Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.

The Saintly Madman : A Study of the Scholarly Reception History of Buile Shuibhne

This study examines the scholarly reception history of an early Irish text, Buile Shuibhne (The Frenzy of Suibhne), by focusing on the various theoretical and methodological presuppositions which have determined the scholars’ understanding of the text’s religious allegorical significance in the course of the 20th century. The reception-oriented inquiry takes the intersubjective aspect of literary interpretation as the basis for accentuating the importance of communally shared presumptions and reading strategies in the explication of interpretive variety. The materials of the study have been divided into four frameworks of interpretation: historical, pre-Christian, Christian and anthropological. This heuristic division does not denote mutually exclusive paradigms, but rather refers to perceived similarities within each group regarding the questions posed, and the evidence adduced, in textual analysis. The historical framework concentrates on the issues of the origins of the tale and the possible historicity of its main protagonist. The pre-Christian framework covers the theories of the shamanic, Indo-European and Celtic elements in the text, whereas the Christian framework includes readings emphasising the biblical, monastic and ascetic aspects of the tale. The anthropological framework in turn focuses on the parallels drawn between the narrative and the universal structure of the rites of passage. In addition to the examination of these four frameworks, the study also links the question of methodology with wider issues of authorship and textual integrity, and critically reconsiders the manner in which J.G. O'Keeffe's 1913 edition of the text has been reified in previous scholarship as a representation of a 12th century authorial original. The overall objective of the present case-study is to relate theoretical conceptions of literary theory, comparative religion and historiography to the study of early Irish narrative material by considering the communal and institutional dimension of meaning-making, and the implications of comparative methodology for historical research. In this aim, the prevailing methodological presuppositions informing the scholarly discourse on Buile Shuibhne are set against the wider context of Celtic Studies scholarship, in order to draw attention to the need to critically reflect upon the operations of knowledge production in future research.

Èto i to v povesti Staruha Daniila Harmsa

This dissertation focuses on the short story Starukha (The Old Woman), one of the last works of the Russian writer Daniil Kharms (1905-1942). The story, written in 1939, is analysed using the Kharmsian concepts èto and to (this and that) as a heuristic interpretative model. The first chapter gives a detailed analysis of this model, as well as a survey of the critical work done to date on Kharms and Starukha. In the second chapter the model is applied to study the different states of consciousness of the male protagonist. This is significant, because he is the "I" of the work, from whose point of view everything is being told. The third chapter takes a closer look at the reality of the world that exists independently of the consciousness of the protagonist. Physical objects can be said to bear - besides their everyday meaning - a hidden symbolic meaning. Similarly, the characters can be considered as representatives of everyday reality and otherworldliness. The fourth chapter deals with the narrative devices of Starukha. The problematics of the relation between fact and fiction plays an essential role in the story. Kharms's use of Ich-Erzählung and different tenses, which contributes to achieving a complicated elaboration of this kind of problematics, is examined in detail. The fifth chapter provides an intertextual reading of Starukha, based on its allusions to the Bible and the Christian tradition. As a result, the whole story can be seen as a kind of meditation on the Passion of Christ. The final chapter examines how the important Kharmsian concepts of the grotesque and the absurd manifest themselves in Starukha. The old woman represents in a grotesque way two opposite systems: the religious and the totalitarian. The absurdity of Starukha can be claimed to be illusory. Therefore, it is better to speak about paradoxicality. Starukha itself is a kind of paradox, in the sense that it tries to say something of the ultimate truth of reality, which inevitably remains ineffable.

Kirurgisen operaation teatteri : Teatterillinen kuva ja ihmismuodon esitettävyys avantgardistisen väkivallan näkökulmasta

