 respektive 5 minuter medan en kontakttid på 2 minuter användes för de fluidiserades AIX-filtren. Reningseffektiviteten undersöktes med avseende på PFOS, diklofenak, citalopram, metoprolol och oxazepam vid två tillfällen motsvarande 100 respektive 5 000 behandlade bäddvolymer i GAK-filtren. Utifrån analyserna beräknades reduktionsgraden över de enskilda filtren samt över varje driftlinje. Driftstabiliteten undersöktes genom att jämföra backspolningsbehovet för de tre driftlinjerna samt genom att undersöka reduktionen av TOC, DOC och UVA över dessa. Resultaten visade på god reduktion av mikroföroreningar över samtliga driftlinjer, där reduktionen var över 80 procent. Vid jämförelse av de olika GAK-filtren uppvisade Cyclecarb 401 högre reduktion av PFOS och diklofenak jämför med Filtrasorb 400. Seriedrift av GAK- och AIX-filter visades komplettera varandra där mikroföroreningar som avskildes sämre av GAK avskildes i efterföljande AIX vilket medför ett mer resurseffektivt användande av adsorbenterna. Stabiliteten med avseende på backspolningsbehov och reduktion var högst för driftlinje tre och viss korrelation mellan TOC och UVA och några mikroföroreningar kunde ses men där fler analyser skulle behövas för att undersöka detta närmare. / Pharmaceuticals and poly- and perfluoroalkyl substances (PFASs) have been detected in outgoing water from wastewater treatment plants (WWTPs) where the presence of these micropollutants (MPs) has received much attention as these substances have been shown to lead to negative effects on the aquatic environment. The traditional treatment techniques currently used at WWTPs have a limited treatment effect on MPs, where an alternative treatment step for advanced treatment of MPs is required to reduce the emission of these to the environment. This study aimed to investigate the reduction of MPs for the combination of granular activated carbon (GAC) and anion exchange resin (AE) and to investigate which design (i.e., upstream flow with fluidized bed or downstream flow with fixed beds), worked most effectively in terms of operational stability. Furthermore, the work aimed to investigate, based on previously collected data from similar column experiments at Uppsala Water and Waste AB, correlation between total organic carbon (TOC), dissolved organic carbon (DOC) and UVA with pharmaceuticals and PFAS that could be used as a follow-up method in the later full-scale treatment step at Kungsängsverket. The column experiments consisted of seven columns which were divided into three operating lines. Operating line one consisted of a GAC filter with Cyclecarb 401 followed by an AE filter with Purolite 694E, similar design was used for operating line two but with Filtrasorb 400 instead of Cyclecarb 401, both lines with downstream flow. The last operating line consisted of Cyclecarb 401 followed by two AE filters with upstream flow and fluidized beds. Outgoing water from Kungsängsverket WWTP was pumped to each operating line through a pre-treatment consisting of two cartridge filters. In the GAC and AE filters with downstream flow a contact time of 15 and 5 minute respectively, was used, while a contact time of 2 minutes was used for the fluidized AE filters. The reduction efficacy was examined for five selected MPs PFOS, diclofenac, citalopram, metoprolol and oxazepam on two occasions corresponding to 100 and 5 000 treated bed volumes in the GAC filters. Based on the analyzes, the degree of reduction was calculated over the individual filters and over each operating line. Operational stability was examined by comparing the backwashing need for the three operating lines and by examining the reduction of TOC, DOC and UVA over these. The results showed a good reduction of MPs for all operating lines, where the reduction was over 80 percent. Cyclecarb 401 showed a higher reduction of PFOS and diclofenac compared to Filtrasorb 400. Series operation of GAC and AE filters was shown to complement each other, where MPs that showed lower separation in GAC were separated in subsequent AE, which results in a more resource-efficient use of the adsorbents. The stability seen to backwashing needs and reduction was highest for operating line three. The correlation study showed correlation between TOC and UVA with some of the MPs, but more samples would be needed to investigate this further.

Normal human urine contains many anions and cations. Ionic concentrations in urine have classically been determined by spectrophotometry of color reactions, flame emission spectrophotometry, atomic absorption spectrophotometry, high performance liquid chromatography, or potentiometry with ion-specific electrodes. Capillary ion electrophoresis (CIE) is a form of capillary electrophoresis which uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Various salts can be added to urine specimens to abnormally elevate ionic concentrations and interfere with either immunoassay urine drug screening procedures or gas chromatographic/mass spectrometric confirmation techniques. Application of CIE for the direct detection of endogenous anions and anionic adulterants in human urine specimens was the purpose of this investigation. CIE was performed using a Waters Quanta 4000 Capillary Electrophoresis System with either direct or indirect ultraviolet absorption detection at 254 nm. CIE of 30 random normal urine specimens and 21 urine specimens suspected of adulteration was performed. Duplicate aliquots were assayed by CIE and by colorimetric technique for nitrite. Sixteen specimens had elevated concentrations of nitrite and/or nitrate. The correlation coefficient between nitrite CIE and colorimetric results was 0.9895. Three specimens had detectable concentrations of chromate and were suspected of being adulterated with "Urine Luck," an adulterant found to contain chromate. Two specimens suspected of being adulterated with bleach were found to only contain chloride, sulfate, and phosphate. CIE is applicable to forensic analysis of urine anion concentrations. CIE can easily quantitate numerous endogenous anions and offers a method to detect and/or confirm anion adulteration of urine specimens.

