NADPH-Dependent Thioredoxin System In Regulation of Chloroplast Functions

Photosynthesis, the process in which carbon dioxide is converted into sugars using the energy of sunlight, is vital for heterotrophic life on Earth. In plants, photosynthesis takes place in specific organelles called chloroplasts. During chloroplast biogenesis, light is a prerequisite for the development of functional photosynthetic structures. In addition to photosynthesis, a number of other metabolic processes such as nitrogen assimilation, the biosynthesis of fatty acids, amino acids, vitamins, and hormones are localized to plant chloroplasts. The biosynthetic pathways in chloroplasts are tightly regulated, and especially the reduction/oxidation (redox) signals play important roles in controlling many developmental and metabolic processes in chloroplasts. Thioredoxins are universal regulatory proteins that mediate redox signals in chloroplasts. They are able to modify the structure and function of their target proteins by reduction of disulfide bonds. Oxidized thioredoxins are restored via the action of thioredoxin reductases. Two thioredoxin reductase systems exist in plant chloroplasts, the NADPHdependent thioredoxin reductase C (NTRC) and ferredoxin-thioredoxin reductase (FTR). The ferredoxin-thioredoxin system that is linked to photosynthetic light reactions is involved in light-activation of chloroplast proteins. NADPH can be produced via both the photosynthetic electron transfer reactions in light, and in darkness via the pentose phosphate pathway. These different pathways of NADPH production enable the regulation of diverse metabolic pathways in chloroplasts by the NADPH-dependent thioredoxin system. In this thesis, the role of NADPH-dependent thioredoxin system in the redox-control of chloroplast development and metabolism was studied by characterization of Arabidopsis thaliana T-DNA insertion lines of NTRC gene (ntrc) and by identification of chloroplast proteins regulated by NTRC. The ntrc plants showed the strongest visible phenotypes when grown under short 8-h photoperiod. This indicates that i) chloroplast NADPH-dependent thioredoxin system is non-redundant to ferredoxinthioredoxin system and that ii) NTRC particularly controls the chloroplast processes that are easily imbalanced in daily light/dark rhythms with short day and long night. I identified four processes and the redox-regulated proteins therein that are potentially regulated by NTRC; i) chloroplast development, ii) starch biosynthesis, iii) aromatic amino acid biosynthesis and iv) detoxification of H₂O₂. Such regulation can be achieved directly by modulating the redox state of intramolecular or intermolecular disulfide bridges of enzymes, or by protecting enzymes from oxidation in conjunction with 2-cysteine peroxiredoxins. This thesis work also demonstrated that the enzymatic antioxidant systems in chloroplasts, ascorbate peroxidases, superoxide dismutase and NTRC-dependent 2-cysteine peroxiredoxins are tightly linked up to prevent the detrimental accumulation of reactive oxygen species in plants.

Chemical characteristics and Kraft pulping of tension wood from Eucalyptus globulus labill

Tension (TW) and opposite wood (OW) of Eucalyptus globulus trees were analyzed for its chemical characteristics and Kraft pulp production. Lignin content was 16% lower and contained 32% more syringyl units in TW than in OW. The increase in syringyl units favoured the formation of β-O-4 bonds that was also higher in TW than in OW (84% vs. 64%, respectively). The effect of these wood features was evaluated in the production of Kraft pulps from both types of wood. At kappa number 16, Kraft pulps obtained from TW demanded less active alkali in delignification and presented slightly higher or similar pulp yield than pulps made with OW. Fiber length, coarseness and intrinsic viscosity were also higher in tension than in opposite pulps. When pulps where refined to 30°SR, TW pulps needed 18% more revolutions in the PFI mill to achieve the same beating degree than OW pulps. Strength properties (tensile, tear and burst indexes) were slightly higher or similar in tension as compared with opposite wood pulps. After an OD0(EO)D1 bleaching sequence, both pulps achieved up to 89% ISO brightness. Bleached pulps from TW presented higher viscosity and low amount of hexenuronic acids than pulps from OW. Results showed that TW presented high xylans and low lignin content that caused a decrease in alkali consumption, increase pulp strength properties and similar bleaching performance as compared with pulps from OW.