Dissertation considers the birth of modernist and avant-gardist authorship as a reaction against mass society and massculture. Radical avant-gardism is studied as figurative violence done against the human form. The main argument claims avant-gardist authorship to be an act of masculine autogenesis. This act demands human form to be worked to an elementary state of disarticulateness, then to be reformed to the model of the artist's own psychophysical and idiosyncratic vision and experience. This work is connected to concrete mass, mass of pigment, charcoal, film, or flesh. This mass of the figure is worked to create a likeness in the nervous system of the spectator. The act of violence against the human figure is intended to shock the spectator. This shock is also a state of emotional and perceptional massification. I use theatrical image as heuristic tool and performance analysis, connecting figure and spectator into a larger image, which is constituted by relationships of mimesis, where figure presents the likeness of the spectator and spectator the likeness of the figure. Likeness is considered as both gestural - social mimetic - and sensuous - kinesthetically mimetic. Through this kind of construction one can describe and contextualize the process of violent autogenesis using particular images as case studies. Avant-gardist author is the author of theatrical image, not particular figure, and through act of massification the nervous system of the spectator is also part of this image. This is the most radical form and ideology of avant-gardist and modernist authorship or imagerial will to power. I construct a model of gestural-mimic performer to explicate the nature of violence done for human form in specific works, in Mann's novella Death in Venice, in Schiele's and Artaud's selfportaits, in Francis Bacon's paintings, in Beckett's shortplat NOT I, in Orlan's chirurgical performance Operation Omnipresense, in Cindy Sherman's Film/Stills, in Diamanda Galás's recording Vena Cava and in Hitchcock's Psycho. Masspsychology constructed a phobic picture of human form's plasticity and capability to be constituted by influencies coming both inside and outside - childhood, atavistic organic memories, urban field of nervous impulses, unconsciousness, capitalist (image)market and democratic masspolitics. Violence is then antimimetic and antitheatrical, a paradoxical situation, considering that massmedias and massaudiences created an enormous fascination about possibilities of theatrical and hypnotic influence in artistic elites. The problem was how to use theatrical image without coming as author under influence. In this work one possible answer is provided: by destructing the gestural-mimetic performer, by eliminating representations of mimic body techniques from the performer of human (a painted figure, a photographed figure, a filmed figure or an acted figure, audiovisual or vocal) figure. This work I call the chirurgical operation, which also indicates co-option with medical portraitures or medico-cultural diagnoses of human form. Destruction of the autonomy of the performer was a parallel process to constructing the new mass media audience as passive, plastic, feminine. The process created an image of a new kind of autotelic masculine author-hero, freed from human form in its bourgeois, aristocratic, classical and popular versions.

Leibniz on Rational Decision-Making

In this study I discuss G. W. Leibniz's (1646-1716) views on rational decision-making from the standpoint of both God and man. The Divine decision takes place within creation, as God freely chooses the best from an infinite number of possible worlds. While God's choice is based on absolutely certain knowledge, human decisions on practical matters are mostly based on uncertain knowledge. However, in many respects they could be regarded as analogous in more complicated situations. In addition to giving an overview of the divine decision-making and discussing critically the criteria God favours in his choice, I provide an account of Leibniz's views on human deliberation, which includes some new ideas. One of these concerns is the importance of estimating probabilities in making decisions one estimates both the goodness of the act itself and its consequences as far as the desired good is concerned. Another idea is related to the plurality of goods in complicated decisions and the competition this may provoke. Thirdly, heuristic models are used to sketch situations under deliberation in order to help in making the decision. Combining the views of Marcelo Dascal, Jaakko Hintikka and Simo Knuuttila, I argue that Leibniz applied two kinds of models of rational decision-making to practical controversies, often without explicating the details. The more simple, traditional pair of scales model is best suited to cases in which one has to decide for or against some option, or to distribute goods among parties and strive for a compromise. What may be of more help in more complicated deliberations is the novel vectorial model, which is an instance of the general mathematical doctrine of the calculus of variations. To illustrate this distinction, I discuss some cases in which he apparently applied these models in different kinds of situation. These examples support the view that the models had a systematic value in his theory of practical rationality.

