Understanding the bioactivity of plant tannins: developments in analysis methods and structure-activity studies

Tannins, typically segregated into two major groups, the hydrolyzable tannins (HTs) and the proanthocyanidins (PAs), are plant polyphenolic secondary metabolites found throughout the plant kingdom. On one hand, tannins may cause harmful nutritional effects on herbivores, for example insects, and hence they work as plants’ defense against plant-eating animals. On the other hand, they may affect positively some herbivores, such as mammals, for example by their antioxidant, antimicrobial, anti-inflammatory or anticarcinogenic activities. This thesis focuses on understanding the bioactivity of plant tannins, their anthelmintic properties and the tools used for the qualitative and quantitative analysis of this endless source of structural diversity. The first part of the experimental work focused on the development of ultra-high performance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) based methods for the rapid fingerprint analysis of bioactive polyphenols, especially tannins. In the second part of the experimental work the in vitro activity of isolated and purified HTs and their hydrolysis product, gallic acid, was tested against egg hatching and larval motility of two larval developmental stages, L1 and L2, of a common ruminant gastrointestinal parasite, Haemonchus contortus. The results indicated clear relationships between the HT structure and the anthelmintic activity. The activity of the studied compounds depended on many structural features, including size, functional groups present in the structure, and the structural rigidness. To further understand tannin bioactivity on a molecular level, the interaction between bovine serum albumin (BSA), and seven HTs and epigallocatechin gallate was examined. The objective was to define the effect of pH on the formation on tannin–protein complexes and to evaluate the stability of the formed complexes by gel electrophoresis and MALDI-TOF-MS. The results indicated that more basic pH values had a stabilizing effect on the tannin–protein complexes and that the tannin oxidative activity was directly linked with their tendency to form covalently stabilized complexes with BSA at increased pH.

Pyridyyliryhmän sisältävien erotusmateriaalien ominaisuudet ja soveltaminen hydrometallurgiassa

Diplomityössä tutkittiin silikarunkoisen, pyridyyliryhmän sisältävän erotusmateriaalin kykyä erottaa selektiivisesti kuparia, nikkeliä, kobolttia ja kadmiumia sinkkisulfaattiliuoksista. Erotuskykyä verrattiin kolonnikokein toiseen kaupalliseen polystyreenidivinyylibentseenirunkoiseen (PS-DVB) erotusmate-riaaliin, jonka funktionaalinen ryhmä on samantyyppinen. Silikarunkoisen erotusmateriaalin happo-emäsominaisuuksia selvitettiin titrauksin. Metallien sitoutumisen kinetiikkaa ja eluointia verrattiin PS-DVB-runkoisen erotus-materiaalin kirjallisuudessa esitettyihin tuloksiin. Lisäksi määritettiin kummankin erotusmateriaalin partikkelikokojakaumat. Silikarunkoisen materiaalin havaittiin turpoavan noin 4 % muutettaessa se vapaastaemäsmuodosta happomuotoon. Turpoaminen oli huomattavasti vähäisempää kuin PS-DVB-runkoisella materiaalilla. Silikarunkoisen erotus-materiaalin titrauksen tuloksena voitiin todeta, että se oli heikosti emäksinen. Titrauskäyrä muistutti PS-DVB-runkoisen erotusmateriaalin titrauskäyrää, vaikka erotusmateriaalien protonoitumisalueet olivat hieman erilaiset. Tämä johtuu siitä, että silikarunkoisen materiaalin funktionaaliset ryhmät poikkeavat rakenteeltaan hieman PS-DVB-runkoisesta materiaalista. Tasapainokokeet osoittivat, että silikarunkoinen erotusmateriaali adsorboi tutkituista metalleista eniten kuparia ja nikkeliä ja vähiten sinkkiä. Kaikkien tutkittujen metallien eluointi silikarunkoisesta erotusmateriaalista onnistui rikkihapolla toisin kuin PS-DVB-runkoisesta erotusmateriaalista, jonka regenerointiin tarvitaan rikkihappoa tai ammoniumhydroksidia riippuen siitä, mitä metalleja siihen on ladattu.

