Gymnasisters läsvanor och skrivvanor : Finlandssvenska gymnasisters åsikter om ämnet modersmål och litteratur

Bakgrunden till denna avhandling är att nivån i studentskrivningarna i modersmål och litteratur har sjunkit under de senaste tio åren bland både finskspråkiga och svenskspråkiga gymnasister i Finland. I och med det startades ett projekt, Språklig mångfald, vars syfte är att undersöka bakgrundsfaktorer till att nivån har sjunkit. Som en del av projektet redogör jag i den här avhandlingen för finlandssvenska gymnasisters läsvanor och skrivvanor, dvs. vad, hur ofta och på vilket språk gymnasisterna läser och skriver. Dessutom har jag undersökt gymnasisters attityder till ämnet modersmål och litteratur. Min studie är baserad på en enkätundersökning bland 81 gymnasister samt på intervjuer med åtta gymnasister. Resultatet visar att gymnasister läser dagstidningar i betydligt större utsträckning än de läser böcker. Speciellt gymnasister med låga betyg i modersmål och litteratur läser sällan eller aldrig skönlitteratur. Skrivkulturen bland gymnasisterna innefattar främst media som textmeddelanden per mobiltelefon och debattinlägg på diskussionsforum på Internet. I dessa texter används oftast ett enkelt och kortfattat språk. Gymnasister med höga betyg i modersmålsämnet läser oftare och skriver flera olika genrer under sin fritid än vad gymnasister med låga betyg gör. Gymnasister med höga betyg läser och skriver även oftare på svenska än vad gymnasister med låga betyg gör. I min studie framkommer det att gymnasisterna anser att modersmålsämnet är viktigt, men inte roligt samt att höga betyg korrelerar med positiva attityder. De som har negativa attityder har ofta haft svårigheter med ämnet och har även svårt att se någon koppling mellan modersmålsämnet och vardagen samt framtiden. Många av dem anser att studentskrivningarna är det enda målet med modersmålsämnet.

Konstteoretikern och livet : Mikael Lybecks Breven till Cecilia i nietzscheansk belysning

I avhandlingen analyseras den finlandssvenska författaren Mikael Lybecks (1864-1925) verk Breven till Cecilia. Denna brevroman hör till Lybecks senare produktion och utkom 1920. Verket har kommit att betraktas som en klassiker inom den finlandssvenska litteraturen. Av författarensövriga produktion är det strängt taget endast Tomas Indal (1911) som haft en jämförbar genomslagskraft. Trots detta har romanen inte varit föremål för en enda mer omfattande studie. l sin Lybeckbiografi relaterar Erik Kihlman i första hand verket till det biografiska och till samhällssituationen vid tiden för romanens tillkomst. Vetenskapen, konsten och livet bildar tillsammans den triangel som utgör romanens stomme. Antiken är starkt närvarande i romanen och med begrepp som härrör från denna era kan man tala om det sokratiska, det apollinska och det dionysiska. Detta är begrepp som Friedrich Nietzsche utgår i från i sitt ungdomsverk Tragedins födelse (Die Geburt der Tragödie aus dem Geiste der Musik) från 1872. Nietzsches vetenskapskritik och hans syn på den högsta konsten som ett brödraförbund mellan Dionysos och Apollon står i förvånansvärt hög grad i samklang med den övergripande tematiken i Lybecks verk. När Nietzsche därtill framhäver musiken som grunden förall konst får många inslag i romanen sin förklaring. l Breven till Cecilia skildras en vändpunkt i huvudpersonen Sven Ingelets liv. Därför ligger tyngdpunkten i den tematiska analysen vid det som gör att verket kan betraktas som en utvecklingsroman. Även om romanen skildrar en tidsperiod på endast ett och ett halvt år så hinnerhuvudpersonen ändå undergå en betydande personlighetsförändring. Nietzsches Tragedinsfödelse med dess ovannämnda grundläggande begrepp lämpar sig ypperligt som referensram för en analys av denna förändring. Avhandlingen har också strukturerats utgående från de nietzscheanska begreppen sokratiskt, dionysiskt och apollinskt vilka alltså återspeglar huvudpersonens utvecklingsfaser. Från att ha varit en renodlad förnuftsmänniska väcks Sven Ingelet genom ett kortvarigt kärleksförhållande till det dionysiska med allt vad det innebär av känslorus och lidande. Att Ingelet nästan helt uppgått i sin roll som konstteoretiker har medfört att hans förhållningssätt till omvärlden utmärks av intresselös betraktelse. Till följd av sin viljesvaghet och bristande handlingskraft måstehan försöka behålla sin älskades gunst genom magiska pseudohandlingar. Detta leder in Ingelet i myternas värld. Hans verklighetsuppfattning får andra dimensioner än den vetenskapliga. Det dionysiska uppvaknandet medför tillsammans med det efterföljande brevskrivandet att Ingeletnår fram till en självkännedom och upphör att vara en främling i tillvaron. Ingelet utvecklar ocksåen ny konstsyn som förutsätter ett samband mellan konsten och livet. Detta apollinska utvecklingsstadium kännetecknas också av sanningsförmedlande drömmar, och av en tilltagande resignation som till slut utmynnar i ett självmord. Nyckelord: brevroman, Dionysos, Apollon, främlingskap, verklighetsflykt

