Untersuchungen zur Fachsprache der Ökologie und des Umweltschutzes im Deutschen und Finnischen : Bezeichnungsvarianten unter einem geschichtlichen, lexikographischen, morphologischen und linguistisch-pragmatischen Aspekt

The work integrates research in the language and terminology of various fields with lexicography, etymology, semantics, word formation, and pragmatics. Additionally, examination of German and Finnish provides the work with perspective of contrastive linguistics and the translation of texts in specialized fields. The work is an attempt to chart the language, vocabulary, different textual types, and essential communication-connected features of this special field. The study is primary concerned with internal communication within the field of ecology, but it also provides a comparison of the public discussion of environmental issues in Germany and Finland. The work attempts to use textual signs to provide a picture of the literary communication used on the different vertical levels in the central text types within the field. The dictionaries in the fields of environmental issues and ecology for the individual text types are examined primarily from the perspective of their quantity and diversity. One central point of the work is to clarify and collect all of the dictionaries in the field that have been compiled thus far in which German and/or Finnish ware included. Ecology and environmental protection are closely linked not only to each other but also to many other scientific fields. Consequently, the language of the environmental field has acquired an abundance of influences and vocabulary from the language of the special fields close to it as well as from that of politics and various areas of public administration. The work also demonstrates how the popularization of environmental terminology often leads to semantic distortion. Traditionally, scientific texts have used the smallest number of expressions, the purpose of which is to appeal to or influence the behavior of the text recipient. Particularly in Germany, those who support or oppose measures to protect the environment have long been making concerted efforts to represent their own views in the language that they use. When discussing controversial issues competing designations for the same referent or concept are used in accordance with the interest group to which the speaker belongs. One of the objectives of the study is to sensitize recipients of texts to notice the euphemistic expressions that occur in German and Finnish texts dealing with issues that are sensitive from the standpoint of environmental policy. One particular feature of the field is the wealth and large number of variants designating the same entry or concept. The terminological doublets formed by words of foreign origin and their German or Finnish language equivalents are quite typical of the field. Methods of corpus linguistics are used to determine the reasons for the large number of variant designations as well as their functionality.

Zur Sättigung der Valenz in den Kleinen Meldungen des Typus Notiz : Eine pragmatisch fundierte Analyse

Valency Realization in Short Excerpts of News Text. A Pragmatics-funded analysis This dissertation is a study of the so-called pragmatic valency. The aim of the study is to examine the phenomenon both theoretically by discussing the research literature and empirically based on evidence from a text corpus consisting of 218 short excerpts of news text from the German newspaper Frankfurter Allgemeine Zeitung. In the theoretical part of the study, the central concepts of the valency and the pragmatic valency are discussed. In the research literature, the valency denotes the relation among the verb and its obligatory and optional complements. The pragmatic valency can be defined as modification of the so-called system valency in the parole, including non-realization of an obligatory complement, non- realization of an optional complement and realization of an optional complement. Furthermore, the investigation of the pragmatic valency includes the role of the adjuncts, elements that are not defined by the valency, in the concrete valency realization. The corpus study investigates the valency behaviour of German verbs in a corpus of about 1500 sentences combining the methodology and concepts of valency theory, semantics and text linguistics. The analysis is focused on the about 600 sentences which show deviations from the system valency, providing over 800 examples for the modification of the system valency as codified in the (valency) dictionaries. The study attempts to answer the following primary question: Why is the system valency modified in the parole? To answer the question, the concept of modification types is entered. The modification types are recognized using distinctive feature bundles in which each feature with a negative or a positive value refers to one reason for the modification treated in the research literature. For example, the features of irrelevance and relevance, focus, world and text type knowledge, text theme, theme-rheme structure and cohesive chains are applied. The valency approach appears in a new light when explored through corpus-based investigation; both the optionality of complements and the distinction between complements and adjuncts as defined in the present valency approach seem in some respects defective. Furthermore, the analysis indicates that the adjuncts outside the valency domain play a central role in the concrete realization of the valency. Finally, the study suggests a definition of pragmatic valency, based on the modification types introduced in the study and tested in the corpus analysis.