Risk Parity: A Modern Tool for Diversification

The purpose of this study is to examine how well risk parity works in terms of risk, return and diversification relative to more traditional minimum variance, 1/N and 60/40 portfolios. Risk parity portfolios were constituted of five risk sources; three common asset classes and two alternative beta investment strategies. The three common asset classes were equities, bonds and commodities, and the alternative beta investment strategies were carry trade and trend following. Risk parity portfolios were constructed using five different risk measures of which four were tail risk measures. The risk measures were standard deviation, Value-at-Risk, Expected Shortfall, modified Value-at-Risk and modified Expected Shortfall. We studied also how sensitive risk parity is to the choice of risk measure. The hypothesis is that risk parity portfolios provide better return with the same amount of risk and are better diversified than the benchmark portfolios. We used two data sets, monthly and weekly data. The monthly data was from the years 1989-2011 and the weekly data was from the years 2000-2011. Empirical studies showed that risk parity portfolios provide better diversification since the diversification is made at the risk level. Risk based portfolios provided superior return compared to the asset based portfolios. Using tail risk measures in risk parity portfolios do not necessarily provide better hedge from tail events than standard deviation.

The Evolution of Securities Lending and Risk Mitigation: A Finnish Market Case Study

Although securities lending is an important function of the financial markets, it has not received that much academic attention. This study examines the evolution of European securities lending and risk management with an emphasis on the development of collateral management, the function responsible for reducing credit risk. The effects of the recent financial instabilities are also considered. The evolution of the Finnish securities lending market is examined in more detail through a case-study. This study can be classified as a constructive qualitative case study. The initial practical knowledge comes from the author's own experience and additional insight and theoretical background is acquired through a literature review. The case study is based on research, semi-structured interviews and a brief analysis of numerical data. The main observation of this study was that securities lending is now recognized as more of an investment management discipline than an operational support function. The recent financial instabilities have resulted in an increased focus on risk and transparency. In securities lending this is directly reflected in collateral management guidelines and procedures. Collateral management has become increasingly technologically developed and automated. Collateral optimization initiatives have been started to make the process more efficient, liquid, and cost effective. Although securities lending is generally an OTC-market with no standard market place, centralized exchange-like models have been introduced. Finnish securities lending has now shifted towards the more common global OTC model. Although the Finnish securities lending industry has developed, and the main laws governing it (tax legislation) have changed, there is still need for development. There are still not many Finnish participants involved and due to legal issues most securities loans are collateralized with cash and not securities (e.g. government bonds).

