Functional regulation of the Neurofibromatosis 2 tumor suppressor merlin

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder manifested by the formation of multiple benign tumors of the nervous system. Affected individuals typically develop bilateral vestibular schwannomas which lead to deafness and balance disorders. The syndrome is caused by inactivation of the NF2 tumor suppressor gene, and mutation or loss of the NF2 product, merlin, is sufficient for tumorigenesis in both hereditary and sporadic NF2-associated tumors. Merlin belongs to the band 4.1 superfamily of cytoskeletal proteins, which also contain the related ezrin, radixin, and moesin (ERM) proteins. The ERM members provide a link between the cell cytoskeleton and membrane by connecting membrane-associated proteins to actin filaments. By stabilizing complexes in the cell cortex, the ERMs modulate morphology, growth, and migration of cells. Despite their structural homology, overlapping subcellular distribution, direct molecular association, and partial overlap of molecular interactions, merlin and ezrin exert opposite effects on cell proliferation. Merlin suppresses cell proliferation, whereas ezrin expression is linked to oncogenic activity. We hypothesized that the regions which differ between the proteins might explain merlin s specificity as a tumor suppressor. We therefore analyzed the regions, which are most diverse between merlin and ezrin; the N-terminal tail and the C-terminus. To determine the properties of the C-terminal region, we studied the two most predominant merlin isoforms together with truncation variants similar to those found in patients. We also focused on the evolutionally conserved C-terminal residues, E545-E547, that harbor disease causing mutations in its corresponding DNA sequence. In addition to inhibiting cell proliferation, merlin regulates cytoskeletal organization. The morphogenic properties of merlin may play a role in tumor suppression, since patient-derived tumor cells demonstrate cytoskeletal abnormalities. We analyzed the mechanisms of merlin-induced extension formation and determined that the C-terminal region of amino acids 538-568 is particularly important for the morphogenic activity. We also characterized the role of C-terminal merlin residues in the regulation of proliferation, phosphorylation, and intramolecular associations. In contrast to previous reports, we demonstrated that both merlin isoforms are able to suppress cell proliferation, whereas C-terminally mutated merlin constructs showed reduced growth inhibition. Phosphorylation serves as a mechanism to regulate the tumor suppressive activity of merlin. The C-terminal serine 518 is phosphorylated in response to both p21-activated kinase (PAK) and protein kinase A (PKA), which inactivates the growth inhibitory function of merlin. However, at least three differentially phosphorylated forms of the protein exist. In this study we demonstrated that also the N-terminus of merlin is phosphorylated by AGC kinases, and that both PKA and Akt phosphorylate merlin at serine 10 (S10). We evaluated the impact of this N-terminal tail phosphorylation, and showed that the phosphorylation state of S10 is an important regulator of merlin s ability to modulate cytoskeletal organization but also regulates the stability of the protein. In summary, this study describes the functional effect of merlin specific regions. We demonstrate that both S10 in the N-terminal tail and residues E545-E547 in the C-terminus are essential for merlin activity and function.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