Vilijonkka ikkunassa - Tove Janssonin muumiteosten kuva, sana ja liike

Tove Jansson (1914--2001) was a Finnish illustrator, author, artist, caricaturist and comic artist. She is best known for her Moomin Books, written in Swedish, which she illustrated herself, and published between 1945 and 1977. My study focuses on the interweaving of images and words in Jansson s picturebooks, novels and short stories situated in the fantasy world of Moomin Valley. In particular, it concentrates on Jansson s development of a special kind of aesthetics of movement and stasis, based upon both illustration and text. The conventions of picturebook art and illustration are significant to both Jansson s visual art and her writing, and she was acutely conscious of them. My analysis of Jansson s work begins by discussing her first published picturebooks and less familiar illustrations (before she began her Moomin books) and I then proceed to discuss her three Moomin picturebooks, The Book about Moomin, Mymble and Little My; Who Will Comfort Toffle?, and The Dangerous Journey. The discussion moves from images to words and from words to images: Barthes s (1982) concept of anchoring and, in particular, what he calls relaying , form a point of reading and viewing Moomin texts and illustrations in a complementary relation, in which the message s unity occurs on a higher level: that of the story, the anecdote, the diegesis . The eight illustrated Moomin novels and one collection of short stories are analysed in a similar manner, taking into account the academic discourse about picturebooks which was developed in the last decade of the 20th century and beginning of the 21st century by, among others, scholars such as Nodelman, Rhedin, Doonan, Thiele, Stephens, Lewis, Nikolajeva and Scott. In her Moomin books, Jansson uses a wide variety of narrative and illustrative styles which are complementary to each other. Each book is different and unique in its own way, but a certain development or progression of mood and representation can be seen when assessing the series as a whole. Jansson s early stories are happy and adventurous but her later Moomin novels, beginning from Moominland Midwinter, focus more on the interiority of the characters, placing them in difficult situations which approximate social reality. This orientation is also reflected in the representation of movement and space. The books which were published first include more obviously descriptive passages, exemplifying the tradition of literary pictorialism. Whereas in Jansson s later work, the space develops into something that is alive which can have an enduring effect on the characters personalities and behaviour. This study shows how the idea of an image a dynamic image -- forms a holistic foundation for Jansson s imagination and work. The idea of central perspective, or frame, for instance, provided inspiration for whole stories or in the way that she developed her characters, as in the case of the Fillyjonk, who is a complex female figure, simultaneously frantic and prim. The idea of movement is central to the narrative art of picturebooks and illustrated texts, particularly in relation to the way that action is depicted. Jansson, however, also develops a specific choreography of characters in which poses and postures signify action, feelings and relationships. Here, I use two ideas from modern dance, contraction and release (Graham), to characterise the language of movement which is evident in Jansson s words and images. In Jansson s final Moomin novels and short stories, the idea of space becomes more and more dynamic and closely linked with characterisation. My study also examines a number of Jansson s early sketches for her Moomin novels, in which movement is performed much more dramatically than in those illustrations which appeared in the last novels to be published.

A sequential analysis of "nú" and "núna" in Icelandic conversation

This thesis is an empirical study of how two words in Icelandic, "nú" and "núna", are used in contemporary Icelandic conversation. My aims in this study are, first, to explain the differences between the temporal functions of "nú" and "núna", and, second, to describe the non-temporal functions of "nú". In the analysis, a focus is placed on comparing the sequential placement of the two words, on their syntactical distribution, and on their prosodic realization. The empirical data comprise 14 hours and 11 minutes of naturally occurring conversation recorded between 1996 and 2003. The selected conversations represent a wide range of interactional contexts including informal dinner parties, institutional and non-institutional telephone conversations, radio programs for teenagers, phone-in programs, and, finally, a political debate on television. The theoretical and methodological framework is interactional linguistics, which can be described as linguistically oriented conversation analysis (CA). A comparison of "nú" and "núna" shows that the two words have different syntactic distributions. "Nú" has a clear tendency to occur in the front field, before the finite verb, while "núna" typically occurs in the end field, after the object. It is argued that this syntactic difference reflects a functional difference between "nú" and "núna". A sequential analysis of "núna" shows that the word refers to an unspecified period of time which includes the utterance time as well as some time in the past and in the future. This temporal relation is referred to as reference time. "Nú", by contrast, is mainly used in three different environments: a) in temporal comparisons, 2) in transitions, and 3) when the speaker is taking an affective stance. The non-temporal functions of "nú" are divided into three categories: a) "nú" as a tone particle, 2) "nú" as an utterance particle, and 3) "nú" as a dialogue particle. "Nú" as a tone particle is syntactically integrated and can occur in two syntactic positions: pre-verbally and post-verbally. I argue that these instances are employed in utterances in which a speaker is foregrounding information or marking it as particularly important. The study shows that, although these instances are typically prosodically non-prominent and unstressed, they are in some cases delivered with stress and with a higher pitch than the surrounding talk. "Nú" as an utterance particle occurs turn-initially and is syntactically non-integrated. By using "nú", speakers show continuity between turns and link new turns to prior ones. These instances initiate either continuations by the same speaker or new turns after speaker shifts. "Nú" as a dialogue particle occurs as a turn of its own. The study shows that these instances register informings in prior turns as unexpected or as a departure from the normal state of affairs. "Nú" as a dialogue particle is often delivered with a prolonged vowel and a recognizable intonation contour. A comparative sequential and prosodic analysis shows that in these cases there is a correlation between the function of "nú" and the intonation contour by which it is delivered. Finally, I argue that despite the many functions of "nú", all the instances can be said to have a common denominator, which is to display attention towards the present moment and the utterances which are produced prior or after the production of "nú". Instead of anchoring the utterances in external time or reference time, these instances position the utterance in discourse internal time, or discourse time.