Ulkomaalaisen yrityksen verkostosuhteet Venäjällä

Tutkimuksen tavoitteena on konkreettisen yritysverkoston avulla selvittää, minkälaisia ovat ulkomaalaisen yrityksen potentiaaliset kumppanit ja yritysten väliset suhteet Venäjällä. Toisena tavoitteena on kuvata käytännön työelämään sopivalla menetelmällä riittävän kokonaisvaltaisesti yritysverkoston kumppanit sekä kumppaneiden ja kärkiyrityksen väliset suhteet. Samalla tarkastellaan yritysten välisten suhteiden riskejä. Selvitysmenetelmällä pyritään nopeuteen ja ketteryyteen ja sen tavoitteena on lisätä ulkomaisen johdon käsitystä yritysverkostosta johdon päätöksentekoa varten. Tutkimus jakaantuu teoria tarkasteluun ja yhden Venäjällä toimivan ulkomaisen yrityksen osto-osaston verkostoa selvittävään empiiriseen osaan. Teoriatarkastelussa selvitetään erilaisia lähestymistapoja ja laajempia viitekehyksiä yritysverkostojen tutkimukseen. Teoriatarkastelun perusteella valittiin viitekehykset yrityskumppaneiden ja yritysten välisen suhteen sekä riskien arviointiin. Empiirisen osan tutkimus toteutettiin haastattelukyselytutkimuksena perustuen näihin viitekehyksiin. Yrityskumppaneita ja yritysten välisiä suhteita tarkastellaan tilastollisin menetelmin ja riskejä arvioidaan laadullisella tutkimusotteella. Tutkimustulokset vastaavat monelta osin liiketoimintakulttuurista ja –ympäristöstä saatua kokemukseen ja tutkimustietoon perustuvaa käsitystä. Verkostoitumisen kulmakivet ovat hyvin toimivat ja määritellyt vaihdannan liiketoiminta- ja tukiprosessit, hyvin tunnetut kumppanit ja tälle perustalle rakennetut sosiaaliset suhteet sekä liiketoimintaympäristöön ja –kulttuuriin soveltuvat yhteistyökompetenssit. Tutkitun yritysverkoston yhteistyösuhteet voidaan jakaa hierarkkiseen organisaatiokumppanuuteen, toimittajakumppanuuteen, luottamukseen perustuvaan markkinaehtoiseen suhteeseen ja opportunistiseen markkinasuhteeseen. Hyviä yhteistyökumppaneita ja –suhteita löytyy taustaltaan erilaisista toimittajista. Tutkimuksessa käytetyt viitekehykset toimivat hyvin erityisesti yrityskumppanin arvioinnissa. Yritysten välisen suhteen arviointiin käytetyn viitekehyksen ongelmat liittyivät lähinnä erilaiseen liiketoimintakulttuuriin ja käsitykseen yritysten välisestä strategisesta suhteesta. Riskit liittyivät toimitussuhteeseen useammin kuin omaan organisaatioon tai toimittajaan.

Metallien eluointi kelatoivasta ioninvaihtimesta

Kelatoivat ioninvaihtimet ovat yleensä makrohuokoisia hartseja, joiden avulla metalleja poistetaan ja otetaan mahdollisesti talteen teollisuuden puhdistettavista jätevirroista. Ne muodostavat metalli-ionien kanssa komplekseja runkomateriaaliin kovalenttisesti liitettyjen aktiivisten funktionaalisten ryhmiensä välityksellä. Ioninvaihtimen selektiivisyys eri metalli-ioneja kohtaan vaihtelee riippuen siihen liitetystä kelatoivasta ryhmästä. Työssä tutkitaan metallien tarttumista kelatoiviin ioninvaihtimiin, sekä niiden eluoitumista adsorbentistaan. Kelatoivat ioninvaihtimet sitovat metalli-ioneja tehokkaasti ligandiensa monihampaisuudesta johtuen. Metallien tarttuminen kelatoivaan ioninvaihtimeen ei kuitenkaan ole yksiselitteistä, vaan siihen vaikuttaa muun muassa pH yhdessä monen muun tekijän kanssa. Ioninvaihtimien selektiivisyyttä tarkastellaan työssä lähinnä kovien ja pehmeiden happojen ja emästen HSAB -teorian näkökulmasta. Regeneroinnilla ioninvaihdin saadaan jälleen alkuperäiseen muotoonsa, minkä jälkeen se voidaan käyttää uudelleen. Yleensä regenerointi suoritetaan kemiallisesti. Tässä työssä ioninvaihtimien regenerointia ja kohdemetallin eluointia tarkastellaan paitsi teoriassa, myös kokeellisessa osuudessa. Kokeellisessa osuudessa tutkitaan kuparin (Cu2+) eluoitumista kelatoivasta Dowex M-4195 kationinvaihtohartsista. Kokeissa hartsi ladattiin kuparilla erillisessä panoksessa kuparin ollessa syöttöliuoksessa kuparisulfaattina. Eluointiliuoksina käytettiin 2 ja 5 molaarista rikkihappoa, sekä 2 molaarista ammoniumhydroksidia. Eluointi suoritettiin panostoimisena kolonniajona ja eluaatista otetut näytteet analysoitiin atomiadsorptiospektrofotometrillä. Analyysitulokset esitetään läpäisykäyrinä, joiden perusteella 2M ammoniumhydroksidi on kolmesta tutkitusta eluentista tehokkain eluoimaan kuparia Dowex M-4195 hartsista.

