Ihanne ja todellisuus : Jäsenyyteen sitoutuminen Suomen evankelis-luterilaisessa kirkossa 1960-luvulta 2000-luvulle

The subject of the study is the ideal and reality of commitment to membership in the Evangelical Lutheran Church of Finland from the 1960s to the 2000s. The research task is to ascertain what manner of commitment the Evangelical Lutheran Church of Finland expects from its members (the ideal) and how in reality membership of the Church is realized (empiria). The research object is also to study the extent to which the ideal of commitment evinced by the Church and the actual relation of commitment to the Church changed during the research period. Additionally, those factors were analysed which influence the relation between the ideal and reality of commitment. In the analysis of the ideal of commitment the research data are official documents of the Evangelical Lutheran Church of Finland. They include confessions of the Church, Catechisms, Christian doctrine, joint strategies and plans of the Church, likewise the Church Act and Church Order. The reality of commitment is explored on the basis of Church membership, participation in parish activity and the private practice of religion, likewise attitude to Christian faith. The empirical data of the study comprise Church statistics, material from Statistics Finland and relevant surveys implemented during the research period. The ideal of commitment alongside membership includes knowing the basic tenets of Christian faith and family life based on prayer and participation in liturgical cycles. A member of the Church is expected to take care of his/her faith by living in participation of the Word and sacrament, bearing responsibility for the parish and faithfully discharging his/her worldly obligations. There have been no major changes in the ideal of commitment during the research period. On the contrary, the reality of commitment has changed. Although the majority of Finns are still members of the Church, there has been a constant decline in their share of the population. The same can be stated with respect to parish life. This has its own strengths, among them Church rites, parish activity around feast days and also work with children and confirmation training. However, the general trend is towards a decline in participation. There has also been a decrease in commitment to belief in God as taught by the Church. On the other hand, private religious observance has not changed at all. From the perspective of commitment the Evangelical Lutheran Church of Finland exists in a state of tension between the theological ideal and sociological empiria. Matters exerting a particular influence over the relation between ideal and reality are communality and varying conceptions of the Church, likewise contextuality and the related private Christianity. Societal change poses a challenge to traditional Church communality. A decline in communality has in turn led to a decline in belonging to the Church. Weakening awareness of membership has undermined the handing down of the tradition among younger generations. Modernization has influence the identity of the Church and brought the Church to an internal divergence. This way it has been able to retain its structure as a folk church but at the same time it has lost its opportunities for the formation of a clear identity. The Church has adjusted to societal change by outward-directed activities (performance) alongside the purely religious message (function). The tension between an unchanged message and a changed operating environment has increased. The challenge of contextuality has led the Church to review parish life, the nature of teaching and activity and the language used by the Church, likewise the cultural modus. Increasingly privatized Christianity challenges above all the theology and teaching of the Church, but also the life of worship and relation to cultural life.

Parisuhdetyössä toimivien vapaaehtoisten motivaatio ja sitoutuminen : Tutkimus HNMKY:n parisuhderyhmien ryhmänjohtajista