La contextualisation du discours radiophonique par des moyens prosodiques. L’exemple de cinq grands philosophes français du XXe siècle

"Radiodiskurssin kontekstualisointi prosodisin keinoin. Esimerkkinä viisi suurta ranskalaista 1900-luvun filosofia" Väitöskirja käsittelee puheen kontekstualisointia prosodisin keinoin. Toisin sanottuna työssä käsitellään sitä, miten puheen prosodiset piirteet (kuten sävelkulku, intensiteetti, tauot, kesto ja rytmi) ohjaavat puheen tulkintaa vanhastaan enemmän tutkittujen sana- ja lausemerkitysten ohella. Työssä keskitytään seitsemään prosodisesti merkittyyn kuvioon, jotka koostuvat yhden tai usean parametrin silmiinpistävistä muutoksista. Ilmiöitä käsitellään sekä niiden akustisten muotojen että tyypillisten esiintymisyhteyksien ja diskursiivisten tehtävien näkökulmasta. Aineisto koostuu radio-ohjelmista, joissa puhuu viisi suurta ranskalaista 1900-luvun filosofia: Gaston Bachelard, Albert Camus, Michel Foucault, Maurice Merleau-Ponty ja Jean-Paul Sartre. Ohjelmat on lähetetty eri radiokanavilla Ranskassa vuosina 1948–1973. Väitöskirjan tulokset osoittavat, että prosodisesti merkityt kuviot ovat moniulotteisia puheen ilmiöitä, joilla on keskeinen rooli sanotun kontekstualisoinnissa: ne voivat esimerkiksi nostaa tai laskea sanotun informaatioarvoa, ilmaista puhujan voimakasta tai heikkoa sitoutumista sanomaansa, ilmaista rakenteellisen kokonaisuuden jatkumista tai päättymistä, jne. Väitöskirja sisältää myös kontrastiivisia osia, joissa ilmiöitä verrataan erääseen klassisessa pianomusiikissa esiintyvään melodiseen kuvioon sekä erääseen suomen kielen prosodiseen ilmiöön. Tulokset viittaavat siihen, että tietynlaista melodista kuviota käytetään samankaltaisena jäsentämiskeinona sekä puheessa että klassisessa musiikissa. Lisäksi tulokset antavat viitteitä siitä, että tiettyjä melodisia muotoja käytetään samankaltaisten implikaatioiden luomiseen kahdessa niinkin erilaisessa kielessä kuin suomessa ja ranskassa. Yksi väitöskirjan osa käsittelee pisteen ja pilkun prosodista merkitsemistä puheessa. Tulosten mukaan pisteellä ja pilkulla on kummallakin oma suullinen prototyyppinsä: piste merkitään tyypillisesti sävelkulun laskulla ja tauolla, ja pilkku puolestaan sävelkulun nousulla ja tauolla. Merkittävimmät tulokset koskevat kuitenkin tapauksia, joissa välimerkki tulkitaan prosodisesti epätyypillisellä tavalla: sekä pisteellä että pilkulla vaikuttaisi olevan useita eri suullisia vastaavuuksia, ja välimerkkien tehtävät voivat muotoutua hyvin erilaisiksi niiden prosodisesta tulkinnasta riippuen.

« Le roman est agréablement traduit » : Étude comparative sur la visibilité des traductions et des traducteurs en Finlande et en France à travers des critiques des livres