Regioselective modification of galactose-containing polysaccharides in aqueous media

Biorefining is defined as sustainable conversion of biomass into marketable products and energy. Forests cover almost one third of earth’s land area, and account for approximately 40% of the total annual biomass production. In forest biorefining, the wood components are, in addition to the traditional paper and board products, converted into chemicals and biofuels. The major components in wood are cellulose, hemicelluloses, and lignin. The main hemicellulose in softwoods, which are of interest especially for the Nordic forest industry, is O-acetyl galactoglucomannan (GGM). GGM can be isolated in industrial scale from the waste waters of the mechanical pulping process, but is not yet today industrially utilized. In order to attain desired properties of GGM for specific end-uses, chemical and enzymatic modifications can be performed. Regioselective modifications of GGM, and other galactose-containing polysaccharides were done by oxidations, and by combining oxidations with subsequent derivatizations of the formed carbonyl or carboxyl groups. Two different pathways were investigated: activation of the C-6 positions in different sugar units by TEMPO-mediated oxidation, and activation of C-6 position in only galactose-units by oxidation catalyzed by the enzyme galactose oxidase. The activated sites were further selectively derivatized; TEMPO-oxidized GGM by a carbodiimide-mediated reaction forming amides, and GO-oxidized GGM by indium-mediated allylation introducing double or triple bonds to the molecule. In order to better understand the reaction, and to develop a MALDI-TOF-MS method for characterization of regioselectively allylated GGM, α-D-galactopyranoside and raffinose were used as model compounds. All reactions were done in aqueous media. To investigate the applicability of the modified polysaccharides for, e.g., cellulose surface functionalization, their sorption onto pulp fibres was studied. Carboxylation affects the sorption tendency significantly; a higher degree of oxidation leads to lower sorption. By controlling the degree of oxidation of the polysaccharides and the ionic strength of the sorption media, high degrees of sorption of carboxylated polysaccharides onto cellulose could, however, be obtained. Anionic polysaccharides were used as templates during laccase-catalyzed polymerization of aniline, offering a green, chemo-enzymatic route for synthesis of conducting polyaniline (PANI) composite materials. Different polysaccharide templates, such as, native GGM, TEMPO-oxidized GGM, naturally anionic κ-carrageenan, and nanofibrillated cellulose produced by TEMPO-oxidation, were assessed. The conductivity of the synthesized polysaccharide/PANI biocomposites varies depending on the polysaccharide template; κ-CGN, the anionic polysaccharide with the lowest pKa value, produces the polysaccharide/PANI biocomposites with the highest conductivity. The presented derivatization, sorption, and polymerization procedures open new application windows for polysaccharides, such as spruce GGM. The modified polysaccharides and the conducting biocomposites produced provide potential applications in biosensors, electronic devices, and tissue engineering.