Nikkelin takaisinuutto karboksyylihappoon perustuvasta uuttoreagenssista

Työssä tutkittiin typpihapon soveltuvuutta nikkelin takaisinuuttoon. Tarkoituksena oli selvittää, millä typpihapon konsentraatioilla orgaaninen faasi, joka koostuu Versatic 10 uuttoreagenssista ja alifaattisesta laimentimesta, alkaa nitrautua tai hapettua ja mitkä ovat mahdolliset sivureaktiot. Lisäksi tutkittiin rikkihapon ja eräiden orgaanisten aineiden kontaminaation vaikutusta uuttoliuokseen. Kirjallisuusosassa kartoitetaan mahdollisten nitrautumisreaktioiden mekanismit, sekä kuvataan laimentimen, uuttoreagenssin ja mahdollisten reaktiotuotteiden ominaisuuksia, sekä niiden mahdollisessa muodostumisessa syntyviä riskejä. Orgaanisen faasin kestotesteissä tutkittavia muuttujia olivat typpi- ja rikkihapon konsentraatio, sekoitusaika, lämpötila, avoin tai suljettu astia sekä vieraiden aineiden kontaminaatio. Kontaminaatiota aiheuttavien orgaanisten materiaalien funktionaaliset ryhmät olivat hydroksi-, karbonyyli- ja amiiniryhmät, joiden lisäksi tutkittiin syklisen yhdisteen kontaminaatiota. Analyyseissä käytettiin FT-IR- spektroskopiaa, jolla tutkittiin reagenssin funktionaalisen ryhmän reaktioita ja uusien ryhmien muodostumista, sekä seurattiin selkeytyksessä erottumattomien typpiyhdisteiden määrää ja laatua orgaanisessa faasissa. Uuttofaasin koostumuksen muutosta seurattiin myös mittaamalla leimahduspisteen muutosta.

Upconverting Phosphor Technology: Exceptional Photoluminescent Properties Light Up Homogeneous Bioanalytical Assays

The aim of the present study was to demonstrate the wide applicability of the novel photoluminescent labels called upconverting phosphors (UCPs) in proximity-based bioanalytical assays. The exceptional features of the lanthanide-doped inorganic UCP compounds stem from their capability for photon upconversion resulting in anti-Stokes photoluminescence at visible wavelengths under near-infrared (NIR) excitation. Major limitations related to conventional photoluminescent labels are avoided, rendering the UCPs a competitive next-generation label technology. First, the background luminescence is minimized due to total elimination of autofluorescence. Consequently, improvements in detectability are expected. Second, at the long wavelengths (>600 nm) used for exciting and detecting the UCPs, the transmittance of sample matrixes is significantly greater in comparison with shorter wavelengths. Colored samples are no longer an obstacle to the luminescence measurement, and more flexibility is allowed even in homogeneous assay concepts, where the sample matrix remains present during the entire analysis procedure, including label detection. To transform a UCP particle into a biocompatible label suitable for bioanalytical assays, it must be colloidal in an aqueous environment and covered with biomolecules capable of recognizing the analyte molecule. At the beginning of this study, only UCP bulk material was available, and it was necessary to process the material to submicrometer-sized particles prior to use. Later, the ground UCPs, with irregular shape, wide size-distribution and heterogeneous luminescence properties, were substituted by a smaller-sized spherical UCP material. The surface functionalization of the UCPs was realized by producing a thin hydrophilic coating. Polymer adsorption on the UCP surface is a simple way to introduce functional groups for bioconjugation purposes, but possible stability issues encouraged us to optimize an optional silica-encapsulation method which produces a coating that is not detached in storage or assay conditions. An extremely thin monolayer around the UCPs was pursued due to their intended use as short-distance energy donors, and much attention was paid to controlling the thickness of the coating. The performance of the UCP technology was evaluated in three different homogeneous resonance energy transfer-based bioanalytical assays: a competitive ligand binding assay, a hybridization assay for nucleic acid detection and an enzyme activity assay. To complete the list, a competitive immunoassay has been published previously. Our systematic investigation showed that a nonradiative energy transfer mechanism is indeed involved, when a UCP and an acceptor fluorophore are brought into close proximity in aqueous suspension. This process is the basis for the above-mentioned homogeneous assays, in which the distance between the fluorescent species depends on a specific biomolecular binding event. According to the studies, the submicrometer-sized UCP labels allow versatile proximity-based bioanalysis with low detection limits (a low-nanomolar concentration for biotin, 0.01 U for benzonase enzyme, 0.35 nM for target DNA sequence).