Constructing a Community : Myths and Realities of the Open Development Model

The open development model of software production has been characterized as the future model of knowledge production and distributed work. Open development model refers to publicly available source code ensured by an open source license, and the extensive and varied distributed participation of volunteers enabled by the Internet. Contemporary spokesmen of open source communities and academics view open source development as a new form of volunteer work activity characterized by hacker ethic and bazaar governance . The development of the Linux operating system is perhaps the best know example of such an open source project. It started as an effort by a user-developer and grew quickly into a large project with hundreds of user-developer as contributors. However, in hybrids , in which firms participate in open source projects oriented towards end-users, it seems that most users do not write code. The OpenOffice.org project, initiated by Sun Microsystems, in this study represents such a project. In addition, the Finnish public sector ICT decision-making concerning open source use is studied. The purpose is to explore the assumptions, theories and myths related to the open development model by analysing the discursive construction of the OpenOffice.org community: its developers, users and management. The qualitative study aims at shedding light on the dynamics and challenges of community construction and maintenance, and related power relations in hybrid open source, by asking two main research questions: How is the structure and membership constellation of the community, specifically the relation between developers and users linguistically constructed in hybrid open development? What characterizes Internet-mediated virtual communities and how can they be defined? How do they differ from hierarchical forms of knowledge production on one hand and from traditional volunteer communities on the other? The study utilizes sociological, psychological and anthropological concepts of community for understanding the connection between the real and the imaginary in so-called virtual open source communities. Intermediary methodological and analytical concepts are borrowed from discourse and rhetorical theories. A discursive-rhetorical approach is offered as a methodological toolkit for studying texts and writing in Internet communities. The empirical chapters approach the problem of community and its membership from four complementary points of views. The data comprises mailing list discussion, personal interviews, web page writings, email exchanges, field notes and other historical documents. The four viewpoints are: 1) the community as conceived by volunteers 2) the individual contributor s attachment to the project 3) public sector organizations as users of open source 4) the community as articulated by the community manager. I arrive at four conclusions concerning my empirical studies (1-4) and two general conclusions (5-6). 1) Sun Microsystems and OpenOffice.org Groupware volunteers failed in developing necessary and sufficient open code and open dialogue to ensure collaboration thus splitting the Groupware community into volunteers we and the firm them . 2) Instead of separating intrinsic and extrinsic motivations, I find that volunteers unique patterns of motivations are tied to changing objects and personal histories prior and during participation in the OpenOffice.org Lingucomponent project. Rather than seeing volunteers as a unified community, they can be better understood as independent entrepreneurs in search of a collaborative community . The boundaries between work and hobby are blurred and shifting, thus questioning the usefulness of the concept of volunteer . 3) The public sector ICT discourse portrays a dilemma and tension between the freedom to choose, use and develop one s desktop in the spirit of open source on one hand and the striving for better desktop control and maintenance by IT staff and user advocates, on the other. The link between the global OpenOffice.org community and the local end-user practices are weak and mediated by the problematic IT staff-(end)user relationship. 4) Authoring community can be seen as a new hybrid open source community-type of managerial practice. The ambiguous concept of community is a powerful strategic tool for orienting towards multiple real and imaginary audiences as evidenced in the global membership rhetoric. 5) The changing and contradictory discourses of this study show a change in the conceptual system and developer-user relationship of the open development model. This change is characterized as a movement from hacker ethic and bazaar governance to more professionally and strategically regulated community. 6) Community is simultaneously real and imagined, and can be characterized as a runaway community . Discursive-action can be seen as a specific type of online open source engagement. Hierarchies and structures are created through discursive acts. Key words: Open Source Software, open development model, community, motivation, discourse, rhetoric, developer, user, end-user

Turvallinen koulu? : Helsinkiläisten peruskoulujen turvallisuuskulttuurista ja siihen vaikuttavista tekijöistä

Objectives. This study concentrated on the aspect of physical safety in the learning environment of primary and secondary schools in Helsinki. Taking a broad perspective, the study analysed the safety culture of Helsinki schools, ranked them with a point rating system, and thoroughly investigated reasons behind their differences. Safety culture in educa-tional institutions has not been studied previously although research related to safety culture in business is abundant and some studies have considered safety culture in the healthcare sector. The study focused on the attitudes and practices of teachers concerning safety. The main research question is as follows: "What factors related to safety culture distinguish schools with an exceptional safety culture from those weaker one?" Research methods. The study used a mixed-methods approach. It s quantitative part charted the safety attitudes, values and expertise of employees in selected schools. The study also analysed physical safety in selected schools through specific safety inspections. The 14 schools in the study were selected randomly, two from each district. Altogether 388 of 475 employees returned a Webropol questionnaire (N= 475, 81,68 %). The qualitative part of the study analysed the reasons behind perceived differences in school s safety cul-ture using theme interviews with school principals. Elements of a phenomenographic research approach were also used. Results and conclusions. According to the study the distinctive features of good safety cultures in the Helsinki schools included a well-developed understanding of safety hazards and the requirements of basic safety, good safety management practices, a developed understanding of the systematic nature of safety, an appreciation of safety evidenced through everyday practices and open and communal safety-related work. The study offers result-based safety culture improvements for teachers, those responsible for school safety, principals, educational administrators, teacher educators and authorities.