Pro gradu -työssä tutkitaan ja vertaillaan käännöskirjallisuuden arvosteluja Ranskassa ja Suomessa. Empiirinen aineisto koostuu kaikista Helsingin Sanomien ja Le Monden vuonna 2003 julkaisemista arvosteluista. Lehdissä oli yhteensä 2691 arvosteltua kirjaa, joista 845 oli käännöksiä. Päätavoite on ollut selvittää näiden valtasanomalehtien kritiikkejä tutkimalla, kummassa maassa kääntäjän ja käännöksen asema on näkyvämpi ja minkälaisia julkaistut käännöskritiikit ovat. Lisäksi tavoitteena on ollut tutkia, kumpi lehdistä on avoimempi vieraskielisiä kirjoja ja käännöksiä kohtaan. Kirja-arvostelujen lähemmälle tutkimiselle luodaan pohjaa perehtymällä kääntäjän ja käännöksen näkyvyyteen liittyviin seikkoihin. Tässä käytetään hyväksi Koskisen (2000) tutkimusta. Tutkimuksessa tarkastellaan myös, millainen on hyvä käännös eri kääntäjien ja tutkijoiden mielestä, sekä muita saman aihepiirin tutkimuksia ja aiheesta vallalla ollutta keskustelua. Lisäksi selvitetään, miksi laadukkaat käännösarvostelut ovat harvinaisia ja mistä tämä johtuu. Analyysivaiheen kvantitatiivisessa osassa perehdytään käännösten määrälliseen osuuteen sekä aineistossa että Ranskan ja Suomen kokonaisjulkaisumäärissä. Aineiston käännösarvostelut luokitellaan niiden sisällön mukaan. Tässä on käytetty soveltuvin osin Gullinin (1998) kehittelemää mallia. Käännösarvostelujen sisältöanalyysissa kiinnitetään huomiota niiden käännöstä ja kääntäjää koskeviin kommentteihin. Arvostelujen laadun ja kriitikkojen käyttämien arvosteluperusteiden pohdinta nojautuu aiheesta aikaisemmin tehtyihin teoreettisiin sekä empiirisiin tutkimuksiin. Pro gradu -työ sisältää myös erillisen katsauksen käännettyjen lastenkirjojen arvosteluihin sekä arvostelujen ulkopuolisiin kääntäjiin ja kääntämiseen liittyviin artikkeleihin. Koko tutkimuksen ajan lähestymistapa on vertaileva Le Monden ja Helsingin Sanomien välillä. Tutkimuksesta selviää, että Le Monde julkaisee huomattavasti enemmän kirja-arvosteluja. Molemmat lehdet sisältävät kuitenkin suhteellisesti yhtä paljon arvosteluja käännöskirjoista. Helsingin Sanomissa on enemmän vieraskielisten teosten arvosteluja, ja käännösten ja kääntäjän asema on huomattavasti näkyvämpi lehden kritiikeissä. Suurin osa Le Monden käännösarvosteluista sisältää vain kääntäjän nimen bibliografisissa tiedoissa. Helsingin Sanomissa vain alle puolet käännöskirjoista on arvosteltu tällä tavoin. Myös kritiikit, joissa kääntäjän nimeä ei mainita ollenkaan, ovat yleisempiä ranskalaislehdessä. Suhteellisen pieni osa käännösarvosteluista arvioi käännöksen laatua. Näille arvioille on ominaista perustelujen ja analyysin puuttuminen ja ne ovat usein lyhyitä. Arviot ovat sävyltään enimmäkseen positiivisia tai neutraaleja. Hyvin yleistä on myös se, että kriitikko sekoittaa kaksi eri asiaa: kääntäjän ja kirjailijan tyylin. Yleisin kriitikkojen käyttämä arviointikriteeri on tutkia käännöksen ja kohdekielen tai käännöksen ja lähtötekstin suhdetta. Monesti arviointikriteeri jää täysin epäselväksi. Helsingin Sanomien kritiikeissä kääntäjiin viitataan huomattavasti useammin myös itse arvostelutekstissä. Lehti tuo näkyvästi esille kääntäjiä muissakin kuin kirja-arvosteluartikkeleissaan. Sen sijaan Le Mondessa ei ole pelkästään kääntäjiä käsitteleviä tekstejä.

Expression of cytochrome P450 enzymes and metabolism of timolol in human ocular tissue

Glaucoma is a group of progressive optic neuropathies causing irreversible blindness if not diagnosed and treated in the early state of progression. Disease is often, but not always, associated with increased intraocular pressure (IOP), which is also the most important risk factor for glaucoma. Ophthlamic timolol preparations have been used for decades to lower increased intraocular pressure (IOP). Timolol is locally well tolerated but may cause e.g. cardiovascular and pulmonary adverse effects due to systemic absorption. It has been reported that approximately 80% of a topically administered eye drop is systemically absorbed. However, only limited information is available on timolol metabolism in the liver or especially in the human eye. The aim of this work was to investigate metabolism of timolol in human liver and human ocular tissues. The expression of drug metabolizing cytochrome P450 (CYP) enzymes in the human ciliary epithelial cells was studied. The metabolism of timolol and the interaction potential of timolol with other commercially available medicines were investigated in vitro using different liver preparations. The absorption of timolol to the aqueous humor from two commercially available products: 0.1% eye gel and 0.5% eye drops and the presence of timolol metabolites in the aqueous humor were investigated in a clinical trial. Timolol was confirmed to be metabolized mainly by CYP2D6 as previously suggested. Potent CYP2D6 inhibitors especially fluoxetine, paroxetine and quinidine inhibited the metabolism of timolol. The inhibition may be of clinical significance in patients using ophthalmic timolol products. CYP1A1 and CYP1B1 mRNAs were expressed in the human ciliary epithelial cells. CYP1B1 was also expressed at protein level and the expression was strongly induced by a known potent CYP1B1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The CYP1B1 induction is suggested to be mediated by aryl hydrocarbon receptor (AHR). Low levels of CYP2D6 mRNA splice variants were expressed in the human ciliary epithelial cells and very low levels of timolol metabolites were detected in the human aqueous humor. It seems that negligible amount of CYP2D6 protein is expressed in the human ocular tissues. Timolol 0.1% eye gel leads to aqueous humor concentration high enough to achieve therapeutic effect. Inter-individual variation in concentrations is low and intraocular as well as systemic safety can be increased when using this product with lower timolol concentration instead of timolol 0.5% eye drops.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Protein cross-linking with oxidative enzymes and transglutaminase : Effects in meat protein systems