Selective inhibition of human tankyrases

Tankyrases belong to the Diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme superfamily, also known as poly(ADP-ribose) polymerases (PARPs). They catalyze a covalent post-translational modification reaction where they transfer ADP-ribose units from NAD+ to target proteins. Tankyrases are involved in many cellular processes and their roles in telomere homeostasis, Wnt signaling and in several diseases including cancers have made them interesting drug targets. In this thesis project, selective inhibition of human tankyrases was studied. A homogeneous fluorescence-based assay was developed to screen the compound libraries. The assay is inexpensive, operationally easy, and performs well according to the statistical analysis. Assay suitability was confirmed by screening a natural product library. Flavone was identified as the most potent inhibitor in the library and this motivated us to screen a larger flavonoid library. Results showed that flavones were indeed the best inhibitor of tankyrases among flavonoids. To further study the structure-activity relationship, a small library of flavones containing single substitution was screened and potency measurements allowed us to generate structure-activity relationship. Compounds containing substitutions at 4´-position were more potent in comparison to other substitutions, and importantly, hydrophobic groups improved isoenzyme selectivity as well as the potency. A flavone derivative containing a hydrophobic isopropyl group (compound 22), displayed 6 nM potency against TNKS1, excellent isoenzyme selectivity and Wnt signaling inhibition. Protein interactions with compounds were studied by solving complex crystal structures of the compounds with TNKS2 catalytic domain. A novel tankyrase inhibitor (IWR-1) was also crystallized in complex with TNKS2 catalytic domain. The crystal structure of TNKS2 in complex with IWR-1 showed that the compound binds to adenosine site and it was the first known ARTD inhibitor of this kind. To date, there is no structural information available about the substrate binding with any of the ARTD family members; therefore NAD+ was soaked with TNKS2 catalytic domain crystals. However, analysis of crystal structure showed that NAD+ was hydrolyzed to nicotinamide. Also, a co-crystal structure of NAD+ mimic compound, EB-47, was solved which was used to deduce some insights about the substrate interactions with the enzyme. Like EB-47, other ARTD1 inhibitors were also shown to inhibit tankyrases. It indicated that selectivity of the ARTD1 inhibitors should be considered as some of the effects in cells could come from tankyrase inhibition. In conclusion, the study provides novel information on tankyrase inhibition and presents new insight into the selectivity and potency of compounds.

The Combined treatment of UV Light and Titanium Dioxide (TiO2) in Grease Filtration Technique

The aim of this thesis was to identify the best grease removal technique with the application of low power of UV light to TiO2 coated grease filters. The treatment with various power series of ozone generating and ozone free lamps to normal grease filters and TiO2 coated grease filters were examined and the obtained results are compared to each other in this paper. The effect of ozone reaction was observed and compared with the effect of TiO2. The experiments were solely based on the photo oxidation and photo catalytic oxidation reactions. TiO2 is a green catalyst used in the photocatalytic reaction. Sunflower oil was used for grease production and tetracholoroethylene as a solvent. Grease samples were collected from the ventilation duct connected to the cooking hood system. Sample extraction was done in ultrasonic bath with the principle of sonication. The sample analysis was done by FTIR machine. The result determining the concentration of grease was the quantification of saturated C-H bonds in the chosen peak group of the spectrum. A very low power of UVC light functions perfectly with the Titanium dioxide. The experimental results have shown the combined treatment of titanium dioxide and UV light is an effective method in grease removal process. The photocatalytic reaction with titanium dioxide is better than photo oxidation reaction with ozone treatment. Photocatalytic reaction is environmentally friendly, energy efficient and economical.

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8 µM, kcat = 5.3 min-1, kcat/Km = 2 min-1 µM-1 and Km = 5.0 µM, kcat = 7.0 min-1, kcat/Km = 1.4 min-1 µM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), a-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by a-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations

From Molecular Blocks To Bioanalytical Assays: Combining Lanthanide Luminescence and Bioaffinity Binders for Protein Detection