Om naturens förvandlingar : Vetenskap, kunskap och frihet i Rudolf Steiners tidiga tänkande. Idéhistoriska perspektiv på Waldorfpedagogiken

TRANSFORMATIONS OF NATURE Science, Knowledge and Freedom in the Early Thinking of Rudolf Steiner. Perspectives on Waldorf Education in the light of the History of Ideas Waldorf Education is based on the worldview that Rudolf Steiner (1861-1925) developed to a wide-ranging anthroposophical movement in the first decades of the 20th century. This thesis takes as its departure the early thinking of Rudolf Steiner that precedes anthroposophy, and its main purpose is to uncover the tradition of ideas represented in Steiner´s early life and which, in different ways, have emerged in the practice of Waldorf Education. Through systematic analysis I attempt to bring to light different aspects of Steiner’s early thinking: his concept of science, his epistemological startingpoints and his philosophy of freedom. By departing from J. W. Goethe’s qualitative concept of science, Steiner strove in his early works to formulate a monistic worldview which appears to be closely related to the Romantic Movement and its philosophy of nature. Characteristic traits of his thinking are, on the one hand, a critique of a one-sided enlightenment and, on the other hand, an aspiration to see the world as a living organic unity. Human beings can, by developing our intuitive faculties, get a deeper understanding of the indissoluble relationship between man and nature. Against this background Steiner´s early thinking can be read as a special kind of romantic development narrative. Steiner’s early thinking also opens the way for romantic perspectives on Waldorf Education. It appears that many central aims and conceptions in Waldorf Education can be illuminated by the epistemological perspective upon which Steiner elaborated early in his life. An organic curriculum, phenomenological didactics and high ideal of freedom can be considered seen as educational applications of conceptions that played an important role in Goethe and his age. Thus, Waldorf Education provides in our contemporary society an exceptional set of educational values: a holistic education with romantic undertones.

Tidigt fullständigt svenskt språkbad i Esbo

Avhandlingen behandlar tidigt fullständigt svenskt språkbad i Esbo. Arbetet utreder vad tidigt fullständigt språkbad innebär samt vad språkbadsföräldrars och språkbadslärares åsikter om språkbadet i Esbo är. Undersökningen omfattar 60 familjer samt samtliga lärare (14 st) som arbetar med språkbad i Esbo. Undersökningen består av två enkäter; en till språkbadsföräldrar och en tillspråkbadslärare. Av de 60 enkäter som sändes ut till språkbadsföräldrarna besvarades 45, dvs. 75%. 12 lärare av 14, dvs. 86%, besvarade den andra enkäten. Det är viktigt att känna till centrala begrepp i anslutning till tvåspråkighet, då man talar om andraspråkstillägnandet i språkbad. I detta arbete presenteras en modell (Sahi, 1999) som åskådliggör centrala begrepp i anslutning till tvåspråkighet. Tidigtfullständigt språkbad är ett program som är planerat att omfatta hela grundskolan. Språkbad är ämnat för enspråkiga majoritetsbarn i ett tvåspråkigt land. Dessa barn tillägnar sig minoritetsspråket i skolan. Språkbadet är inte enbart ett program, utan även en metod. Skillnaden mellan språkbad och traditionell undervisning i språk är att man i språkbadet betonar kommunikation framom grammatik. Målet medspråkbadet är funktionell tvåspråkighet. Många faktorer inverkar på andraspråkstillägnandet i språkbadet. I detta arbete betonas attitydernas och motivationens betydelse. Språkbad kräver en medveten och aktiv insats av föräldrarna. Vidare betonar litteraturen som behandlar språkbad undervisningsmetodernas betydelse samt språkbadslärarens viktiga roll. Föräldrarna är nöjda med språkbadsprogrammet, eftersom det givit goda resultat och motsvarat förväntningarna. Enligt föräldrarna är språkbad ett mjukt, modernt och effektivt program. Föräldrarna anser att barnen lär sig badspråket på ett naturligt sätt och även får en öppnare inställning till språk och kulturer. Föräldrarna betonar lärarnas viktiga insatser samt kontinuitetens betydelse. Vidare tycker föräldrarna att språkbadet borde inledas i ett tidigare skede och att eleverna borde ha mera kontakt med badspråket utanför skolan. Målet med att barnen går i språkbad är, enligt föräldrarna, att barnen ska förstå och tala språket flytande samtkänna kulturen som hör ihop med språket. Både föräldrarna och lärarna tycker att språkbadsklasserna är för stora och att det är brist på material. Lärarna anser att man mer än tidigare borde informera allmänheten, beslutsfattare samt föräldrar om verksamheten. Därtill tycker lärarna att samarbetet mellan daghem och skola samt specialundervisningen borde utvecklas. Lärarna betonar att språkbad inte lämpar sig för alla barn. Lärarna anser sig främst behöva praktiska råd av andra som arbetar med språkbad samt teoretisk kunskap. Både föräldrarna och lärarna tycker att språkbadsverksamheten i Esbo borde koordineras och att språkbadselevernas prestationer noggrant borde följas upp. Nyckelord: Tvåspråkighet, Språkbad, Andraspråkstillägnande Keywords: Bilingualism, Immersion, Second Language Aquisition.