The effects of tyrosinase, laccase and transglutaminase (TG) were studied in different meat protein systems. The study was focused on the effects of the enzymes on the gel formation properties of myofibrils, and on the textural and water-holding properties of the heated meat systems. The cross-linking efficiency of a novel Trichoderma reesei tyrosinase was compared to that of the commercial Agaricus bisporus tyrosinase. Trichoderma tyrosinase was found to be superior compared to the Agaricus enzyme in its protein cross-linking efficiency and in the incorporation of a small molecule into a complex proteinaceous substrate. Tyrosinase, laccase and TG all polymerised myofibrillar proteins, but laccase was also found to cause protein fragmentation. A positive connection between covalent cross-link and gel formation was observed with tyrosinase and TG. Laccase was able to increase the gel formation only slightly. With an excessive laccase dosage the gel formation declined due to protein fragmentation. Tyrosinase, laccase and TG had different effects on the texture and water-holding of the heated chicken breast meat homogenates. Tyrosinase improved the firmness of the homogenate gels free of phosphate and with a low amount of meat. TG improved the firmness of all studied homogenates. Laccase weakened the gel firmness of the low-meat, low-salt and low-salt/phosphate homogenates and maintained the firmness on the control level in the homogenate free of phosphate. Tyrosinase was the only enzyme capable of reducing the weight loss in the homogenates containing a low amount of meat and a low amount of NaCl. TG was the only enzyme that could positively affect the firmness of the homogenate gel containing both low NaCl and phosphate amounts. In pilot scale the test products were made of coarsely ground chicken breast fillet with a moderate amount of salt. Increasining the amount of meat, salt and TG contents favoured the development of firmness of the test products. The evaporation loss decreased slightly along with increasing TG and NaCl amounts in the experimental conditions used, indicating a positive interaction between these two factors. In this work it was shown that tyrosinase, laccase and TG affected the same myofibrillar proteins, i.e. myosin and troponin T. However, these enzymes had distinguishable effects on the gel formation of a myofibril system as well as on the textural and water-holding properties of the finely ground meat homogenates, reflecting distinctions at least in the reaction mechanisms and target amino acid availability in the protein substrates for these enzymes.

Characterization of lignin-modifying enzymes of the selective white-rot fungus Physisporinus rivulosus

White-rot fungi are wood degrading organisms that are able to decompose all wood polymers; lignin, cellulose and hemicellulose. Especially the selective white-rot fungi that decompose preferentially wood lignin are promising for biopulping applications. In biopulping the pretreatment of wood chips with white-rot fungi enhances the subsequent pulping step and substantially reduces the refining energy consumption in mechanical pulping. Because it is not possible to carry out biopulping in industrial scale as a closed process it has been necessary to search for new selective strains of white-rot fungi which naturally occur in Finland and cause selective white-rot of Finnish wood raw-material. In a screening of 300 fungal strains a rare polypore, Physisporinus rivulosus strain T241i isolated from a forest burn research site, was found to be a selective lignin degrader and promising for the use in biopulping. Since selective lignin degradation is apparently essential for biopulping, knowledge on lignin-modifying enzymes and the regulation of their production by a biopulping fungus is needed. White-rot fungal enzymes that participate in lignin degradation are laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP) and hydrogen peroxide forming enzymes. In this study, P. rivulosus was observed to produce MnP, laccase and oxalic acid during growth on wood chips. In liquid cultures manganese and veratryl alcohol increased the production of acidic MnP isoforms detected also in wood chip cultures. Laccase production by P. rivulosus was low unless the cultures were supplemented with sawdust and charred wood, the components of natural growth environment of the fungus. In white-rot fungi the lignin-modifying enzymes are typically present as multiple isoforms. In this study, two MnP encoding genes, mnpA and mnpB, were cloned and characterized from P. rivulosus T241i. Analysis of the N-terminal amino acid sequences of two purified MnPs and putative amino acid sequence of the two cloned mnp genes suggested that P. rivulosus possesses at least four mnp genes. The genes mnpA and mnpB markedly differ from each other by the gene length, sequence and intron-exon structure. In addition, their expression is differentially affected by the addition of manganese and veratryl alcohol. P. rivulosus produced laccase as at least two isoforms. The results of this study revealed that the production of MnP and laccase was differentially regulated in P. rivulosus, which ensures the efficient lignin degradation under a variety of environmental conditions.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Enzymes with radical tendencies: the PFL family