Modeling of electrolyte sorption – from phase equilibria to dynamic separation systems

In this thesis, general approach is devised to model electrolyte sorption from aqueous solutions on solid materials. Electrolyte sorption is often considered as unwanted phenomenon in ion exchange and its potential as an independent separation method has not been fully explored. The solid sorbents studied here are porous and non-porous organic or inorganic materials with or without specific functional groups attached on the solid matrix. Accordingly, the sorption mechanisms include physical adsorption, chemisorption on the functional groups and partition restricted by electrostatic or steric factors. The model is tested in four Cases Studies dealing with chelating adsorption of transition metal mixtures, physical adsorption of metal and metalloid complexes from chloride solutions, size exclusion of electrolytes in nano-porous materials and electrolyte exclusion of electrolyte/non-electrolyte mixtures. The model parameters are estimated using experimental data from equilibrium and batch kinetic measurements, and they are used to simulate actual single-column fixed-bed separations. Phase equilibrium between the solution and solid phases is described using thermodynamic Gibbs-Donnan model and various adsorption models depending on the properties of the sorbent. The 3-dimensional thermodynamic approach is used for volume sorption in gel-type ion exchangers and in nano-porous adsorbents, and satisfactory correlation is obtained provided that both mixing and exclusion effects are adequately taken into account. 2-Dimensional surface adsorption models are successfully applied to physical adsorption of complex species and to chelating adsorption of transition metal salts. In the latter case, comparison is also made with complex formation models. Results of the mass transport studies show that uptake rates even in a competitive high-affinity system can be described by constant diffusion coefficients, when the adsorbent structure and the phase equilibrium conditions are adequately included in the model. Furthermore, a simplified solution based on the linear driving force approximation and the shrinking-core model is developed for very non-linear adsorption systems. In each Case Study, the actual separation is carried out batch-wise in fixed-beds and the experimental data are simulated/correlated using the parameters derived from equilibrium and kinetic data. Good agreement between the calculated and experimental break-through curves is usually obtained indicating that the proposed approach is useful in systems, which at first sight are very different. For example, the important improvement in copper separation from concentrated zinc sulfate solution at elevated temperatures can be correctly predicted by the model. In some cases, however, re-adjustment of model parameters is needed due to e.g. high solution viscosity.

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.

Terpenoid transformations over gold catalysts

Nowadays biomass transformation has a great potential for the synthesis of value-added compounds with a wide range of applications. Terpenoids, extracted from biomass, are inexpensive and renewable raw materials which often have a biological activity and are widely used as important organic platform molecules in the development of new medicines as well as in the synthesis of fine chemicals and intermediates. At the same time, special attention is devoted to the application of gold catalysts to fine chemical synthesis due to their outstanding activity and/or selectivity for transformations of complex organic compounds. Conversion of renewable terpenoids in the presence of gold nanoparticles is one of the new and promising directions in the transformation of biomass to valuable chemicals. In the doctoral thesis, different kinds of natural terpenoids, such as α-pinene, myrtenol and carvone were selected as starting materials. Gold catalysts were utilized for the promising routes of these compounds transformation. Investigation of selective α-pinene isomerization to camphene, which is an important step in an industrial process towards the synthesis of camphor as well as other valuable substrates for the pharmaceutical industry, was performed. A high activity of heterogeneous gold catalysts in the Wagner-Meerwein rearrangement was demonstrated for the first time. Gold on alumina carrier was found to reach the α-pinene isomerization conversion up to 99.9% and the selectivity of 60-80%, thus making this catalyst very promising from an industrial viewpoint. A detailed investigation of kinetic regularities including catalyst deactivation during the reaction was performed. The one-pot terpene alcohol amination, which is a promising approach to the synthesis of valuable complex amines having specific physiological properties, was investigated. The general regularities of the one-pot natural myrtenol amination in the presence of gold catalysts as well as a correlation between catalytic activity, catalyst redox treatment and the support nature were obtained. Catalytic activity and product distribution were shown to be strongly dependent on the support properties, namely acidity and basicity. The gold-zirconia (Au/ZrO2) catalyst pretreated under oxidizing atmosphere was observed to be rather active, resulting in the total conversion of myrtenol and the selectivity to the corresponding amine of about 53%. The reaction kinetics was modelled based on the mechanistic considerations with the catalyst deactivation step incorporated in the mechanism. Carvone hydrogenation over a gold catalyst was studied with the general idea of investigating both the activity of gold catalysts in competitive hydrogenation of different functional groups and developing an approach to the synthesis of valuable carvone derivatives. Gold was found to promote stereo- and chemoselective carvone hydrogenation to dihydrocarvone with a predominant formation of the trans-isomer, which generally is a novel synthetic method for an industrially valuable dihydrocarvone. The solvent effect on the catalytic activity as well as on the ratio between trans- and cis-dihydrocarvone was evaluated.