Tutkimuksen tarkoituksena on tarkastella kristillisessä parisuhdetyössä toimivien, HNMKY:n vapaaehtoisten ryhmänjohtajien motivaatiota ja sitoutumista sekä näiden välistä yhteyttä. Tutkimuksessa selvitettiin, keitä HNMKY:n avioliittoleirin vapaaehtoiset ryhmänjohtajat ovat, mitkä ovat heidän motivaatiot ryhmänvetäjinä sekä, mihin he ovat sitoutuneet ja, mikä heitä sitouttaa. Tutkimuksessa tarkasteltiin erityisesti arvoihin ja organisaatioon sitoutumista. Tutkimuksen empiirinen aineisto koostui Paremman avioliiton perheleirien 2007 ja 2008 vapaaehtoisien parisuhderyhmien ryhmänjohtajina toimineilta kerätystä kyselyaineistosta (N=163). Tutkimusote oli kvantitatiivinen. Teoreettinen viitekehys muodostui motiivien osalta Claryn ja kumppaneiden kehittelemästä The Volunteer Functions Inventory -mallista (VFI), joka on kehitetty vapaaehtoisten motiivien mittaamiseen sekä muusta aiemmasta motiivitutkimuksesta. Sitoutumisen osalta teoreettinen viitekehys muodostui vapaaehtoisten sitoutumiseen liittyvästä teoriasta sekä aiemmasta sitoutumisen tutkimuksesta. Parisuhdetyön ryhmänvetäjiltä löydettiin faktorianalyyseilla aiemmasta tutkimuksesta osittain poiketen neljä erilaista motivaatiota: 1) Oman parisuhteen hoito, 2) Auttamisen arvot, 3) Itselle saaminen sekä 4) Toimiminen yhdessä. Merkittävin vapaaehtoisuuteen motivoiva tekijä oli Oman parisuhteen hoito, joka sisälsi oman parisuhteen hoidon lisäksi omien vaikeuksien käsittelyä sekä vertaistukeen liittyviä motivaatioita. Auttamisen arvot sisälsivät toisten auttamisen tärkeyden sekä kristilliset arvot. Itselle saaminen piti sisällä oppimismotiiveja sekä omat vaikeudet motiiveja. Toimiminen yhdessä käsittää itsetuntomotiiveja ja sosiaalisia motiiveja. Näistä Itselle saaminen ja Toimiminen yhdessä liittyivät merkittävästi toisiinsa. Ryhmänvetäjiltä löydettiin faktorianalyyseilla neljä erilaista sitotuttavaa tekijää: 1) Saatu tuki, 2) Osa identiteettiä, 3) Palkitsevuus sekä 4) Onnistuminen auttamisessa. Merkittävin syy vapaaehtoisuuteen sitoutumiseen on Saatu tuki, joka sisältää sekä HNMKY:ltä että myös muilta ryhmänjohtajilta saadun tuen. Osa identiteettiä sisältää sen, että ryhmänjohtamisesta on tullut osa minua, ja että toiminta on vahvistanut itsetuntoa sekä sopii elämäntilanteeseen. Tähän sisältyi myös toisten auttamisen tärkeys. Sitouttavista tekijöistä vahvimmin toisiinsa liittyvät Saatu tuki ja Osa identiteettiä. Motivaatio ja sitoutuminen liittyvät monella tavoin toisiinsa. Merkittävimmin toisiinsa liittyvät motivaatiossa Auttamisen arvot ja sitoutumisessa Osa identiteettiä. Näille ihmisille auttamisen arvoissa tärkeää ovat kristillisten arvojen edistäminen parisuhdetyössä sekä merkittävien asioiden tekeminen itselle tärkeässä tehtävässä. Heitä motivoi toisten auttaminen ja sama asia sitouttaa heitä myös vapaaehtoiseen parisuhdetyöhön. Niitä ryhmänjohtajia, joita motivoi vapaaehtoistoimintaan oma parisuhteen hoito, sitouttaa tähän työhön sen palkitsevuus. Niitä, joita motivoivat Auttamisen arvot tai Itselle saaminen, sitouttaa Saatu tuki, Osa identiteettiä ja Palkitsevuus. Kun motivoivana tekijänä on Toiminen yhdessä, silloin sitouttaa Saatu tuki ja Osa identiteettiä. Ryhmänjohtajat ovat erittäin sitoutuneita HNMKY:n arvoihin ja opetukseen. Vastaajille on erittäin merkittävää kristillisten arvojen edistäminen parisuhdetyössä. Kristillisyyden ja sen arvojen merkityksellisyys, auttamisen sekä oman parisuhteen ja perheen tärkeys liittyvät toisiinsa. Avainsanat – Nyckelord Vapaaehtoistyö, motiivit, sitoutuminen, parisuhde Säilytyspaikka – Förvaringställe Teologisen tiedekunnan kirjasto