Novel insights on functions of myotilin/palladin family members

Poikkijuovaisen luuranko- ja sydänlihaksen supistumisyksikkö, sarkomeeri, koostuu tarkoin järjestyneistä aktiini- ja myosiinisäikeistä. Rakenne eroaa muista solutyypeistä, joissa aktiinisäikeistö muovautuu jatkuvasti ja sen järjestyminen säätelee solun muotoa, solujakautumista, soluliikettä ja solunsisäisten organellien kuljetusta. Myotilin, palladin ja myopalladin kuuluvat proteiiniperheeseen, jonka yhteispiirteenä ovat immunoglobuliinin kaltaiset (Igl) domeenit. Proteiinit liittyvät aktiinitukirankaan ja niiden arvellaan toimivan solutukirangan rakenne-elementteinä ja säätelijöinä. Myotilinia ja myopalladinia ilmennetään poikkijuovaisessa lihaksessa. Sen sijaan palladinin eri silmukointimuotoja tavataan monissa kudostyypeissä kuten hermostossa, ja eri muodoilla saattaa olla solutyypistä riippuvia tehtäviä. Poikkijuovaisessa lihaksessa kaikki perheen jäsenet sijaitsevat aktiinisäikeitä yhdistävässä Z-levyssä ja ne sitovat Z-levyn rakenneproteiinia, -aktiniinia. Myotilingeenin pistemutaatiot johtavat periytyviin lihastauteihin, kun taas palladinin mutaatioiden on kuvattu liittyvän periytyvään haimasyöpään ja lisääntyneeseen sydäninfarktin riskiin. Tässä tutkimuksessa selvitettin myotilinin ja pallainin toimintaa. Kokeissa löydettiin uusia palladinin 90-92kDa alatyyppiin sitoutuvia proteiineja. Yksi niistä on aktiinidynamiikkaa säätelevä profilin. Profilinilla on kahdenlaisia tehtäviä; se edesauttaa aktiinisäikeiden muodostumista, mutta se voi myös eristää yksittäisiä aktiinimolekyylejä ja edistää säikeiden hajoamista. Solutasolla palladinin ja profilinin sijainti on yhtenevä runsaasti aktiinia sisältävillä solujen reuna-alueilla. Palladinin ja profilinin sidos on heikko ja hyvin dynaaminen, joka sopii palladinin tehtävään aktiinisäideiden muodostumisen koordinoijana. Toinen palladinin sitoutumiskumppani on aktiinisäikeitä yhteensitova -aktiniini. -Aktiniini liittää solutukirangan solukalvon proteiineihin ja ankkuroi solunsisäisiä viestintämolekyylejä. Sitoutumista välittävä alue on hyvin samankaltainen palladinissa ja myotilinissa. Luurankolihaksen liiallinen toistuva venytys muuttaa Z-levyjen rakennetta ja muotoa. Prosessin aikana syntyy uusia aktiinifilamenttejä sisältäviä tiivistymiä ja lopulta uusia sarkomeereja. Löydöstemme perusteella myotilinin uudelleenjärjestyminen noudattaa aktiinin muutoksia. Tämä viittaa siihen, että myotilin liittää yhteen uudismuodostuvia aktiinisäikeitä ja vakauttaa niitä. Myotilin saattaa myös ankkuroida viesti- tai rakennemolekyylejä, joiden tehtävänä on edesauttaa Z-levyjen uudismuodostusta. Tulostemme perusteella arvelemme, että myotilin toimii Z-levyjen rakenteen vakaajana ja aktiinisäikeiden säätelijänä. Palladinin puute johtaa sikiöaikaiseen kuolemaan hiirillä, mutta myotilinin puutoksella ei ole samanlaisia vaikutuksia. Tuotettujen myotilin poistogeenisten hiirten todetiin syntyvän ja kehittyvän normaalisti eikä niillä esiintynyt rakenteellisia tai toiminnallisia häiriöitä. Toisaalta aiemmissa kokeissa, joissa hiirille on siirretty ihmisen lihastautia aikaansaava myotilingeeni, nähdään samankaltaisia kuin sairailla ihmisillä. Näin ollen muuntunut myotilin näyttä olevan lihaksen toiminnalle haitallisempi kuin myotilinin puute. Myotilinin ja palladinin yhteisvaikutusta selvittääksemme risteytimme myotilin poistegeenisen hiiren ja hiirilinjan, joka ilmentää puutteellisesti palladinin 200 kDa muotoa. Puutteellisesti 200 kDa palladinia ilmentävien hiirten sydänlihaksessa todettiin vähäisiä hienorakenteen muutoksia, mutta risteytetyillä hiirillä tavattiin rakenteellisia ja toiminnallisia muutoksia myös luurankolihaksessa. Tulosten perusteella voidaan todeta, että palladinin 200 kDa muoto säätelee sydänlihassolujen rakennetta. Luurankolihaksessa sen sijaan myotilinilla ja palladinilla näyttäisi olevan päällekkäisiä tehtäviä.