The effect of structure on the dilute solution properties of branched polysaccharides studied with SEC and AsFlFFF

Cereal arabinoxylans, guar galactomannans, and dextrans produced by lactic acid bacteria(LAB) are a structurally diverse group of branched polysaccharides with nutritional and industrial functions. In this thesis, the effect of the chemical structure on the dilute solution properties of these polysaccharides was investigated using size-exclusion chromatography(SEC) and asymmetric flow field-flow fractionation (AsFlFFF) with multiple-detection. The chemical structures of arabinoxylans were determined, whereas galactomannan and dextran structures were studied in previous investigations. Characterization of arabinoxylans revealed differences in the chemical structures of cereal arabinoxylans. Although arabinoxylans from wheat, rye, and barley fiber contained similar amounts of arabinose side units, the substitution pattern of arabinoxylans from different cereals varied. Arabinoxylans from barley husks and commercial low-viscosity wheat arabinoxylan contained a lower number of arabinose side units. Structurally different dextrans were obtained from different LAB. The structural effects on the solution properties could be studied in detail by modifying pure wheat and rye arabinoxylans and guar galactomannan with specific enzymes. The solution characterization of arabinoxylans, enzymatically modified galactomannans, and dextrans revealed the presence of aggregates in aqueous polysaccharide solutions. In the case of arabinoxylans and dextrans, the comparison of molar mass data from aqueous and organic SEC analyses was essential in confirming aggregation, which could not be observed only from the peak or molar mass distribution shapes obtained with aqueous SEC. The AsFlFFF analyses gave further evidence of aggregation. Comparison of molar mass and intrinsic viscosity data of unmodified and partially debranched guar galactomannan, on the other hand, revealed the aggregation of native galactomannan. The arabinoxylan and galactomannan samples with low or enzymatically extensively decreased side unit content behaved similarly in aqueous solution: lower molar mass samples stayed in solution but formed large aggregates, whereas the water solubility of the higher-molar-mass samples decreased significantly. Due to the restricted solubility of galactomannans in organic solvents, only aqueous galactomannan solutions were studied. The SEC and AsFlFFF results differed for the wheat arabinoxylan and dextran samples. Column matrix effects and possible differences in the separation parameters are discussed, and a problem related to the non-established relationship between the separation parameters of the two separation techniques is highlighted. This thesis shows that complementary approaches in the solution characterization of chemically heterogeneous polysaccharides are needed to comprehensively investigate macromolecular behavior in solution. These results may also be valuable when characterizing other branched polysaccharides.

Lasten sitoutuneisuus toimintaan : kvantitatiivinen havainnointitutkimus

Children s involvement is a key quality factor in Early Years Education. As a process variable it concentrates on children s actions and experiences. The involved children are operating in their zone of proximal development. The aim of this study was to find out how the children involved themselves in the Finnish day care centres. The problems of the study were: (1) how the children are involved in different situations between the hours 8.00 and 12.00, (2) how do the skills of children whose involvement level is high differ from the skills of children whose involvement level is low and (3) how do the learning environments of the children whose involvement level is high differ from the learning environments of the children whose involvement level is low? The research method was observation and children s involvement levels were assessed using LIS-YC Scale. In addition, the kindergarten teachers evaluated the children s skills and the team workers did the evaluations of the educational settings. The data used in this study was a part of the Orientaation lähteillä research. The 802 children, who took part in the study, were from 48 different groups of eight different municipalities in Central Uusimaa. There were 18358 observations of children s involvement and the quantitative data was analyzed using correlation, cross tabulation and t-test. Children s involvement was an average at a moderate level. The involvement levels were the highest during playing time and adult guided tasks and lowest during eating and basic care situations. The level of involvement was higher if the children were adaptable, proactive, self-motivated and good players. The involvement lever was lower if the children needed some special care. The children s involvement is supported if the educators had at least once a week a meeting and if children s confidence and identity construction was frequently considered in educational discussions. Furthermore, the appreciation of the ethical issues and positive atmosphere appeared to confirm the involvement. The children s involvement is decreased if the educators had been perpetually short of time or resources or there has been lack of joy and humour in the group.

