No description available.

No description available.

Although enzymes show great potential for use in industrial applications, their implementation from a practical perspective is still somewhat limited by various shortcomings in the area of enzyme immobilization. The use of silica sol-gels for protein entrapment has been studied extensively over the past 15 years or so. However, our understanding of the interactions between the immobilization matrix and the entrapped biomolecules is still relatively poor. Non-invasive in situ spectroscopic characterization is a promising approach to gain a better understanding of the fundamentals governing sol-gel immobilization. This thesis describes the application of Fourier transform infrared (FTIR) microscopy, two dimensional (2D) FTIR and fluorescence spectroscopy to characterize the immobilization matrix, entrapped model proteins and their interactions. Hydroperoxide lyase (HPL [E.C. 4.1.2.]) was chosen as a potential model protein for sol-gel entrapment. HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and the novel water soluble 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. The highest HPL activity was achieved under aqueous conditions with high salt and low surfactant concentrations and LA-OOH as the substrate. A significant interaction between salt and surfactant as well as salt and substrate was identified and a hypothesis to explain the basis of the interaction phenomena is presented. To analyze sol-gels with spectroscopic techniques, a sample format amenable to these techniques was needed. Therefore, a spin-coating technique for the preparation of aluminum or glass supported sol-gel thin films containing immobilized protein and a varying degree of the organically modified precursor propyltrimethoxysilane (PTMS) was developed. This approach produced samples that were suitable for chemical mapping using FTIR microscopy or fluorescence spectroscopic investigations. A data analysis method was developed to extract information on chemical speciation and distribution from FTIR data matrices obtained through FTIR microscopy. Results indicate that sol-gel thin films are not homogeneous on the microscopic level. Instead, they are heterogeneous with a clustering in the distribution of the model proteins studied (lysozyme [E.C. 3.2.1.17], lipase [E.C. 3.1.1.3] and bovine serum albumin (BSA)) at the scale investigated. The appearance of these clusters was found to depend on the type of protein entrapped, as well in some cases on the composition of the sol-gel. Moreover, the PTMS distribution was positively correlated with the protein distribution in the case of lipase and negatively correlated in the case of lysozyme and BSA. Additionally, sol-gels with a higher PTMS content appeared to conserve protein structure in areas where lipase clustered. Lysozyme and BSA, on the other hand, seemed to retain their structures in high concentration clusters better at lower PTMS content. A hypothesis taking into account the surface hydrophobicity of the proteins and the sol-gel composition as the basis for these phenomena is proposed. Fluorescence spectroscopy revealed that the PTMS content of the sol-gels had a direct effect on the physical properties of the immobilized proteins as evidenced by a blue shift of the intrinsic tryptophan (TRP) fluorescence. Temperature-dependent fluorescence spectroscopy revealed that the amount of TRP quenching was inversely proportional to the PTMS content of the sol-gel, suggesting that there were varying amounts of water available for quenching for the different immobilized enzyme systems. Analysis of the sol-gels by 2D FTIR spectroscopy with a focus on the amide A region using Gaussian peak deconvolution revealed two different species of water for the 50 % PTMS thin film sol-gels with BSA that could be described as fully and not fully H-bonded. It was also found that these species of water showed different removal profiles during thermal treatment. 2D FTIR of the amide I region followed by absorbance difference spectrum evaluation revealed that the temperature stability of the three model proteins was also sol-gel composition dependent. A hypothesis that the surface characteristics of the proteins determine the nature of the composition dependence is presented.