Suunnittelunäkemys metsäorganisaatioissa ja sen vaikutus tietojärjestelmälle asetettaviin vaatimuksiin

Department of Forest Resource Management in the University of Helsinki has in years 2004?2007 carried out so-called SIMO -project to develop a new generation planning system for forest management. Project parties are organisations doing most of Finnish forest planning in government, industry and private owned forests. Aim of this study was to find out the needs and requirements for new forest planning system and to clarify how parties see targets and processes in today's forest planning. Representatives responsible for forest planning in each organisation were interviewed one by one. According to study the stand-based system for managing and treating forests continues in the future. Because of variable data acquisition methods with different accuracy and sources, and development of single tree interpretation, more and more forest data is collected without field work. The benefits of using more specific forest data also calls for use of information units smaller than tree stand. In Finland the traditional way to arrange forest planning computation is divided in two elements. After updating the forest data to present situation every stand unit's growth is simulated with different alternative treatment schedule. After simulation, optimisation selects for every stand one treatment schedule so that the management program satisfies the owner's goals in the best possible way. This arrangement will be maintained in the future system. The parties' requirements to add multi-criteria problem solving, group decision support methods as well as heuristic and spatial optimisation into system make the programming work more challenging. Generally the new system is expected to be adjustable and transparent. Strict documentation and free source code helps to bring these expectations into effect. Variable growing models and treatment schedules with different source information, accuracy, methods and the speed of processing are supposed to work easily in system. Also possibilities to calibrate models regionally and to set local parameters changing in time are required. In future the forest planning system will be integrated in comprehensive data management systems together with geographic, economic and work supervision information. This requires a modular method of implementing the system and the use of a simple data transmission interface between modules and together with other systems. No major differences in parties' view of the systems requirements were noticed in this study. Rather the interviews completed the full picture from slightly different angles. In organisation the forest management is considered quite inflexible and it only draws the strategic lines. It does not yet have a role in operative activity, although the need and benefits of team level forest planning are admitted. Demands and opportunities of variable forest data, new planning goals and development of information technology are known. Party organisations want to keep on track with development. One example is the engagement in extensive SIMO-project which connects the whole field of forest planning in Finland.