Luottamusmiesten sitoutuminen yritykseen ja ammattijärjestöönsä

Tutkimuksen tavoitteena on kartoittaa luottamusmiesten organisaatio- ja ammattijärjestösitoutumiseen liittyviä tekijöitä sekä kuvata eri tavoin sitoutuneita ryhmiä. Keskeisiä tutkimuskysymyksiä ovat siten: Rapauttavatko hyvät esimies-alaissuhteet ja sujuviksi koetut neuvottelusuhteet työnantajan ja ammattiyhdistyksen välillä perustaa luottamusmiesten ammattijärjestösitoutumiselta? Mitkä tekijät vaikuttavat luottamusmiehen organisaatio- ja ammattijärjestösitoutumisen taustalla? Tutkimustulokset osoittavat, että oman johtajan/esimiehen ja luottamusmiehen välinen molemminpuolinen hyväksyntä ja siitä syntynyt motivoituneisuus ja työtyytyväisyys yhdistyneenä pitkän työsuhteen antamaan turvallisuuden tunteeseen ovat voimakkaammin pääkonttorien luottamusmiesten organisaatiositoutumisen taustalla. Kun taas kentän luottamusmiesten työssään kokema epävarmuus, oman työn autonomian puute, haasteeton työnjohtoilmasto tuntuivat aiheuttavan enemmän kritiikkiä työorganisaatiota kohtaan ja suuntaavan samaistumista oman ammattijärjestön suuntaan. Enemmistö tutkimuksen luottamusmiehistä oli kuitenkin kaksoissitoutuneita ja he kokivat ammattiliittonsa ja työnantajansa välisten suhteiden perustuvan yhteistyöhön ja luottamukseen organisaation johtoon. Luottamusmiesten sitoutuminen professioon ja työnantajaan näyttäisi olevan toinen vaihtoehtoinen tulevaisuuden kaksoissidoksen malli.

Suomalaisten sopimustuottajien luottamus ja sitoutuminen yhteistyösuhteeseen

Tutkimuksen tavoitteena oli analysoida luottamukseen ja sitoutumiseen vaikuttavien taustatekijöiden merkitystä kalkkunantuottajien ja Länsi-Kalkkunan liikesuhteessa, sekä sitä miten sitoutuminen ja luottamus vaikuttavat tuottajien haluun jatkaa liikesuhteessa. Tutkimusta varten tutustuttiin laajasti luottamusta ja sitoutumista käsittelevään kirjallisuuteen ja alan kvalitatiivisiin sekä kvantitatiivisiin tutkimuksiin ja tutkimuksessa päätettiin keskittyä testaamaan mallia, jota oli muokattu hieman Zineldinin ja Jonssonin (2000) tutkimuksessaan kehittämästä mallista. Malliin lisättiin tuottajien jatkohalukkuus. Tutkimus oli osa Kotimaisten kalkkunantuottajien kilpailukyvyn kehittäminen –hanketta ja toimiesiselvityksenä kokonaisvaltaisen kalkkunaketjun kehittämisstrategian löytämiseksi. Teoriaosassa perehdyttiin ensin yritysyhteistyöhön ja sen keskeisiin käsitteisiin. Lisäksi tarkasteltiin luottamusta, sitoutumista ja niiden taustatekijöitä sekä keskeisiä käsitteitä ja määritelmiä. Lopuksi esiteltiin tutkimuksessa testattu teoreettinen viitekehys. Empiirinen osio perustui tutkimuksessa tehtyyn kyselytutkimukseen ja siitä saatuun aineistoon. Kyselyyn vastanneita kalkkunantuottajia oli 37. Kyselylomakkeessa oli yhteensä 75 osiota, joista suurin osa oli väittämiä ja lisäksi lomakkeessa oli muutamia avoimia vastausmahdollisuuksia ja monivalintakysymyksiä. Kysely toteutettiin kevään 2011 aikana. Aineistoa analysoinnissa käytettiin apuna frekvenssi- ja prosenttijakaumia sekä korrelaatioita muuttujien ja summamuuttujien välisen yhteyden tutkimisessa. Lisäksi teoriaosassa esitetyn mallin testaukseen käytettiin regressioanalyysiä. Aineisto jaettiin vielä luottamuksen ja sitoutumisen mukaan osiin, joita tarkasteltiin regressioanalyysissä saatujen tulosten avulla tarkemmin. Tutkimustulosten mukaan kommunikaatiolla, tyytyväisyydellä yhteistyösuhteeseen sekä kumppanin koetulla opportunismin ja mukautumisen tasolla oli voimakkaimmat korrelaatiot luottamukseen ja sitoutumiseen. Regressioanalyyseillä testatun mallin vahvimmaksi selittäjäksi nousi kommunikaatio, mutta vaikutusta luottamukseen todettiin olevan myös kumppanin koetulla opportunismilla ja tyytyväisyydellä yhteistyösuhteeseen. Aineistosta voitiin tunnistaa luottajat ja ei-luottajat sekä sitoutuneet ja ei-sitoutuneet tuottajat. Tulevan muutosstrategian eteenpäin viemistä voi vaikeuttaa se, että tuottajista noin 49 % ei luottanut yhteistyökumppaniin. Yhteistyösuhteeseen oli sitoutunut noin 74 % tuottajista, mikä voi helpottaa yhteistyötä ja tulevan muutosstrategian läpivientiä tulevaisuudessa. Tulevaa muutosprosessia voi olla mahdollista helpottaa ja luottamuksen tasoa nostaa ei-luottavien joukossa panostamalla avoimempaan, tuottajille sopivampaan ja vaatimuksia vastaavaan kommunikaatioon.