Although enzymes show great potential for use in industrial applications, their implementation from a practical perspective is still somewhat limited by various shortcomings in the area of enzyme immobilization. The use of silica sol-gels for protein entrapment has been studied extensively over the past 15 years or so. However, our understanding of the interactions between the immobilization matrix and the entrapped biomolecules is still relatively poor. Non-invasive in situ spectroscopic characterization is a promising approach to gain a better understanding of the fundamentals governing sol-gel immobilization. This thesis describes the application of Fourier transform infrared (FTIR) microscopy, two dimensional (2D) FTIR and fluorescence spectroscopy to characterize the immobilization matrix, entrapped model proteins and their interactions. Hydroperoxide lyase (HPL [E.C. 4.1.2.]) was chosen as a potential model protein for sol-gel entrapment. HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and the novel water soluble 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. The highest HPL activity was achieved under aqueous conditions with high salt and low surfactant concentrations and LA-OOH as the substrate. A significant interaction between salt and surfactant as well as salt and substrate was identified and a hypothesis to explain the basis of the interaction phenomena is presented. To analyze sol-gels with spectroscopic techniques, a sample format amenable to these techniques was needed. Therefore, a spin-coating technique for the preparation of aluminum or glass supported sol-gel thin films containing immobilized protein and a varying degree of the organically modified precursor propyltrimethoxysilane (PTMS) was developed. This approach produced samples that were suitable for chemical mapping using FTIR microscopy or fluorescence spectroscopic investigations. A data analysis method was developed to extract information on chemical speciation and distribution from FTIR data matrices obtained through FTIR microscopy. Results indicate that sol-gel thin films are not homogeneous on the microscopic level. Instead, they are heterogeneous with a clustering in the distribution of the model proteins studied (lysozyme [E.C. 3.2.1.17], lipase [E.C. 3.1.1.3] and bovine serum albumin (BSA)) at the scale investigated. The appearance of these clusters was found to depend on the type of protein entrapped, as well in some cases on the composition of the sol-gel. Moreover, the PTMS distribution was positively correlated with the protein distribution in the case of lipase and negatively correlated in the case of lysozyme and BSA. Additionally, sol-gels with a higher PTMS content appeared to conserve protein structure in areas where lipase clustered. Lysozyme and BSA, on the other hand, seemed to retain their structures in high concentration clusters better at lower PTMS content. A hypothesis taking into account the surface hydrophobicity of the proteins and the sol-gel composition as the basis for these phenomena is proposed. Fluorescence spectroscopy revealed that the PTMS content of the sol-gels had a direct effect on the physical properties of the immobilized proteins as evidenced by a blue shift of the intrinsic tryptophan (TRP) fluorescence. Temperature-dependent fluorescence spectroscopy revealed that the amount of TRP quenching was inversely proportional to the PTMS content of the sol-gel, suggesting that there were varying amounts of water available for quenching for the different immobilized enzyme systems. Analysis of the sol-gels by 2D FTIR spectroscopy with a focus on the amide A region using Gaussian peak deconvolution revealed two different species of water for the 50 % PTMS thin film sol-gels with BSA that could be described as fully and not fully H-bonded. It was also found that these species of water showed different removal profiles during thermal treatment. 2D FTIR of the amide I region followed by absorbance difference spectrum evaluation revealed that the temperature stability of the three model proteins was also sol-gel composition dependent. A hypothesis that the surface characteristics of the proteins determine the nature of the composition dependence is presented.

Universiẗat, Diss.--Paderborn, 1999.

Universiẗat, Diss., 2000--Köln.

Antioxidants are widely used to protect organic materials from damages caused by autoxidation, an oxidation process that occurs under normal aerobic conditions. In this thesis, novel multifunctional organoselenium and organotellurium antioxidants were designed, synthesized, and evaluated in search for compounds with better radical-trapping capacity, regenerability, and hydroperoxide-decomposing ability. Selenium was incorporated into ebselenols and hydroxy-2,3-dihydrobenzo[b]selenophenes and tellurium into diaryl disulfides and aryltellurophenols. All newly developed antioxidants were evaluated in a chlorobenzene/water two-phase lipid peroxidation system containing suitable co-antioxidants in the aqueous phase. Ebselenol carrying a hydroxyl group (OH) ortho to selenium showed a two-fold longer inhibition time than the reference α-tocopherol in the presence of aqueous-phase ascorbic acid. 2,3-Dihydrobenzo[b]selenophenes carrying a 5- or 7-OH outperformed α-tocopherol both when it comes to radical-trapping capacity and regenerability. Alkyltellurothiophenols, in situ formed from their corresponding disulfides by tris(2-carboxyethyl)phosphine, were also efficient regenerable radical-trapping antioxidants. The consumption of N-acetylcysteine in the water phase was followed and found to be limiting for the duration of the inhibition. The hydroperoxide-decomposing ability of all organoselenium antioxidants was evaluated. Ebselenols were often better glutathione peroxidase mimics than the parent. In an effort to find out more about antioxidant mechanisms, aryltellurophenols carrying electron donating and electron withdrawing groups in the phenolic or aryltelluro parts were synthesized and OH bond dissociation enthalpies, BDEO-Hs, were calculated. Compounds carrying electron donating groups in the phenolic or aryltelluro part of the molecule showed the best radical-trapping capacity. Deuterium labelling experiments suggested that hydrogen atom transfer could be the rate-limiting step in the antioxidant mechanism.