Semantic Classes in Topic Detection and Tracking

Topic detection and tracking (TDT) is an area of information retrieval research the focus of which revolves around news events. The problems TDT deals with relate to segmenting news text into cohesive stories, detecting something new, previously unreported, tracking the development of a previously reported event, and grouping together news that discuss the same event. The performance of the traditional information retrieval techniques based on full-text similarity has remained inadequate for online production systems. It has been difficult to make the distinction between same and similar events. In this work, we explore ways of representing and comparing news documents in order to detect new events and track their development. First, however, we put forward a conceptual analysis of the notions of topic and event. The purpose is to clarify the terminology and align it with the process of news-making and the tradition of story-telling. Second, we present a framework for document similarity that is based on semantic classes, i.e., groups of words with similar meaning. We adopt people, organizations, and locations as semantic classes in addition to general terms. As each semantic class can be assigned its own similarity measure, document similarity can make use of ontologies, e.g., geographical taxonomies. The documents are compared class-wise, and the outcome is a weighted combination of class-wise similarities. Third, we incorporate temporal information into document similarity. We formalize the natural language temporal expressions occurring in the text, and use them to anchor the rest of the terms onto the time-line. Upon comparing documents for event-based similarity, we look not only at matching terms, but also how near their anchors are on the time-line. Fourth, we experiment with an adaptive variant of the semantic class similarity system. The news reflect changes in the real world, and in order to keep up, the system has to change its behavior based on the contents of the news stream. We put forward two strategies for rebuilding the topic representations and report experiment results. We run experiments with three annotated TDT corpora. The use of semantic classes increased the effectiveness of topic tracking by 10-30\% depending on the experimental setup. The gain in spotting new events remained lower, around 3-4\%. The anchoring the text to a time-line based on the temporal expressions gave a further 10\% increase the effectiveness of topic tracking. The gains in detecting new events, again, remained smaller. The adaptive systems did not improve the tracking results.

Efficient search for statistically significant dependency rules in binary data

Analyzing statistical dependencies is a fundamental problem in all empirical science. Dependencies help us understand causes and effects, create new scientific theories, and invent cures to problems. Nowadays, large amounts of data is available, but efficient computational tools for analyzing the data are missing. In this research, we develop efficient algorithms for a commonly occurring search problem - searching for the statistically most significant dependency rules in binary data. We consider dependency rules of the form X->A or X->not A, where X is a set of positive-valued attributes and A is a single attribute. Such rules describe which factors either increase or decrease the probability of the consequent A. A classical example are genetic and environmental factors, which can either cause or prevent a disease. The emphasis in this research is that the discovered dependencies should be genuine - i.e. they should also hold in future data. This is an important distinction from the traditional association rules, which - in spite of their name and a similar appearance to dependency rules - do not necessarily represent statistical dependencies at all or represent only spurious connections, which occur by chance. Therefore, the principal objective is to search for the rules with statistical significance measures. Another important objective is to search for only non-redundant rules, which express the real causes of dependence, without any occasional extra factors. The extra factors do not add any new information on the dependence, but can only blur it and make it less accurate in future data. The problem is computationally very demanding, because the number of all possible rules increases exponentially with the number of attributes. In addition, neither the statistical dependency nor the statistical significance are monotonic properties, which means that the traditional pruning techniques do not work. As a solution, we first derive the mathematical basis for pruning the search space with any well-behaving statistical significance measures. The mathematical theory is complemented by a new algorithmic invention, which enables an efficient search without any heuristic restrictions. The resulting algorithm can be used to search for both positive and negative dependencies with any commonly used statistical measures, like Fisher's exact test, the chi-squared measure, mutual information, and z scores. According to our experiments, the algorithm is well-scalable, especially with Fisher's exact test. It can easily handle even the densest data sets with 10000-20000 attributes. Still, the results are globally optimal, which is a remarkable improvement over the existing solutions. In practice, this means that the user does not have to worry whether the dependencies hold in future data or if the data still contains better, but undiscovered dependencies.

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.

Golgi proteomics : Identification of a novel cartilage-specific Golgi protein GoPro49