New [18F]Tracers for Alzheimer's Disease Imaging. Labeling Synthesis And Biological Testing

Alzheimer’s disease (AD) is the most common form of dementia. Characteristic changes in an AD brain are the formation of β-amyloid protein (Aβ) plaques and neurofibrillary tangles, though other alterations in the brain have also been connected to AD. No cure is available for AD and it is one of the leading causes of death among the elderly in developed countries. Liposomes are biocompatible and biodegradable spherical phospholipid bilayer vesicles that can enclose various compounds. Several functional groups can be attached on the surface of liposomes in order to achieve long-circulating target-specific liposomes. Liposomes can be utilized as drug carriers and vehicles for imaging agents. Positron emission tomography (PET) is a non-invasive imaging method to study biological processes in living organisms. In this study using nucleophilic 18F-labeling synthesis, various synthesis approaches and leaving groups for novel PET imaging tracers have been developed to target AD pathology in the brain. The tracers were the thioflavin derivative [18F]flutemetamol, curcumin derivative [18F]treg-curcumin, and functionalized [18F]nanoliposomes, which all target Aβ in the AD brain. These tracers were evaluated using transgenic AD mouse models. In addition, 18F-labeling synthesis was developed for a tracer targeting the S1P3 receptor. The chosen 18F-fluorination strategy had an effect on the radiochemical yield and specific activity of the tracers. [18F]Treg-curcumin and functionalized [18F]nanoliposomes had low uptake in AD mouse brain, whereas [18F]flutemetamol exhibited the appropriate properties for preclinical Aβ-imaging. All of these tracers can be utilized in studies of the pathology and treatment of AD and related diseases.

Functional Genomic Approaches for Deciphering Plant-Virus Interactions

Plant-virus interactions are very complex in nature and lead to disease and symptom formation by causing various physiological, metabolic and developmental changes in the host plants. These interactions are mainly the outcomes of viral hijacking of host components to complete their infection cycles and of host defensive responses to restrict the viral infections. Viral genomes contain only a small number of genes often encoding for multifunctional proteins, and all are essential in establishing a viral infection. Thus, it is important to understand the specific roles of individual viral genes and their contribution to the viral life cycles. Among the most important viral proteins are the suppressors of RNA silencing (VSRs). These proteins function to suppress host defenses mediated by RNA silencing and can also serve in other functions, e.g. in viral movement, transactivation of host genes, virus replication and protein processing. Thus these proteins are likely to have a significant impact on host physiology and metabolism. In the present study, I have examined the plant-virus interactions and the effects of three different VSRs on host physiology and gene expression levels by microarray analysis of transgenic plants that express these VSR genes. I also studied the gene expression changes related to the expression of the whole genome of Tobacco mosaic virus (TMV) in transgenic tobacco plants. Expression of the VSR genes in the transgenic tobacco plants causes significant changes in the gene expression profiles. HC-Pro gene derived from the Potyvirus Y (PVY) causes alteration of 748 and 332 transcripts, AC2 gene derived from the African cassava mosaic virus (ACMV) causes alteration of 1118 and 251transcripts, and P25 gene derived from the Potyvirus X (PVX) causes alterations of 1355 and 64 transcripts in leaves and flowers, respectively. All three VSRs cause similar up-regulation in defense, hormonally regulated and different stress-related genes and down-regulation in the photosynthesis and starch metabolism related genes. They also induce alterations that are specific to each viral VSR. The phenotype and transcriptome alterations of the HC-Pro expressing transgenic plants are similar to those observed in some Potyvirus-infected plants. The plants show increased protein degradation, which may be due to the HC-Pro cysteine endopeptidase and thioredoxin activities. The AC2-expressing transgenic plants show a similar phenotype and gene expression pattern as HC-Pro-expressing plants, but also alter pathways related to jasmonic acid, ethylene and retrograde signaling. In the P25 expressing transgenic plants, high numbers of genes (total of 1355) were up-regulated in the leaves, compared to a very low number of down-regulated genes (total of 5). Despite of strong induction of the transcripts, only mild growth reduction and no other distinct phenotype was observed in these plants. As an example of whole virus interactions with its host, I also studied gene expression changes caused by Tobacco mosaic virus (TMV) in tobacco host in three different conditions, i.e. in transgenic plants that are first resistant to the virus, and then become susceptible to it and in wild type plants naturally infected with this virus. The microarray analysis revealed up and down-regulation of 1362 and 1422 transcripts in the TMV resistant young transgenic plants, and up and down-regulation of a total of 1150 and 1200 transcripts, respectively, in the older plants, after the resistance break. Natural TMV infections in wild type plants caused up-regulation of 550 transcripts and down-regulation of 480 transcripts. 124 up-regulated and 29 down-regulated transcripts were commonly altered between young and old TMV transgenic plants, and only 6 up-regulated and none of the down-regulated transcripts were commonly altered in all three plants. During the resistant stage, the strong down-regulation in translation-related transcripts (total of 750 genes) was observed. Additionally, transcripts related to the hormones, protein degradation and defense pathways, cell division and stress were distinctly altered. All these alterations may contribute to the TMV resistance in the young transgenic plants, and the resistance may also be related to RNA silencing, despite of the low viral abundance and lack of viral siRNAs or TMV methylation activity in the plants.