Sedimentary zooplankton remains as indicators of lake ecological quality and trophic structure

To protect and restore lake ecosystems under threats posed by the increasing human population, information on their ecological quality is needed. Lake sediments provide a data rich archive that allows identification of various biological components present prior to anthropogenic alterations as well as a constant record of changes. By providing a longer dimension of time than any ongoing monitoring programme, palaeolimnological methods can help in understanding natural variability and long-term ecological changes in lakes. As zooplankton have a central role in the lake food web, their remains can potentially provide versatile information on past trophic structure. However, various taphonomic processes operating in the lakes still raise questions concerning how subfossil assemblages reflect living communities. This thesis work aimed at improving the use of sedimentary zooplankton remains in the reconstruction of past zooplankton communities and the trophic structure in lakes. To quantify interspecific differences in the accumulation of remains, the subfossils of nine pelagic zooplankton taxa in annually laminated sediments were compared with monitoring results for live zooplankton in Lake Vesijärvi. This lake has a known history of eutrophication and recovery, which resulted from reduced external loading and effective fishing of plankti-benthivorous fish. The response of zooplankton assemblages to these known changes was resolved using annually laminated sediments. The generality of the responses observed in Lake Vesijärvi were further tested with a set of 31 lakes in Southern Finland, relating subfossils in surface sediments to contemporary water quality and fish density, as well as to lake morphometry. The results demonstrated differential preservation and retention of cladoceran species in the sediment. Daphnia, Diaphanosoma and Ceriodaphnia were clearly underrepresented in the sediment samples in comparison to well-preserved Bosmina species, Chydorus, Limnosida and Leptodora. For well-preserved species, the annual net accumulation rate was similar to or above the expected values, reflecting effective sediment focusing and accumulation in the deepest part of the lake. The decreased fish density and improved water quality led to subtle changes in zooplankton community composition. The abundance of Diaphanosoma and Limnosida increased after the reduction in fish density, while Ceriodaphnia and rotifers decreased. The most sensitive indicator of fish density was the mean size of Daphnia ephippia and Bosmina (E.) crassicornis ephippia and carapaces. The concentration of plant-associated species increased, reflecting expanding littoral vegetation along with increasing transparency. Several of the patterns observed in Lake Vesijärvi could also be found within the set of 31 lakes. According to this thesis work, the most useful cladoceran-based indices for nutrient status and planktivorous fish density in Finnish lakes were the relative abundances of certain pelagic taxa, and the mean size of Bosmina spp. carapaces, especially those of Bosmina (E.) cf. coregoni. The abundance of plant-associated species reflected the potential area for aquatic plants. Lake morphometry and sediment organic content, however, explained a relatively high proportion of the variance in the species data, and more studies are needed to quantify lake-specific differences in the accumulation and preservation of remains. Commonly occurring multicollinearity between environmental variables obstructs the cladoceran-based reconstruction of single environmental variables. As taphonomic factors and several direct and indirect structuring forces in lake ecosystems simultaneously affect zooplankton, the subfossil assemblages should be studied in a holistic way before making final conclusions about the trophic structure and the change in lake ecological quality.

Timotein (Phleum pratense ssp. pratense L.) alkuperän vaikutus kylmänkestävyyteen, vernalisaatiovaatimukseen sekä kasvuun ja kehitykseen

Nurmiheinien merkitys maailmanlaajuisesti on merkittävä, sillä noin 69 % maapallon peltopinta-alasta on pysyvää laidunmaata tai niittyä. Suomessa nurmien osuus on noin 29 %, ja tuotanto perustuu pääosin intensiiviseen säilörehuntuotantoon. Yleisin nurmiheinälaji Suomessa on timotei (Phleum pratense ssp. pratense L.). Timotei on talvenkestävä ja soveltuu siksi pohjoisiin kasvuoloihin. Timoteilajikkeita jalostettaessa pohjoista alkuperää olevia vanhempaislinjoja käytetään hyvän talvenkestävyyden varmistamiseksi, eteläisiä tavoiteltaessa nopeaa kasvurytmiä. Ilmaston muutoksen ennustetaan lisäävän erilaisia äärioloja kuten myrskyjä ja sateita. Vuorokauden keskilämpötila nousee ja kasvukausi pidentyy. Lisäksi talvet muuttuvat sateisemmiksi. Muutokset näkyvät erityisesti pohjoisissa kasvuympäristöissä. Tutkimuksessa haluttiinkin selvittää eri alkuperää edustavien timoteilajikkeiden ja linjojen kylmänkestävyyttä, kasvu-, ja kehitysnopeutta sekä vernalisaation vaikutusta. Lisäksi tutkittiin syysviljojen vernalisaatiovasteen mittaamiseen käytettyjen menetelmien soveltuvuutta nurmille. Tutkimukseen kuului kaksivuotinen peltokoe sekä kasvatuskaappikoe. Vernalisaatio nopeutti timotein kasvua ja kehitystä. Tutkimuksen perusteella eteläistä alkuperää olevilla lajikkeilla kasvu- ja kukintavalmius oli olemassa ilman vernalisaatiota. Pohjoisilla lajikkeilla oli suurempi vernalisaatiovaste ja niiden kukkiminen ja kasvu nopeutui vernalisaation myötä. Vernalisaatiolla oli vaikutusta myös kasvuston rakenteeseen. Generatiivisten versojen määrä lisääntyi vernalisaation myötä, kun taas vegetatiivisten versojen määrä väheni. Kylmänkestävyys oli tutkimuksen perusteella riippuvainen syksyn karaistumisjakson pituudesta sekä jakson lämpösummasta (FH-COLD). Korkea keskilämpötila ja lyhyt karaistumisjakso heikensivät kylmänkestävyyttä. Vastaavasti karaistumiskauden lämpötilan ollessa välillä 0 °C:ta ja + 5 °C:ta ja jakson pituuden kasvaessa kylmänkestävyys lisääntyi. Tutkimuksen perusteella vernalisaatiolla oli selvä vaikutus timotein kasvuun ja kehitykseen. Pohjoista alkuperää olevat timoteit reagoivat vernalisaatioon eteläisiä enemmän. Osa pohjoisista linjoista vaati vernalisaation generatiivisten versojen muodostumiseen. Syysviljojen vernalisaatiovasteen mittausmenetelmät soveltuvat osin myös puhtaiden timoteilajikkeiden vernalisaation seurantaan.