Mikroplaster har hittills studerats nästan exklusivt i marin miljö och kunskapen om den ekologiska effekten av plast i våra landbaserade ekosystem är begränsad. Detta trots att det är sannolikt att landmiljöer är de primära utsläppskällorna av plastavfall, och att plastavfall ackumuleras i marina miljöer i anslutning till urbana miljöer och jordbruk. Av den totala mängden avfall som släpps ut eller dumpas i miljön kan plast vara upp till 54% av den totala viktmassan. I jordbruk och deponier i Europa har det i det översta lagret jord ner till ett djup av 10 cm uppmäts så mycket som 607 plastpartiklar per kilo jord. De huvudsakliga källorna till plastpartiklar i jord är slam från avloppsvatten och plastpartiklar avsatta från markdukar och plastöverdrag använda i jordbruksprodukter och trädgårdsprodukter. Det beräknas vara ca 63 000 - 430 000 ton mikroplast som tillförs jordbruket i Europa årligen via gödsel. Hästgödsel står för 10% av den totala mängden gödsel från boskap som används i jordbruk i Sverige, och den totala mängden hästgödsel är uppskattad till 2,7 miljoner ton per år. Hästar utfodras med stråfoder paketerat i sträckfilm tillverkat av polyeten, och hö paketerat med bindgarn tillverkat av polypropen. Utfodring sker även delvis ur fodernät tillverkade av polymerer som polypropen, nylon och terylen (PET; polyester). Syftet med denna studie är att undersöka om mikroplaster förekommer i hästträck genom att reducera organiskt material i 30% väteperoxid och analysera proverna med ljusmikroskop. Resultatet av studien visade flertalet partiklar i form av små mikroplaster identifierade i primär och sekundär form i samtliga undersökta prover. Kvantifieringen av partiklar i proverna visade signifikant skillnad i medelvärde mellan två fodertyper. Förekomsten av mikroplast i alla undersökta prover i denna studie indikerar att det finns ett behov av ytterligare forskning om mikroplast i djurgödsel för att förstå effekten av olika gödselmedel som används i jordbruk. / Microplastics have been studied almost exclusively in marine environments, and knowledge about ecological impacts of microplastics in land-based ecosystems is limited. This is despite the fact, that marine environments are likely to accumulate plastic waste from land environments, and that the land environments are the primary sources of global plastic contamination. Of the total amount of waste that is discharged or dumped into the environment, plastic account for up to 54% of the total mass. In agriculture land and landfills in Europe, the top layer of soil down to a depth of 10 cm it has been measured as much as 607 plastic particles per kilo soil. The main sources of plastic particles in our soil are sludge from wastewater deposits and mulching films and plastic coatings used in agricultural and garden products. 63 000 – 430 000 tonnes of microplastics is deposited on agriculture land in Europe annually via fertilizers. Horse manure accounts for 10% of the total amount of livestock manure used in agriculture in Sweden. The estimated total amount of horse manure is 2.7 million tonnes per year. Horses are fed with coarse fodder packaged in plastic film made of polyethylene, and hay packed with twine made of polypropylene. Feeding also takes place partly from slow feeding nets made of polymers such as polypropylene, nylon and terylene (PET; polyester). The aim of this study is to investigate whether microplastics are present in horse manure. Manure from horses with different fodder combinations is analysed for presence of microplastic. Organic matter was degraded with 30% hydrogen peroxide and all the samples were examined with light microscopy. The results of the study showed several particles in the form of small microplastics identified in primary and secondary form in all samples. The quantification of particles in the samples showed a significant difference in mean values between two fodder types. The presence of microplastic in all samples in this study indicate that there is a need for further research on microplastics in animal manure, in order to understand the effect of different fertilizers used in agriculture.