The first glycyl radical in an enzyme was described 20 years ago and since then the family of glycyl radical enzymes (GREs) has expanded to include enzymes catalysing five chemically distinct reactions. The type enzymes of the family, anaerobic ribonucleotide reductase (RNRIII) and pyruvate formate lyase (PFL) had been studied long before it was known that they are GREs. Spectroscopic measurements on the radical and an observation that exposure to oxygen irreversibly inactivates the enzymes by cleavage of the protein proved that the radical is located on a particular glycine residue, close to the C-terminus of the protein. Both anaerobic RNRIII and PFL, are important for many anaerobic and facultative anaerobic bacteria as RNRIII is responsible for the synthesis of DNA precursors and PFL catalyses a key metabolic reaction in glycolysis. The crystal structures of both were solved in 1999 and they revealed that, although the enzymes do not share significant sequence identity, they share a similar structure - the radical site and residues necessary for catalysis are buried inside a ten stranded $\ualpha $/$\ubeta $-barrel. GREs are synthesised in an inactive form and are post-translationally activated by an activating enzyme which uses S-adenosyl methionine and an iron-sulphur cluster to generate the radical. One of the goals of this thesis work was to crystallise the activating enzyme of PFL. This task is challenging as, like GREs, the activating component is inactivated by oxygen. The experiments were therefore carried out in an oxygen free atmosphere. This is the first report of a crystalline GRE activating enzyme. Recently several new GREs have been characterised, all sharing sequence similarity to PFL but not to RNRIII. Also, the genome sequencing projects have identified many PFL-like GREs of unknown function, usually annotated as PFLs. In the present thesis I describe the grouping of these PFL family enzymes based on the sequence similarity and analyse the conservation patterns when compared to the structure of E. coli PFL. Based on this information an activation route is proposed. I also report a crystal structure of one of the PFL-like enzymes with unknown function, PFL2 from Archaeoglobus fulgidus. As A. fulgidus is a hyperthermophilic organism, possible mechanisms stabilising the structure are discussed. The organisation of an active site of PFL2 suggests that the enzyme may be a dehydratase. Keywords: glycyl radical, enzyme, pyruvate formate lyase, x-ray crystallography, bioinformatics

Characterisation, cloning and production of industrially interesting enzymes: gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans

The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.

Acidic pH and acidic enzymes in atherosclerosis

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids and fibrous connective tissue in the arterial wall. Recently, it has been suggested that decrease in the pH of extracellular fluid of the arterial intima may enhance LDL accumulation by increasing binding of the LDL to matrix proteoglycans and also by making the plaque more favorable for acidic enzymes to be active. Many lysosomal acidic enzymes have been found in atherosclerotic plaques. In this thesis, we were able to induce secretion of lysosomal acidic cathepsin F from human monocyte-derived macrophages by stimulation with angiotensin II. We also showed that LDL pre-proteolyzed with cathepsin S was more prone to subsequent hydrolytic modifications by lipases. Especially acidic secretory sphingomyelinase was able to hydrolyze pre-proteolyzed LDL even at neutral pH. We also showed that the proteolyzed and lipolyzed LDL particles were able to bind more efficiently to human aortic proteoglycans. In addition, the role of extracellular acidic pH on the ability of macrophages to internalize LDL was studied. At acidic pH, the production of cell surface proteoglycans in macrophages was increased as well as the binding of native and modified LDL to cell surface proteoglycans. Furthermore, macrophages cultured at acidic pH showed increased internalization of modified and native LDL leading to foam cell formation. This thesis revealed various mechanisms by which acidic pH can increase LDL retention and accumulation in the arterial intima and has the potential to increase the progression of atherosclerosis.