Laatutyöhön sitoutumisen edellytykset ammattikorkeakoulussa

The goal of this research was to establish the necessary conditions under which individuals are prepared to commit themselves to quality assurance work in the organisation of a Polytechnic. The conditions were studied using four main concepts: awareness of quality, commitment to the organisation, leadership and work welfare. First, individuals were asked to describe these four concepts. Then, relationships between the concepts were analysed in order to establish the conditions for the commitment of an individual towards quality assurance work (QA). The study group comprised the entire personnel of Helsinki Polytechnic, of which 341 (44.5%) individuals participated. Mixed methods were used as the methodological base. A questionnaire and interviews were used as the research methods. The data from the interviews were used for the validation of the results, as well as for completing the analysis. The results of these interviews and analyses were integrated using the concurrent nested design method. In addition, the questionnaire was used to separately analyse the impressions and meanings of the awareness of quality and leadership, because, according to the pre-understanding, impressions of phenomena expressed in terms of reality have an influence on the commitment to QA. In addition to statistical figures, principal component analysis was used as a description method. For comparisons between groups, one way variance analysis and effect size analysis were used. For explaining the analysis methods, forward regression analysis and structural modelling were applied. As a result of the research it was found that 51% of the conditions necessary for a commitment to QA were explained by an individual’s experience/belief that QA was a method of development, that QA was possible to participate in and that the meaning of quality included both product and process qualities. If analysed separately, other main concepts (commitment to the organisation, leadership and work welfare) played only a small part in explaining an individual’s commitment. In the context of this research, a structural path model of the main concepts was built. In the model, the concepts were interconnected by paths created as a result of a literature search covering the main concepts, as well as a result of an analysis of the empirical material of this thesis work. The path model explained 46% of the necessary conditions under which individuals are prepared to commit themselves to QA. The most important path for achieving a commitment stemmed from product and system quality emanating from the new goals of the Polytechnic, moved through the individual’s experience that QA is a method of the total development of quality and ended in a commitment to QA. The second most important path stemmed from the individual’s experience of belonging to a supportive work community, moved through the supportive value of the job and through affective commitment to the organisation and ended in a commitment to QA. The third path stemmed from an individual’s experiences in participating in QA, moved through collective system quality and through these to the supportive value of the job to affective commitment to the organisation and ended in a commitment to QA. The final path in the path model stemmed from leadership by empowerment, moved through collective system quality, the supportive value of the job and an affective commitment to the organisation, and again, ended in a commitment to QA. As a result of the research, it was found that the individual’s functional department was an important factor in explaining the differences between groups. Therefore, it was found that understanding the processing of part cultures in the organisation is important when developing QA. Likewise, learning-teaching paradigms proved to be a differentiating factor. Individuals thinking according to the humanistic-constructivistic paradigm showed more commitment to QA than technological-rational thinkers. Also, it was proved that the QA training program did not increase commitment, as the path model demonstrated that those who participated in training showed 34% commitment, whereas those who did not showed 55% commitment. As a summary of the results it can be said that the necessary conditions under which individuals are prepared to commit themselves to QA cannot be treated in a reductionistic way. Instead, the conditions must be treated as one totality, with all the main concepts interacting simultaneously. Also, the theoretical framework of quality must include its dynamic aspect, which means the development of the work of the individual and learning through auditing. In addition, this dynamism includes the reflection of the paradigm of the functions of the individual as well as that of all parts of the organisation. It is important to understand and manage the various ways of thinking and the cultural differences produced by the fragmentation of the organisation. Finally, it seems possible that the path model can be generalised for use in any organisation development project where the personnel should be committed.