Measuring protein biomarkers from sample matrix, such as plasma, is one of the basic tasks in clinical diagnostics. Bioanalytical assays used for the measuring should be able to measure proteins with high sensitivity and specificity. Furthermore, multiplexing capability would also be advantageous. To ensure the utility of the diagnostic test in point-of-care setting, additional requirements such as short turn-around times, ease-ofuse and low costs need to be met. On the other hand, enhancement of assay sensitivity could enable exploiting novel biomarkers, which are present in very low concentrations and which the current immunoassays are unable to measure. Furthermore, highly sensitive assays could enable the use of minimally invasive sampling. In the development of high-sensitivity assays the label technology and affinity binders are in pivotal role. Additionally, innovative assay designs contribute to the obtained sensitivity and other characteristics of the assay as well as its applicability. The aim of this thesis was to study the impact of assay components on the performance of both homogeneous and heterogeneous assays. Applicability of two different lanthanide-based label technologies, upconverting nanoparticles and switchable lanthanide luminescence, to protein detection was explored. Moreover, the potential of recombinant antibodies and aptamers as alternative affinity binders were evaluated. Additionally, alternative conjugation chemistries for production of the labeled binders were studied. Different assay concepts were also evaluated with respect to their applicability to point-of-care testing, which requires simple yet sensitive methods. The applicability of upconverting nanoparticles to the simultaneous quantitative measurement of multiple analytes using imaging-based detection was demonstrated. Additionally, the required instrumentation was relatively simple and inexpensive compared to other luminescent lanthanide-based labels requiring time-resolved measurement. The developed homogeneous assays exploiting switchable lanthanide luminescence were rapid and simple to perform and thus applicable even to point-ofcare testing. The sensitivities of the homogeneous assays were in the picomolar range, which are still inadequate for some analytes, such as cardiac troponins, requiring ultralow limits of detection. For most analytes, however, the obtained limits of detection were sufficient. The use of recombinant antibody fragments and aptamers as binders allowed site-specific and controlled covalent conjugation to construct labeled binders reproducibly either by using chemical modification or recombinant technology. Luminescent lanthanide labels were shown to be widely applicable for protein detection in various assay setups and to contribute assay sensitivity.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

NIR-Vis Up-Conversion Luminescence in the Yb3+,Er3+ Doped Y2O2S, ZrO2, and NaYF4 Nanomaterials

Since the discovery of the up-conversion phenomenon, there has been an ever increasing interest in up-converting phosphors in which the absorption of two or more low energy photons is followed by emission of a higher energy photon. Most up-conversion luminescence materials operate by using a combination of a trivalent rare earth (lanthanide) sensitizer (e.g. Yb or Er) and an activator (e.g. Er, Ho, Tm or Pr) ion in a crystal lattice. Up-converting phosphors have a variety of potential applications as lasers and displays as well as inks for security printing (e.g. bank notes and bonds). One of the most sophisticated applications of lanthanide up-conversion luminescence is probably in medical diagnostics. However, there are some major problems in the use of photoluminescence based on the direct UV excitation in immunoassays. Human blood absorbs strongly UV radiation as well as the emission of the phosphor in the visible. A promising way to overcome the problems arising from the blood absorption is to use a long wavelength excitation and benefit from the up-conversion luminescence. Since there is practically no absorption by the whole-blood in the near IR region, it has no capability for up-conversion in the excitation wavelength region of the conventional up-converting phosphor based on the Yb3+ (sensitizer) and Er3+ (activator) combination. The aim of this work was to prepare nanocrystalline materials with high red (and green) up-conversion luminescence efficiency for use in quantitative whole-blood immunoassays. For coupling to biological compounds, nanometer-sized (crystallite size below 50 nm) up-converting phosphor particles are required. The nanocrystalline ZrO2:Yb3+,Er3+, Y2O2S:Yb3+,Er3+, NaYF4:Yb3+,Er3+ and NaRF4-NaR’F4 (R: Y, Yb, Er) materials, prepared with the combustion, sol-gel, flux, co-precipitation and solvothermal synthesis, were studied using the thermal analysis, FT-IR spectroscopy, transmission electron microscopy, EDX spectroscopy, XANES/EXAFS measurements, absorption spectroscopy, X-ray powder diffraction, as well as up-conversion and thermoluminescence spectroscopies. The effect of the impurities of the phosphors, crystallite size, as well as the crystal structure on the up-conversion luminescence intensity was analyzed. Finally, a new phenomenon, persistent up-conversion luminescence was introduced and discussed. For efficient use in bioassays, more work is needed to yield nanomaterials with smaller and more uniform crystallite sizes. Surface modifications need to be studied to improve the dispersion in water. On the other hand, further work must be carried out to optimize the persistent up-conversion luminescence of the nanomaterials to allow for their use as efficient immunoassay nanomaterials combining the advantages of both up-conversion and persistent luminescence.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.