NK-and T-cell function in chronic myeloid leukemia patients treated with interferon-? monotherapy

Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.

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.

Hevosen mahahaava - sijainti, vakavuusasteet ja altistavat tekijät

Mahahaava on hyvin yleinen ongelma hevosilla; sen prevalenssi vaihtelee 48-100 % välillä. Mahahaava johtuu epätasapainosta mahalaukkua suojaavien tekijöiden ja mahalaukun limakalvoa ärsyttävien tekijöiden välillä. Tarkkaa syytä mahahaavan syntymiselle ei tunneta ja tietoa altistavista tekijöistä ja niiden merkityksestä on melko vähän. Liikunnan, ruokinnan ja stressin epäillään vaikuttavan mahahaavan syntyyn. Tutkimuksessa tähystettiin 45 Yliopistolliseen Hevossairaalaan mahahaavaan viittaavien oireiden vuoksi tutkittavaksi tullutta hevosta. Hevosilta tutkittiin mahalaukun eri alueet ja mikäli mahdollista, myös duodenum. Haavaumat pisteytettiin niiden syvyyden, laajuuden ja limakalvon ärtyneisyyden perusteella 0-4 asteikolla. Hevosista kerättiin perustiedot ja omistajia haastateltiin kyselykaavakkeen avulla hevosten elintavoista ja olosuhteista. Hevoset olivat iältään 1-22 vuotiaita ja mukana oli tammoja, ruunia ja oreja. Suurin osa hevosista oli puoliverisiä (47%), mutta mukana oli myös suomenhevosia, lämminverisiä ja poneja. Käyttötarkoitukseltaan hevoset olivat ratsuja, ravureita tai levossa olevia hevosia. Hevosista 29% oli kilpahevosia. 31% hevosista oli aiemmin hoidettu mahahaavan vuoksi. Kaikilta hevosilta yhtä lukuunottamatta löytyi mahahaava. Haavaumien vakavuus vaihteli 1. - 3. asteen välillä. Suurin osa haavaumista sijaitsi pyloruksessa ja siellä oli myös eniten vakavia haavaumia. Lievimmät muutokset olivat muulla rauhasalueella; siellä todettiin määrällisesti vähiten haavaumia ja suurin osa todetuista haavaumista oli lieviä. Yllättävän monelta (60%) hevoselta löytyi haavauma duodenumista. Antrumin ja pyloruksen välillä todettiin korrelaatio; mikäli antrumissa oli haavauma, löytyi haavauma todennäköisesti myös pyloruksesta. Samanlainen korrelaatio todettiin muun rauhasalueen ja duodenumin välillä; mikäli muulla rauhasalueella todettiin haavauma, niin myös duodenumista löytyi haavauma. Vakavuusasteen ja altistavien tekijöiden, kuten korsirehumäärän, ruokintakertojen tai ulkoilun välillä ei todettu yhteyttä, Tutkimuksen perusteella ei siis voida arvioida, mitkä tekijät vaikuttavat mahahaavan syntyyn. Myöskään oireiden ja haavaumien välillä ei todettu yhteyttä. Mikään tietty oire ei siis kerro haavauman sijainnista tai vakavuusasteesta. Tutkimuksen aineisto oli hyvin valikoitunutta; kaikilla hevosilla oli indikaatio tulla tähystykseen. Tutkimuksessa ei ollut mukana oireetonta vertailuryhmää. Lisäksi aineisto oli useiden ominaisuuksien suhteen hyvin monimuotoinen. Tarvitaan lisää tutkimuksia, jotta mahahaavaan liitetyistä altistavista tekijöistä tai oireista voitaisiin tehdä tarkempia johtopäätöksiä.