Erityistä tukea tarvitsevan taaperon päiväkotiryhmään sopeutuminen : Monitapaustutkimus vuorovaikutussuhteista ja niiden rakentumisesta

How toddlers with special needs adjust to the daycare setting A multiple case study of how the relationships with adults and children are built The aim in this study is to describe how toddlers with special needs adjust to daycare. The emotional well-being and involvement in daycare activities of toddlers are especially investigated in this study. The relationship and how it is built between an adult and a child, a child and a child is examined. The daycare is examined through the socio-cultural theory as a pedagogical institution, where the child adapts by participating in social and cultural activities with the others. The development of the child is the result of the experiences that are gained through the constant relationship between the child, the family and social context. By the attachment theory the inner self-regulation, that allows the child safely adapt to new situations, develops most in the relationship between the child under 3years of age and the attending adult. The relationships between toddlers in daycare are usually built by the coincidental encounters in play and daily activities. In these relationships, the toddler gets the information of themselves and the other children. The complexity of the rules in the setting that organize the social action is challenging for the children and they need constant support from the adults. The participants of the study were five toddlers with special needs. When applying to daycare they were less than three years old and they got the specialist statement for their special needs, and the reference for daycare. The children were observed by recording their attending in the daycare once in the 3-4 months from the first day in daycare. Approximately 15 hours of material that was analysed with the Transana-program. The qualitative material was analysed by first collecting a descriptive model that explains and theorises the phenomenon. By the summery of the narrative it is placed a hypothesis that is tested by quantitative methods using correlations and variance analyses and general linear modeling that is used to count the differences between repeated measures and connections between different variables. The results of the study are built theoretically for the consistent conception between the theory and the findings in research. The toddlers in the study were all dependent on the support given by the adults in all the situations in the daycare. They could not associate with the other children without the support of the adults and their involvement in activities was low. The engagement of an adult in interaction was necessary for the children’s involvement in activities, and the co-operation with the other children. The engagement of teachers was statistically significantly higher than the engagement of other professions.

Kouluruokailuhenkilöstö matkalla kasvattajaksi

The National Board of Education in Finland has defined school catering as a part of education in comprehensive schools. School catering is defined in Finnish law and is financed by the municipalities. The emphasis on supplementary education in the school curriculum obliges the whole staff to learn new professional roles and to co-operate in the school com-munity. An in-service training programme, Smart Catering Staff (SCS) was designed and put into practice among school catering staff. The aim of the one-year SCS programme was to add to and encourage the development of the know-how of catering personnel regarding new challenges in the school community as well as to enhance and empower their educator’s role in the school. The content of the programme consisted of four two-day intensive teaching sessions as well as assignments which were carried out between theese sessions. The main contents of the SCS programme were personal and organizational learning, school curriculum concerning school catering, school culture and co-operation, customer service, and the role of catering staff as educators. The data in this action research was gathered from 2000 to 2008. The results show that the SCS programme can change the work community culture in that the catering staff is more encouraged to participate in the development projects of the school, and can see themselves more as educators alongside the teaching staff. It is concluded that in school communities all adults can be educators, but to achieve this aim empowering in-service training is needed. The school catering staff’s commitment to participation in the education align with the educational goals of the school unite children’s and teens safety nets. This can affect for example the growth of communal responsibility and prevention of alienation. Key words: School Catering, Smart School Catering Staff, Expanded Education, School Community, Action Research