Glutathione Transferases as Detoxification Agents: Structural and Functional Studies

Glutathione transferases (GSTs) are a diverse family of enzymes that catalyze the glutathione-dependent detoxification of toxic compounds. GSTs are responsible for the conjugation of the tripeptide glutathione (GSH) to a wide range of electrophilic substrates. These include industrial pollutants, drugs, genotoxic carcinogen metabolites, antibiotics, insecticides and herbicides. In light of applications in biomedicine and biotechnology as cellular detoxification agents, detailed structural and functional studies of GSTs are required. Plant tau class GSTs play crucial catalytic and non-catalytic roles in cellular xenobiotic detoxification process in agronomically important crops. The abundant existence of GSTs in Glycine max and their ability to provide resistance to abiotic and biotic stresses such as herbicide tolerance is of great interest in agriculture because they provide effective and suitable tools for selective weed control. Structural and catalytic studies on tau class GST isoenzymes from Glycine max (GmGSTU10-10, GmGSTU chimeric clone 14 (Sh14), and GmGSTU2-2) were performed. Crystal structures of GmGSTU10-10 in complex with glutathione sulfenic acid (GSOH) and Sh14 in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were determined by molecular replacement at 1.6 Å and 1.75 Å, respectively. Major structural variations that affect substrate recognition and catalytic mechanism were revealed in the upper part of helix H4 and helix H9 of GmGSTU10-10. Structural analysis of Sh14 showed that the Trp114Cys point mutation is responsible for the enhanced catalytic activity of the enzyme. Furthermore, two salt bridges that trigger an allosteric effect between the H-sites were identified at the dimer interface between Glu66 and Lys104. The 3D structure of GmGSTU2-2 was predicted using homology modeling. Structural and phylogenetic analysis suggested GmGSTU2-2 shares residues that are crucial for the catalytic activity of other tau class GSTs–Phe10, Trp11, Ser13, Arg20, Tyr30, Leu37, Lys40, Lys53, Ile54, Glu66 and Ser67. This indicates that the catalytic and ligand binding site in GmGSTU2-2 are well-conserved. Nevertheless, at the ligandin binding site a significant variation was observed. Tyr32 is replaced by Ser32 in GmGSTU2-2 and thismay affect the ligand recognition and binding properties of GmGSTU2-2. Moreover, docking studies revealed important amino acid residues in the hydrophobic binding site that can affect the substrate specificity of the enzyme. Phe10, Pro12, Phe15, Leu37, Phe107, Trp114, Trp163, Phe208, Ile212, and Phe216 could form the hydrophobic ligand binding site and bind fluorodifen. Additionally, side chains of Arg111 and Lys215 could stabilize the binding through hydrogen bonds with the –NO2 groups of fluorodifen. GST gene family from the pathogenic soil bacterium Agrobacterium tumefaciens C58 was characterized and eight GST-like proteins in A. tumefaciens (AtuGSTs) were identified. Phylogenetic analysis revealed that four members of AtuGSTs belong to a previously recognized bacterial beta GST class and one member to theta class. Nevertheless, three AtuGSTs do not belong to any previously known GST classes. The 3D structures of AtuGSTs were predicted using homology modeling. Comparative structural and sequence analysis of the AtuGSTs showed local sequence and structural characteristics between different GST isoenzymes and classes. Interactions at the G-site are conserved, however, significant variations were seen at the active site and the H5b helix at the C-terminal domain. H5b contributes to the formation of the hydrophobic ligand binding site and is responsible for recognition of the electrophilic moiety of the xenobiotic. It is noted that the position of H5b varies among models, thus providing different specificities. Moreover, AtuGSTs appear to form functional dimers through diverse modes. AtuGST1, AtuGST3, AtuGST4 and AtuGST8 use hydrophobic ‘lock–and–key’-like motifs whereas the dimer interface of AtuGST2, AtuGST5, AtuGST6 and AtuGST7 is dominated by polar interactions. These results suggested that AtuGSTs could be involved in a broad range of biological functions including stress tolerance and detoxification of toxic compounds.