Dioxygenase-cytochrome P450 (DOX-CYP) fusion enzymes are present in diverse human and plant pathogenic fungi. They oxygenate fatty acids to lipid mediators which have regula­tory functions in fungal development and toxin production. These enzymes catalyze the for­mation of fatty acid hy­droperoxides which are subsequently converted by the P450 activities or reduced to the corresponding alcohols. The N-terminal DOX domains show catalytic and structural homology to mammalian cyclooxygenases, which belong to the most thoroughly studied human enzymes. 7,8-Linoleate diol synthase (LDS) of the plant pathogenic fungus Gaeumannomyces graminis was the first characterized member of the DOX-CYP fusion enzyme family. It catalyzes the conversion of linoleic acid to 8R-hydroperoxylinoleic acid (HPODE) and subse­quently to 7S,8S-dihy­droxylinoleic acid by its DOX and P450 domains, respectively. By now, several enzymes with homology to 7,8-LDS have been identified in im­portant fungi, e.g., psi fac­tor-producing oxygenase (ppo)A, ppoB, and ppoC, of Aspergillus nidulans and A. fumigatus. By cloning and recombinant expression, ppoA of A. fumigatus was identi­fied as 5,8-LDS. Partial expression of the 8R-DOX domains of 5,8-LDS of A. fumigatus and 7,8-LDS of G. graminis yielded active protein which demonstrates that the DOX activities of LDS are independent of their P450 domains. The latter domains were shown to contain a conserved motif with catalytically important amide residues. As judged by site-directed mutagene­sis studies, 5,8- and 7,8-LDS seem to facilitate heterolytic cleavage of the oxygen-oxygen bond of 8R-HPODE by aid of a glutamine and an asparagine residue, respectively. Cloning and expression of putative DOX-CYP fusion proteins of A. terreus and Fusarium oxysporum led to the discovery of novel enzyme activities, e.g., linoleate 9S-DOX and two allene oxide synthases (AOS), specific for 9R- and 9S-HPODE, respectively. The fungal AOS are present in the P450 domains of two DOX-CYP fusion enzymes and show higher se­quence homology to LDS than to plant AOS and constitute therefore a novel class of AOS. In summary, this thesis describes the discovery of novel fatty acid oxy­genases of human and plant pathogenic fungi and the characterization of their reaction mechanisms.