Kummimaatilatoiminta opettajien ja maatilojen sekä innovaatiotutkimuksen näkökulmista

Tutkimuksen tarkoituksena on selvittää Finfood - Suomen Ruokatieto ry:n alullepanemassa kummimaatilatoiminnassa mukana olleiden opettajien ja maatilojen käsityksiä ja kokemuksia toiminnasta sekä tarkastella kummimaatilatoimintaa innovaationa. Kummimaatilatoiminta liittyy Finfoodin internetissä julkaisemaan oppimateriaaliin suomalaisen ruoan tiestä. Kummimaatilatoiminnan tavoitteena on, että mahdollisimman monella peruskoululuokalla olisi oma kummimaatila, missä luokka voisi vierailla vuosittain, joka säilyisi luokalla mahdollisimman pitkään ja jonka kanssa luokka suunnittelee oman yhteistyönsä. Kummimaatilatoiminnan avulla pyritään omakohtaisten kokemusten ja elämysten kautta lisäämään nuorten tietoa maaseudun elämästä. Tutkimuksen tausta -osassa tarkastellaan koulun ja yhteiskunnan muiden tahojen yhteistyötä, esitellään tutkimuksia koulun ja teollisuusyritysten yhteistyöstä sekä koulun ja maatalouden yhteistyöhön liittyvää materiaalia. Lisäksi tarkastellaan innovaatiota ja innovaation diffuusiota. Koska kummimaatilatoiminta on uusi toimintamuoto, jossa toisena osapuolena on koulu, tutkimuksessa käsitellään myös kasvatuksen muutoksia (educational change). Tutkimusta käsitellään tapaus- ja toimintatutkimuksena. Tutkimusaineistona ovat kummimaatilatoiminnan alkuvaiheessa syksyllä 1997 mukaan lähteneiden seitsemän opettajan ja kuuden maatilan teemahaastattelut. Tutkimuksessa on haastateltu myös Finfoodin edustajaa. Aineisto kerättiin kevään ja syksyn 1998 aikana. Innovaatiotarkastelun pohjana ovat lisäksi tutkijan omat kokemukset toiminnasta sekä erilaiset kokoukset ja keskustelut. Opettajien ja maatilojen kokemuksista selvitettiin heidän toiminnan aloittamiseen johtaneita motiivejaan, toiminnalle asettamiaan tavoitteita, toiminnassa havaitsemiaan ongelmia sekä käsityksiä toiminnan onnistumisen avaintekijöistä ja toiminnan kehittämisestä. Suuria näkemyseroja ei opettajien ja maatilojen kokemusten välillä ollut. Osapuolet pitivät maaseutua tärkeänä ja halusivat tarjota lapsille kokemuksia elämästä siellä. Yhteisenä tavoitteena opettajilla ja maatiloilla oli, että toiminta olisi mahdollisimman laajaa, ei pelkkiä retkipäiviä maalle. Opettajat korostivat myös vierailujen toiminnallisuutta. Maatilat toivoivat voivansa lisätä toiminnalla myös omaa tietämystään ja ammattitaitoaan. Käytännön asiat kuten matkat olivat eniten ongelmia aiheuttaneita asioita. Muina mahdollisina ongelmina mainittiin asenteet ja tiedotus. Toiminnan avaintekijöinä pidettiin suunnittelua, molempien osapuolten motivoituneisuutta ja aktiivisuutta, vierailujen toiminnallisuutta, tiedottamista ja toiminnan laajentamista. Kehittämisideat liittyivät toiminnan laajentamiseen ja monipuolistamiseen. Tämän tutkimuksen perusteella kummimaatilatoimintaa on ryhdytty kehittämään niin, että koko koululla yksittäisen luokan sijaan on yhteinen kummimaatila. Innovaatiotarkastelu osoitti, että kummimaatilatoiminnan kehittäminen ja levittäminen on pitkä prosessi, jossa koordinaattorin rooli on tärkeä. Osallistujien sitoutuminen toimintaan, osallistujien tukeminen sekä heidän kokemustensa hyödyntäminen on myös tärkeää. Tärkeimmät lähteet tutkimuksen taustateorian kannalta olivat Rogersin, Fullanin ja Hargreavesin teokset. Avainsanat: kummimaatilatoiminta, innovaatio, innovaation diffuusio, muutos Keywords: godfarms, farm visits, innovation, diffusion of innovation, educational change