Ydinvoimalaitoksen tuotannon ylläpitoprosessin suorituskyvyn mittaaminen

Kilpailun kiristyminen on pakottanut ydinvoimalaitoksia parantamaan tehokkuuttaan etsimällä uusia toimintatapoja. Heräte työn teettämiseen syntyi tutkimuksen case-kohteen Loviisan voimalaitoksen kiinnostuksesta Balanced Scorecard (BSC) -johtamisjärjestelmää,sen käyttöönoton mahdollisia vaikutuksia sekä BSC:n ja prosessiajattelun yhdistämistä kohtaan. Tutkimuksen tavoitteena on rakentaa Loviisan voima-laitoksen tuotannon ylläpitoprosessille BSC-mittaristo. Tämä edellyttää selvitystä siitä, mitä erityispiirteitä ydinvoimalaitoksiin liittyy strategisen suorituskyvyn mittaamisen kohteena. Lisäksi tavoitteena on selvittää, mikä tulisi prosessikohtaisten tuloskorttien rooli olla Loviisan voimalaitoksen BSC-järjestelmässä. Tavoitteenaon myös muodostaa suositus toimintamallista, jolla BSC voitaisiin ottaa käyttöön Loviisan voimalaitoksella, sekä selvittää, mitä vaikutuksia käyttöönotolla voiolla. Ydinvoimalaitoksen erityispiirteitä ovat muutokset toimintaympäristössä, viranomais-valvonta, toiminnan pitkäjänteisyys, laaja osaamis- ja tietotarve sekä turvallisuuden ja tiettyjen sidosryhmäsuhteiden merkityksen korostuminen. Johtuen erityispiirteistä Kaplanin ja Nortonin alkuperäistä asiakasnäkökulmaa muutetaan kattamaan sidosryhmät laajemmin. Tuotannon ylläpitoprosessin tuloskortissa vähiten painottuva näkö-kulma on henkilöstön ja uudistumisen näkökulma. Osa laitostason kriittisistä menestystekijöistä todetaan prosessin kannalta epäolennaisiksi. Prosessikohtaiset tuloskortit osoittautuvat vaikeasti hyödynnettäviksi linjaorganisaation ohjaamisessa. Strategiakartta todetaan hyväksi työvälineeksi BSC:nlaadinnassa. Toivasen projektimalli arvioidaan sopivaksi välineeksi mahdolliseen BSC:n käyttöönottoon Loviisan voimalaitoksella. Henkilöstön rooli ja erityispiirteiden vaikutukset tulee kuitenkin tarkistaa ennen mallin käyttöä. BSC-järjestelmän käyttöönoton arvioidaan selkeyttävän voimalaitoksen mittaristokokonaisuutta sekä parantavan syy-seuraussuhteiden hahmottamista ja alempien tasojen tavoitteiden kytkentää laitostason tavoitteisiin.