Anion intercalation in the graphite positive electrode of a dual-ion battery requires high potential (> 4.3 V vs Li+/Li), which aggravates parasitic reactions involving electrolyte decomposition and Al corrosion, manifesting in poor coulombic efficiency, cycle life, and quick self-discharge. This study aims to investigate the stability of anion-intercalated graphite electrodes in a 4 M solution of lithium bis(fluorosulfonyl)imide (LiFSI) in ethyl methyl carbonate (EMC) using both in-situ and ex-situ Raman spectroscopy. The concentrated electrolyte is essential as it limits parasitic reactions at the cathode-electrolyte interface (CEI) occurring in parallel to anion intercalation. Using electrochemical methods including cyclic voltammetry, and post-mortem electron microscopy it was confirmed that the Al current collector is largely stable at potentials as high as 5.2 V in the electrolyte under consideration; no dissolved Al species were detected using EDX characterization. Results from the cyclic voltammetry study also indicate that parasitic reactions can be mitigated when the cut-off potential is limited to 5.0 V leading to higher coulombic efficiency (CE = 94 %) and more stable discharge capacity (85.17 mAh g-1). However, extending the potential to 5.1 and 5.2 V results in the discharge capacity increasing by almost 20 mAh g-1, though at the expense of the coulombic efficiency, which decreases from 94 to 76 %. Upon raising the cut-off potential to 5.3 V, the CE significantly decreased (20.62 %) as a result of extensive solvent decomposition ultimately leading to much quicker capacity fading.  Based on SEM images taken after 50 cycles, graphite particles did not sustain any structural or morphological change during cycling regardless of the cut-off potentials applied. Further tests were conducted on Li-graphite DIBs using galvanostatic methods in the range from 3 to 5 V, and at different specific currents (20, 50, and 100 mA g-1). Though the cells exhibited good performance in terms of capacity retention, and cycle life at all currents, the coulombic efficiency tended to decrease as the test currents were lowered. This observation confirms the presence of parasitic reactions which are only visible when the experimental timescale is sufficiently long. At 50 and 100 mA g-1, the CE reached > 98 % which further verifies the kinetic aspect of electrolyte decomposition reactions. It is evident that self-discharge sustained in the course of open-circuit potential (OCP) relaxation of the fully charged cell can reveal the stability of the electrolyte and the anion-intercalated graphite. Raman spectroscopy measurements conducted in-situ and ex-situ on graphite electrodes charged and discharged to a series of potential cut-offs reveal the existence of self-discharge leading to extraction of anions from the graphite particles. This was demonstrated through the spectral appearance of E2g2(i) band next to E2g2(b) band at a fully intercalated state, as opposed to the in-situ spectrum, which only showed one intercalated band (E2g2(b)). It can be concluded that concentrated electrolytes (such as 4 M LiFSI in EMC) only provide kinetic stability and are unable to entirely inhibit parasitic reactions at the interface. This further highlights the need for electrolyte additives that can create a more stable interfacial passivation layer on the positive electrode so that more reversible anion intercalation can be attained.

Background: Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. Results: In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. Conclusion: The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

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Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride (AETAC) and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion exchanger and quaternary amine as a strong anion exchanger, respectively. An alternative weak anion exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. The dynamic binding capacities of the three monoliths were comparable or superior to values that have been reported for various other monoliths. Chromatographic performances were also similar to those provided by a modified poly(GMA-co-ethylene glycol dimethacrylate) monolith. Separations of standard proteins were achieved under gradient elution conditions using these monolithic columns. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, laborious surface modifications were avoided and the PEGDA crosslinker improved the biocompatibility of the monolithic backbone. Protein separations by capillary size exclusion chromatography (SEC) require a monolith that is biocompatible, has sufficient pore volume, has the appropriate pore size distribution, and is rigid. Most polymer monoliths have not possessed a biomodal pore-size distribution, i.e., especially with one distribution in the macropore region and the other in the mesopore region. Furthermore, non-specific adsorption of proteins in these stationary phases has persisted as a major unresolved problem. To overcome these difficulties, a porous poly[polyethylene glycol methyl ether acrylate (PEGMEA)-co-PEGDA] monolith which can resist adsorption of both acidic and basic proteins when using an aqueous buffer without any organic solvent additives was developed. Based on this biocompatible monolith, surfactants were introduced as porogens with the hope of significantly increasing the mesopore volume within the polymer. Two types of surfactants were studied, including poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO and Brij. Pore size distributions were examined using a well-defined molecular weight range series of proteins and peptides by inverse size exclusion chromatography, which indicated relatively large volume percentages of mesopores and micropores. The two new monoliths demonstrated different SEC behaviors, low nonspecific adsorption of proteins, and high mechanical rigidity. High density lipoprotein (HDL) is a heterogeneous class of lipoprotein particles with subspecies that differ in apolipoprotein and lipid composition, size, density, and charge. In this work, I developed a new capillary SEC method for size separation of native HDL particles from plasma using a capillary packed with BioSep-SEC-4000 particles, Three major sizes of HDL particles were separated. Additionally, capillary SEC and capillary strong anion-exchange chromatography of non-delipidated HDL were accomplished using poly(PEGMEA-co-PEGDA) and poly(AETAC-co-PEGDA) monoliths. These new LC methods using packed and monolithic stationary phases provided rapid separation of HDLs and excellent reproducibility.