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

The Mechanisms of Hedgehog Signal Transduction

Studies in both vertebrates and invertebrates have identified proteins of the Hedgehog (Hh) family of secreted signaling molecules as key organizers of tissue patterning. Initially discovered in Drosophila in 1992, Hh family members have been discovered in animals with body plans as diverse as those of mammals, insects and echinoderms. In humans three related Hh genes have been identified: Sonic, Indian and Desert hedgehog (Shh, Ihh and Dhh). Transduction of the Hh signal to the cytoplasm utilizes an unusual mechanism involving consecutive repressive interactions between Hh and its receptor components, Patched (Ptc) and Smoothened (Smo). Several cytoplasmic proteins involved in Hh signal transduction are known in Drosophila, but mammalian homologs are known only for the Cubitus interruptus (Ci) transcription factor (GLI(1-3)) and for the Ci/GLI-associated protein, Suppressor of Fused (Su(fu)). In this study I analyzed the mechanisms of how the Hh receptor Ptc regulates the signal transducer Smo, and how Smo relays the Shh signal from the cell surface to the cytoplasm ultimately leading to the activation of GLI transcription factors. In Drosophila, the kinesin-like protein Costal2 (Cos2) is required for suppression of Hh target gene expression in the absence of ligand, and loss of Cos2 causes embryonic lethality. Cos2 acts by bridging Smo to the Ci. Another protein, Su(Fu) exerts a weak suppressive influence on Ci activity and loss of Su(Fu) causes subtle changes in Drosophila wing pattern. This study revealed that domains in Smo that are critical for Cos2 binding in Drosophila are dispensable for mammalian Smo function. Furthermore, by analyzing the function of Su(Fu) and the closest mouse homologs of Cos2 by protein overexpression and RNA interference I found that inhibition of the Hh response pathway in the absence of ligand does not require Cos2 activity, but instead critically depends on the activity of Su(Fu). These results indicate that a major change in the mechanism of action of a conserved signaling pathway occurred during evolution, probably through phenotypic drift made possible by the existence in some species of two parallel pathways acting between the Hh receptor and the Ci/GLI transcription factors. In a second approach to unravel Hh signaling we cloned > 90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase-activity deficient mutants. Using this kinome resource as a screening tool, two kinases, MAP3K10 and DYRK2 were found to regulate Shh signaling. DYRK2 directly phosphorylated and induced the proteasome dependent degradation of the key Hh-pathway regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Latent TGF-β binding proteins -3 and -4 : transcriptional control and extracellular matrix targeting