Chromatographic analysis of lignans supplemented in foods

Fytoestrogeenit ovat kasvimateriaalista peräisin olevia yhdisteitä, joilla on ihmisen estrogeeni-hormonin kaltaista aktiivisuutta. Fytoestrogeenit voidaan jakaa kolmeen pääryhmään, joista yksi merkittävä ryhmä on lignaanit. Lignaaneilla on todettu olevan antioxidatiivisia, antiviraalisia ja antibakteriaalisia ominaisuuksia. Niillä on todettu olevan myös positiivisia vaikutuksia hormoniperäisten syöpien ehkäisyssä. Näiden ominaisuuksien vuoksi lignaaneja pyritään hyödyntämään esimerkiksi aktiivisina ainesosina funktionaalisissa elintarvikkeissa. Tässä työssä tutkittiin lignaanin, hydroksimatairesinolin (HMRlignanTM) kemiallisia ominaisuuksia ja soveltuvuutta eri ruoka-aineisiin. Työn tarkoituksena oli selvittää ruoka-aineisiin lisätyn hydroksimatairesinolin kemiallista pysyvyyttä erilaisissa säilytys- ja prosessointioloissa sekä tutkia lignaanin liukoisuutta erilaisiin liuottimiin. Hydroksimatairesinolin analysoimiseksi ruoka-aineista käytettiin korkean erotuskyvyn omaavaa nestekromatografista menetelmää. Menetelmä validoitiin ennen varsinaista analysointia ICH-ohjeiston mukaisesti. Validoinnissa tutkittiin kromatografiamenetelmän spesifisyys, lineaarisuus, tarkkuus, oikeellisuus sekä detektointi- ja määritysrajat tutkittavalle lignaanille. Käytetty menetelmä soveltui hyvin lignaanin analysoimiseen ruoka-aineista. Hydroksimatairesinolin vesiliukoisuuden todettiin olevan noin 1 mg/ml. Tutkimukset osoittivat hydroksimatairesinolin olevan stabiili alle 50ºC:en lämpötiloissa. Korkeammissa lämpötiloissa hydroksimatairesinoli oli stabiili jauhemuodossa lisättynä.

Facilitating coordination improvement efforts in cross-functional process development programs

Business process improvement is a common approach in increasing the effectiveness of an organization. It can be seen as an effort to increase coordination between units. Process improvement has proved to be challenging, and most management consultation firms facilitate organizations in this kind of initiatives. Cross-functional improvement is one of the main areas for internal consultants as well. However, the needs, challenges and means of cross-functional help have been rarely discussed in the literature. The objective of this thesis is on one hand to present a conceptual and descriptive framework to help understand the challenges of facilitating coordination improvement efforts in cross-functional improvement programs, and on the other hand to develop and test feasible solutions for some facilitation situations. The research questions are: 1. Why and in what kind of situations do organizations need help in developing coordination in cross-functional processes? 2. How can a facilitator help organizations in improving coordination to develop cross-functional processes? The study consists of two parts. The first part is an overview of the dissertation, and the second part comprises six research publications. The theoretical background for the study are the differentiation causing challenges in cross-functional settings, the coordination needed to improve processes, change management principles, methods and tools, and consultation practises. Three of the publications introduce tools for helping in developing prerequisites, planning responsibilities and supporting learning during the cross-functional program. The three other papers present frameworks to help understand and analyse the improvement situation. The main methodological approaches used in this study are design science research, action research and case research. The research data has been collected from ten cases representing different kinds of organizations, processes and developing situations. The data has been collected mainly by observation, semi-structured interviews and questionnaires. The research contributes to the rare literature combining coordination theories and process improvement practises. It also provides additional understanding of a holistic point of view in process improvement situations. The most important contribution is the addition to the theories of facilitating change in process improvement situations. From the managerial point of view, this study gives advice to managers and consultants in planning and executing cross-functional programs. The main factors increasing the need for facilitation are the challenges for differentiation, challenges of organizational change in general, and the novelty of initiatives and improvement practices concerning process development. Organizations need help in creating the prerequisites to change, in planning initiatives, easing conflict management and collaboration between groups, as well as supporting the learning of cross-functional improvement. The main challenges of facilitation are combining the different roles as a consultant, maintaining the ownership for the improvement project with the client, and supporting learning in the client organization.