"Totta kai pyrin koko ajan tuntemaan oman kroppani siellä" : Kolmen orkesteriviulistin kokemuksia ruumiillisuudestaan.

Tutkielmassa esitellään kolmen orkesteriviulistin kokemuksia ja käsityksiä omasta ruumiillisuudestaan muusikon työssään. Muusikkolähtöisyys, laajemmin tarkasteltuna tekijälähtöinen tutkimus, on nykyisin yhä tärkeämpi osa musiikintutkimusta. Muusikkolähtöistä tutkimusta on Suomessakin kirjoitettu jonkin verran, harvoin kuitenkaan ruumiillisuusteeman kautta. Haastatellut viulistit ovat pääkaupunkiseudun suurista orkestereista. Muusikon työtä voidaan tarkastella monista eri lähtökohdista. Tässä työssä pyritään ottamaan huomioon ihmisen oma kokemus ja ruumiillinen sidos maailmaan, ja näin se sitoutuu fenomenologisiin lähtökohtiin, erityisesti Maurice Merleau-Pontyn ajatteluun. Michel Foucault ja Elizabeth Grosz edustavat tämän työn kannalta sitä ajattelutapaa, jonka hengessä pidän muusikon työtä historiallisesti rakentuneena ja muutoksenalaisena prosessina. Groszin teoretisointi keskittyy subjektin ruumiillisuuteen ja tapoihin käsitteellistää ruumis länsimaisessa ajattelussa. Haastattelumateriaali jakaantuu kahteen päälukuun. Ensimmäisessä käsitellään soittajien omaa yksityistä tilaa. Harjoittelun oma tila liittyy ruumiillisuuteen sikäli, että ruumiillinen toistotyö ja viulistisen identiteetin muotoutuminen tapahtuu pitkälti yksityisesti, tietoisena julkisen tilan vaatimuksista. Orkesteriviulistien sosiaaliseen tilaan liittyy toisaalta vuorovaikutus muiden muusikoiden kanssa harjoituksissa ja toisaalta muusikoiden ja yleisön kohtaaminen konserteissa. Sosiaaliseen tilaan liittyviä ruumiillisuuteen kytkeytyviä ilmiöitä käsitellään erillään, toisessa analyysiluvussa. Tutkielma tuo esiin välähdyksiä erilaisista ruumiillisuuteen ja viulunsoittoon kytkeytyvistä ilmiöistä. Kvalitatiivisen ja fenomenologisen tutkimuksen hengessä haastateltujen erilaiset kokemukset on pyritty esittelemään pyrkimättä universalisoimaan niitä. Haastattelumateriaali osoittaa viulistisen ruumiillisuuden liittyvän merkittävällä tavalla muusikkojen käsityksiin itsestään. Haastateltujen kokemukset tuovat lisää tietoa orkesteriviulistin työstä.