The Golgi complex is a central organelle of the secretory pathway, responsible for a range of post-translational modifications, as well as for membrane traffic to the plasma membrane and to the endosomal-lysosomal pathway. In addition, this organelle has roles in cell migration, in the regulation of traffic, and as a mitotic check point. The structure of the Golgi complex is highly dynamic and able to respond to the amount of cargo being transported and the stage of the cell cycle. The Golgi proteome reflects the functions and structure of this organelle, and can be divided into three major groups: the Golgi resident proteins (e.g. modification enzymes), the Golgi matrix proteins (involved in structure and tethering events), and trafficking proteins (e.g. vesicle coat proteins and Rabs). The Golgi proteome has been studied on several occasions, from both rat liver and mammary gland Golgi membranes using proteomic approaches, but still little more than half of the estimated Golgi proteome is known. Nevertheless, methodological improvements and introduction of shotgun proteomics have increased the number of identified proteins, and especially the number of identified transmembrane proteins. Cartilage, even though not a typical tissue in which to study membrane traffic, secretes large amounts of extracellular matrix proteins that are extensively modified, especially by amino acid hydroxylation, glycosylation and sulfation. Furthermore, the cartilage ECM contains several, large oligomeric proteins (such as collagen II) that are difficult to assemble and transport. Indeed, cartilage has been shown to be susceptible to changes both in secretory pathway (e.g. the COPII coat assembly) and in post-translational modifications (e.g. heparan sulfate formation). Dental follicle, and the periodontal ligament (PDL) that it forms, are another type of connective tissue, and they have a role in anchoring teeth to bone. This anchorage is achieved by numerous matrix fibres that connect the bone matrix with the cementum. These tissues have in common the secretion of large matrix molecules. In this study the Golgi proteome was analysed from purified, stacked Golgi membranes isolated from rat liver. The identified, extensive proteome included a protein similar to Ab2-095, or Golgi protein 49kDa (GoPro49), which was shown to localise to the Golgi complex as an EGFP fusion protein. Surprisingly, in situ hybridisation showed the GoPro49 expression to be highly restricted to different mesenchymal tissues, especially in cartilage, and this expression pattern was clearly developmentally regulated. In addition to cartilage, GoPro49 was also expressed in the dental follicle, but was not observed in the mature PDL. Importantly, GoPro49 is the first specific marker for the dental follicle. Endogenous GoPro49 protein co-localised with β-COP in both chondrosarcoma and primary dental follicle cell lines. The COPI staining in these cells was highly dynamic, showing a number of tubules. This may reflect the type of secretory cargo they secrete. Currently GoPro49 is the only Golgi protein with such a restricted expression pattern.

Perspectives on knowledge creating inquiry in higher education

Higher education is faced with the challenge of strengthening students competencies for the constantly evolving technology-mediated practices of knowledge work. The knowledge creation approach to learning (Paavola et al., 2004; Hakkarainen et al., 2004) provides a theoretical tool to address learning and teaching organized around complex problems and the development of shared knowledge objects, such as reports, products, and new practices. As in professional work practices, it appears necessary to design sufficient open-endedness and complexity for students teamwork in order to generate unpredictable and both practically and epistemologically challenging situations. The studies of the thesis examine what kinds of practices are observed when student teams engage in knowledge creating inquiry processes, how the students themselves perceive the process, and how to facilitate inquiry with technology-mediation, tutoring, and pedagogical models. Overall, 20 student teams collaboration processes and productions were investigated in detail. This collaboration took place in teams or small groups of 3-6 students from multiple domain backgrounds. Two pedagogical models were employed to provide heuristic guidance for the inquiry processes: the progressive inquiry model and the distributed project model. Design-based research methodology was employed in combination with case study as the research design. Database materials from the courses virtual learning environment constituted the main body of data, with additional data from students self-reflections and student and teacher interviews. Study I examined the role of technology mediation and tutoring in directing students knowledge production in a progressive inquiry process. The research investigated how the scale of scaffolding related to the nature of knowledge produced and the deepening of the question explanation process. In Study II, the metaskills of knowledge-creating inquiry were explored as a challenge for higher education: metaskills refers to the individual, collective, and object-centered aspects of monitoring collaborative inquiry. Study III examined the design of two courses and how the elaboration of shared objects unfolded based on the two pedagogical models. Study IV examined how the arranged concept-development project for external customers promoted practices of distributed, partially virtual, project work, and how the students coped with the knowledge creation challenge. Overall, important indicators of knowledge creating inquiry were the following: new versions of knowledge objects and artifacts demonstrated a deepening inquiry process; and the various productions were co-created through iterations of negotiations, drafting, and versioning by the team members. Students faced challenges of establishing a collective commitment, devising practices to co-author and advance their reports, dealing with confusion, and managing culturally diverse teams. The progressive inquiry model, together with tutoring and technology, facilitated asking questions, generating explanations, and refocusing lines of inquiry. The involvement of the customers was observed to provide a strong motivation for the teams. On the evidence, providing team-specific guidance, exposing students to models of scientific argumentation and expert work practices, and furnishing templates for the intended products appear to be fruitful ways to enhance inquiry processes. At the institutional level, educators do well to explore ways of developing collaboration with external customers, public organizations or companies, and between educational units in order to enhance educational practices of knowledge creating inquiry.