Fractionation of Hen Egg and Oat Lipids with supercritical Fluids Chemical and functional Properties of Fractions

Hen eggs and oats (Avena Sativa) are important materials for the food industry. Today, instead of merely satisfying the feeling of hunger, consumers are asking for healthier, biologically active and environmentally friendly products. The growing awareness of consumers’ increasing demands presents a great challenge to the food industry to develop more sustainable products and utilise modern and effective techniques. The modification of yolk fatty acid composition by means of feed supplements is well understood. Egg yolk phospholipids are polar lipids and are used in several applications including food, cosmetics, pharmaceuticals, and special nutrients. Egg yolk phospholipids are excellent emulsifiers, typically sold as mixtures of phospholipids, triacylglycerols, and cholesterol. However, highly purified and characterised phospholipids are needed in several sophisticated applications. Industrial fractionation of phospholipids is usually based on organic solvents. With these fractionation techniques, some harmful residues of organic solvents may cause problems in further processing. The objective of the present study was to investigate the methods to improve the functional properties of eggs, to develop techniques to isolate the fractions responsible for the specific functional properties of egg yolk lipids, and to apply the developed techniques to plant-based materials, too. Fractionation techniques based on supercritical fluids were utilised for the separation of the lipid fractions of eggs and oats. The chemical and functional characterisation of the fractions were performed, and the produced oat polar lipid fractions were tested as protective barrier in encapsulation processes. Modifying the fatty acid compositions of egg yolks with different types of oil supplements in feed had no affect on their functional or sensory properties. Based on the results of functional and sensory analysis, it is evident that eggs with modified fatty acid compositions are usable in several industrial applications. These applications include liquid egg yolk products used in mayonnaise and salad dressings. Egg yolk powders were utilised in different kinds of fractionation processes. The precipitation method developed in this study resembles the supercritical anti-solvent method, which is typically used in the pharmaceutical industry. With pilot scale supercritical fluid processes, non-polar lipids and polar lipids were successfully separated from commercially produced egg yolk powder and oat flakes. The egg and oat-based polar lipid fractions showed high purities, and the corresponding delipidated fractions produced using supercritical techniques offer interesting starting materials for the further production of bioactive compounds. The oat polar lipid fraction contained especially digalactosyadiacylglycerol, which was shown to have valuable functional properties in the encapsulation of probiotics.

Regulation of photosynthetic electron transfer in plant chloroplasts

This thesis focuses on the molecular mechanisms regulating the photosynthetic electron transfer reactions upon changes in light intensity. To investigate these mechanisms, I used mutants of the model plant Arabidopsis thaliana impaired in various aspects of regulation of the photosynthetic light reactions. These included mutants of photosystem II (PSII) and light harvesting complex II (LHCII) phosphorylation (stn7 and stn8), mutants of energy-dependent non-photochemical quenching (NPQ) (npq1 and npq4) and of regulation of photosynthetic electron transfer (pgr5). All of these processes have been extensively investigated during the past decades, mainly on plants growing under steady-state conditions, and therefore many aspects of acclimation processes may have been neglected. In this study, plants were grown under fluctuating light, i.e. the alternation of low and high intensities of light, in order to maximally challenge the photosynthetic regulatory mechanisms. In pgr5 and stn7 mutants, the growth in fluctuating light condition mainly damaged PSI while PSII was rather unaffected. It is shown that the PGR5 protein regulates the linear electron transfer: it is essential for the induction of transthylakoid ΔpH that, in turn, activates energy-dependent NPQ and downregulates the activity of cytochrome b6f. This regulation was shown to be essential for the photoprotection of PSI under fluctuations in light intensity. The stn7 mutants were able to acclimate under constant growth light conditions by modulating the PSII/PSI ratio, while under fluctuating growth light they failed in implementing this acclimation strategy. LHCII phosphorylation ensures the balance of the excitation energy distribution between PSII and PSI by increasing the probability for excitons to be trapped by PSI. LHCII can be phosphorylated over all of the thylakoid membrane (grana cores as well as stroma lamellae) and when phosphorylated it constitutes a common antenna for PSII and PSI. Moreover, LHCII was shown to work as a functional bridge that allows the energy transfer between PSII units in grana cores and between PSII and PSI centers in grana margins. Consequently, PSI can function as a quencher of excitation energy. Eventually, the LHCII phosphorylation, NPQ and the photosynthetic control of linear electron transfer via cytochrome b6f work in concert to maintain the redox poise of the electron transfer chain. This is a prerequisite for successful plant growth upon changing natural light conditions, both in short- and long-term.