'Luonto' ympäristöarvojen tastalla

Tutkielman tavoitteena on selvittää sanan ‘luonto’ merkitystä ja käyttöä ympäristöfilosofisessa keskustelussa. Mielenkiinnon kohteena on erityisesti se, voidaanko tällä käsitteellä perustella ympäristöarvoja ja voidaanko näillä arvoilla katsoa olevan moraalista velvoittavaa voimaa. Sanalle ‘luonto’ löytyy kaksi kilpailevaa määritelmää, joita käytetään ympäristöfilosofisissa teorioissa rinnakkain tai niistä valitaan toinen. Näiden luonto-käsitteen määritelmien voidaan katsoa olevan läpikäydyn aineiston perusteella yleisesti hyväksyttyjä. Niitä ei kuitenkaan perustella huolella, vaan tyydytään vallitsevaan käsitykseen sitä kyseenalaistamatta. Tästä syystä tämän tutkielman kysymyksenasettelulle on ympäristöfilosofiassa tarvetta. Tutkielma on luonteeltaan analyyttinen. Lähdeaineistoksi on valittu historiallisten auktoriteettien lisäksi modernin ympäristöfilosofian puhutuimpia ja kiistellyimpiä näkökulmia. Historiallisten auktoriteettien tärkein ominaisuus tämän tutkielman kannalta on niiden riippumattomuus modernista ympäristöajattelusta, jolla on omat ennakkokäsityksensä ja asenteensa painolastinaan. Keskeisinä lähteinä ovat: John O’Neillin, Alan Hollandin ja Andrew Lightin Environmental Values; John Stuart Millin On Nature ja Robert Elliotin Faking Nature. J. S. Millin On Nature -essee, jossa luonto-käsitteelle löydetään ympäristöfilosofiassa yhä käytettävät kaksi määritelmää. Näitä ‘luonnon’ määritelmiä syvennetään modernien ympäristöfilosofien ajatuksilla. O.Neill ym. teoksesta on tähän tutkielmaan otettu rakenne sen esittämän kysymyksenasettelun mukaisesti. Lisäksi se tarjoaa tutkielman esittämään ympäristöarvojen moraalisen velvoittavuuden ongelmaan yhden ratkaisun. Elliotin artikkeli on tälle työlle keskeinen kahdesta syystä: ensinnäkin se toimii argumentaation kehityksen vastinparina, sillä sen sisältö on tämän tutkielman näkökulmasta ristiriitainen. Toiseksi, se onnistuu kuvaamaan ympäristöön liitettävien arvojen luonteen vahvalla intuitiivisella vertauksella taiteeseen. Näiden taidevertausten ansioista Elliotin artikkeli on kiistatta yksi modernin ympäristöfilosofian lainatuimmista ja kiistellyimmistä. Elliotin argumentaatiolinjaa jatkavat ajattelijat syventävät näitä käsityksiä entisestään ja myös O’Neill ym. saavat siitä vaikutteita omaan ajatteluunsa. Tämän tutkielman aineiston valossa voidaan todeta, että luonnolle löydetään kaksi eriävää määritelmää. Ensinnäkin ‘luonto’ määritellään tieteellisesti luonnonlaeiksi. Toisessa määritelmässään ‘luonto’ ymmärretään ei-inhimilliseksi ympäristöksi. Näiden määritelmien voidaan sanoa eroavan ensisijaisesti siinä, että jälkimmäisen määritelmän mukaisella luonnolla voidaan katsoa olevan arvoa. Tämä arvo on luonteeltaan joko välillistä tai välitöntä riippuen siitä, arvostetaanko sitä jonkin toisen arvon saavuttamisen välineenä vai sen itsensä tähden itseisarvoisesti. Näiden erilaisten ympäristöarvon lajien tunnistaminen on niiden moraalisen velvoittavuuden kannalta keskeinen tekijä. Osa ympäristöfilosofisista teorioista lähtee siitä, että luonnolla olisi objektiivinen arvonsa silloinkin kun inhimillinen moraalisubjekti ei sitä ole arvostamassa. Toisen tärkeänä huomiona on, että mikäli ‘luonto’ määritellään ei-inhimilliseksi ympäristöksi, kaikki inhimilliset luonnonsuojelutoimet osoittautuvat mahdottomiksi. Ne muuttaisivat tuon ei-inhimillisen ympäristön osaksi inhimillistä ympäristöä. Jotta moraaliselle toimijalle ympäristöarvoilla olisi merkitystä, niiden olisi oltava tämän agentin tunnistettavissa. Tutkielman aineiston valossa voidaan todeta, että vaikka ei-inhimillisellä ympäristöllä tällaista arvoa olisikin, ei se voisi ohjata moraalista toimintaamme. Lisäksi voidaan todeta, ettei tässä työssä esitetyt ympäristöfilosofiset teoriat tätä luonnonsuojelun paradoksi onnistu kiertämään. Tämän työn tärkein johtopäätös on, ettei luontoa voida suojella. Ympäristöfilosofia Etiikka

Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.