Extracellular matrix (ECM) is a complex network of various proteins and proteoglycans which provides tissues with structural strength and resilience. By harvesting signaling molecules like growth factors ECM has the capacity to control cellular functions including proliferation, differentiation and cell survival. Latent transforming growth factor β (TGF-β) binding proteins (LTBPs) associate fibrillar structures of the ECM and mediate the efficient secretion and ECM deposition of latent TGF-β. The current work was conducted to determine the regulatory regions of LTBP-3 and -4 genes to gain insight into their tissue-specific expression which also has impact on TGF-β biology. Furthermore, the current research aimed at defining the ECM targeting of the N-terminal variants of LTBP-4 (LTBP-4S and -4L), which is required to understand their functions in tissues and to gain insight into conditions in which TGF-β is activated. To characterize the regulatory regions of LTBP-3 and -4 genes in silico and functional promoter analysis techniques were employed. It was found that the expression of LTBP-4S and -4L are under control of two independent promoters. This finding was in accordance with the observed expression patterns of LTBP-4S and -4L in human tissues. All promoter regions characterized in this study were TATAless, GC-rich and highly conserved between human and mouse species. Putative binding sites for Sp1 and GATA family of transcription factors were recognized in all of these regulatory regions. It is possible that these transcription factors control the basal expression of LTBP-3 and -4 genes. Smad binding element was found within the LTBP-3 and -4S promoter regions, but it was not present in LTBP-4L promoter. Although this element important for TGF-β signaling was present in LTBP-4S promoter, TGF-β did not induce its transcriptional activity. LTBP-3 promoter activity and mRNA expression instead were stimulated by TGF-β1 in osteosarcoma cells. It was found that the stimulatory effect of TGF-β was mediated by Smad and Erk MAPK signaling pathways. The current work explored the ECM targeting of LTBP-4S and identified binding partners of this protein. It was found that the N-terminal end of LTBP-4S possesses fibronectin (FN) binding sites which are critical for its ECM targeting. FN deficient fibroblasts incorporated LTBP-4S into their ECM only after addition of exogenous FN. Furthermore, LTBP-4S was found to have heparin binding regions, of which the C-terminal binding site mediated fibroblast adhesion. Soluble heparin prevented the ECM association of LTBP-4S in fibroblast cultures. In the current work it was observed that there are significant differences in the secretion, processing and ECM targeting of LTBP-4S and -4L. Interestingly, it was observed that most of the secreted LTBP-4L was associated with latent TGF-β1, whereas LTBP-4S was mainly secreted as a free form from CHO cells. This thesis provides information on transcriptional regulation of LTBP-3 and -4 genes, which is required for the deeper understanding of their tissue-specific functions. Further, the current work elucidates the structural variability of LTBPs, which appears to have impact on secretion and ECM targeting of TGF-β. These findings may advance understanding the abnormal activation of TGF-β which is associated with connective tissue disorders and cancer.

Molecular Interactions Underlying Neuronal Ceroid Lipofuscinoses CLN1 and CLN5

Disorders resulting from degenerative changes in the nervous system are progressive and incurable. Both environmental and inherited factors affect neuron function, and neurodegenerative diseases are often the sum of both factors. The cellular events leading to neuronal death are still mostly unknown. Monogenic diseases can offer a model for studying the mechanisms of neurodegeneration. Neuronal ceroid lipofuscinoses, or NCLs, are a group of monogenic, recessively inherited diseases affecting mostly children. NCLs cause severe and specific loss of neurons in the central nervous system, resulting in the deterioration of motor and mental skills and leading to premature death. In this thesis, the focus has been on two forms of NCL, the infantile NCL (INCL, CLN1) and the Finnish variant of late infantile NCL (vLINCLFin, CLN5). INCL is caused by mutations in the CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. In the Finnish variant of late infantile NCL (vLINCLFin), the CLN5 gene is defective, but the function of the encoded CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases by focusing on the molecular interactions of the defective proteins. In this work, the first interaction partner for PPT1, the mitochondrial F1-ATP synthase, was described. This protein has been linked to HDL metabolism in addition to its well-known role in the mitochondrial energy production. The connection between PPT1 and the F1-ATP synthase was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. The levels of F1-ATP synthase subunits were increased on the surface of Ppt1-deficient neurons when compared to controls. We also detected several changes in lipid metabolism both at the cellular and systemic levels in Ppt1-deficient mice when compared to controls. The interactions between different NCL proteins were also elucidated. We were able to detect novel interactions between CLN5 and other NCL proteins, and to replicate the previously reported interactions. Some of the novel interactions influenced the intracellular trafficking of the proteins. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism introduces a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and to develop strategies for therapies.