Em condições inflamatórias do sistema vascular, altas concentrações de mieloperoxidase somada à presença do ácido úrico, sugerem a formação local do oxidante hidroperóxido de urato. A ação desse peróxido já foi demonstrada sobre glutationa e peroxirredoxinas, tornando plausível a possibilidade de que outras proteínas tiólicas também pudessem ser alvo de oxidação. A proteína dissulfeto isomerase é uma ditiol-dissulfeto oxidoredutase e chaperona, localizada principalmente no retículo endoplasmático, onde participa do enovelamento de proteínas nascentes. Além disso, um pool dessas enzimas foi identificado na superfície da célula e no meio extracelular (secretada) e parece ser especialmente importante em eventos vasculares como ativação e agregação de plaquetas, trombose e remodelamento vascular. Primeiramente, foi investigado se o hidroperóxido de urato era capaz de oxidar a PDI. Pelo ensaio do DTNB foi verificado que os tióis livres da proteína eram consumidos após reação com o peróxido e, em seguida, por nLC-MS/MS os resíduos de cisteínas dos sítios catalíticos foram identificados como os principais alvos de oxidação. Embora não tenham sido verificadas outras modificações além de dissulfetos, foi observado que o tratamento com hidroperóxido promoveu agregação e inativação da proteína. Os estudos subsequentes envolveram uma linhagem de células endoteliais (HUVECs). Análises preliminares de citotoxicidade (detecção da atividade da enzima lactato desidrogenase no sobrenadante e incorporação de sondas fluorescentes ao DNA) mostraram que tratamentos com concentrações de até 400 µM de hidroperóxido de urato não são letais às células em cultura. Usando alquilantes impermeáveis à membrana celular foi mostrado que o hidroperóxido de urato oxida não só a proteína dissulfeto isomerase, mas também proteínas tiólicas totais expressas na superfície das HUVECs. Experimentos de wound healing foram feitos para avaliação da capacidade de migração das células mediante o tratamento com hidroperóxido de urato, mas nenhuma diferença foi observada. Contudo, a incubação das células com os agentes oxidantes hidroperóxido de urato e diamida, inibidores de PDI e integrina e um alquilante de tiol, resultaram, pelo menos nos trinta primeiros minutos, em menor capacidade de adesão das células à fibronectina. Além disso, as células tratadas com hidroperóxido de urato se tornaram mais sensíveis ao destacamento da placa de cultura e apresentaram alteração na morfologia. O tratamento com o peróxido também afetou a homeostase redox das HUVECs, observado pela diminuição da razão GSH/GSSG. Finalmente foram apresentadas evidênciasindiretas de que o ácido úrico é substrato da peroxidasina, uma heme peroxidase abundantemente expressa no sistema vascular. Primeiro, pelo ensaio do Amplex Red foi observado que a presença de ácido úrico na mistura reacional resultou em menor taxa de oxidação do reagente. Depois, por LC-MS/MS, também em amostra na qual o ácido úrico estava presente, foi identificado o hidroxiisourato, álcool resultante da decomposição do hidroperóxido de urato. Todo o conjunto de dados deverá contribuir para o maior entendimento da participação do hidroperóxido de urato em processos oxidativos vasculares − especialmente a oxidação de proteínas − que pode ser um dos mecanismos responsáveis pela alteração da função endotelial e da homeostase vascular. / During vascular inflammatory conditions, high amounts of myeloperoxidase added to the presence of uric acid, suggest the local formation of urate hydroperoxide. Its oxidative action has already been demonstrated on glutathione and peroxiredoxins, making plausible the possibility that other thiol proteins could also be a target for oxidation. The protein disulfide isomerase is a dithiol-disulfide oxidoreductase and chaperone, located mainly in the endoplasmic reticulum, where it is involved in the correct folding of nascent proteins. Also, a pool of these enzymes has been identified in cell surface and the extracellular (secreted) milieu and appears to be important in vascular events, such as platelet activation and aggregation, thrombosis and vascular remodeling. First, it was investigated whether urate hydroperoxide was capable of oxidizing PDI. By the DTNB assay, it was found that the free thiols of the protein were consumed after reaction with the peroxide and then, by nLC-MS / MS, the active redox cysteine residues were identified as the main oxidation targets. Although no modifications other than disulfides have been found, hydroperoxide treatment has been shown to promote protein aggregation and inactivation. Subsequent studies involved an endothelial cell line (HUVECs). Preliminary cytotoxicity analyzes (detection of lactate dehydrogenase enzyme activity in the supernatant and incorporation of fluorescent probes into DNA) have shown that treatments with concentrations up to 400 µM are not lethal to cells in culture. Then, using alkylating agents impermeable to the cell membrane, urate hydroperoxide was shown to oxidize not only PDI but also total thiol proteins expressed on HUVECs surface. Wound healing experiments were performed to evaluate cell migration after treatment with urate hydroperoxide, but no difference was observed. However, incubation of the cells with the oxidizing agents urate hydroperoxide and diamide, inhibitors of both PDI and integrin and a thiol alkylator, resulted, at least for the first thirty minutes, in reduced cell adhesion to fibronectin. In addition, cells treated with urate hydroperoxide became more sensitive to detachment from the culture dish and exhibited alterations in morphology. Treatment with the peroxide also affected the redox homeostasis of the HUVECs, observed by a decrease in the GSH / GSSG ratio. Finally, indirect evidence was presented that uric acid is a substrate of peroxidasin, a heme peroxidase abundantly expressed in the vascular system. First, with the Amplex Red assay it was observed that the presence of uric acid in the reaction mixture resulted in lower oxidation rates of the reagent. Then, by LC-MS / MS, hydroxyisourate, which is the alcohol derived from urate hydroperoxide decomposition, was also identified in samples containing uric acid. Taken together, the data presented should contribute to a better understanding of the involvement of urate hydroperoxide in vascular oxidative processes − especially protein oxidation − that may be one mechanism associated to disturbances in endothelial function and vascular homeostasis.