I konstens namn - scenen som arbetsmiljö : Arbetsplatsmobbning ur skådespelarperspektiv

Workplace bullying is a topic of current interest in Finland. Workplace bullying is found in all professions, including the artistic ones. This thesis aims to explore workplace bullying from the view of the Finland-Swedish actors as a phenomenon that within dramatic art is difficult to define due to the fact that the body and emotions of an actor constitute his or her working tools. The research aims to deepen the understanding of the actors’ working situation, and particularly of the difficulties and problems actors face when exercising their job. The research problems are: What forms of bullying are the actors exposed to? Who is bullying? How is the bullying received by the actors, and what are the possible consequences? The theoretical orientation of this thesis is based upon dialogical philosophy where phenomenology, hermeneutics and dialog meet in an orientation where the unseen is emphasized and made visible. Artistic leadership should be based upon a pedagogic understanding that by an open and equal dialog with the Other recognizes human diversity. The narrative research was undertaken by using an interview guide for the interviews with eleven actors, six women and five men with the voice of a sixth man represented by an article. The interviews, each on average 118 minutes, were recorded and transcribed. The method of discursive analysis was initiated by numerous reflective readings based on analytic induction. The inductive part of the analysis consisted of mapping out the individual experiences of bullying where after the process of finding connecting common features in the extensive material took place. The coded data was then deductively grouped together according to the research problems, and subgroups were formed for deeper description. The research findings show that workplace bullying is an everyday occurrence within the field of dramatic art. Actors are bullied by theatre managers and directors as well as by colleagues and other personnel. The main areas of bullying is depreciation of one’s professional skills, the existing jargon, sexual harassment, collective bullying and bullying because of personal qualities. A significant finding concerning this problem was the existing culture of silence. Even if actually seeing and hearing a colleague being bullied, few stood up to defend the person being bullied because of fear of retaliation. Even the person actually being the object for bullying found it difficult to take any actions.

Datorstödda studier i hälso- och socialvård : MuPSiNet-projektet

The aim of the study was to explore why the MuPSiNet project - a computer and network supported learning environment for the field of health care and social work - did not develop as expected. To grasp the problem some hypotheses were formulated. The hypotheses regarded the teachers' skills in and attitudes towards computing and their attitudes towards constructivist study methods. An online survey containing 48 items was performed. The survey targeted all the teachers within the field of health care and social work in the country, and it produced 461 responses that were analysed against the hypotheses. The reliability of the variables was tested using the Cronbach alpha coefficient and t-tests. Poor basic computing skills among the teachers combined with a vulnerable technical solution, and inadequate project management combined with lack of administrative models for transforming economic resources into manpower were the factors that turned out to play a decisive role in the project. Other important findings were that the teachers had rather poor skills and knowledge in computing, computer safety and computer supported instruction, and that these skills were significantly poorer among female teachers who were in majority in the sample. The fraction of teachers who were familiar with software for electronic patient records (EPR) was low. The attitudes towards constructivist teaching methods were positive, and further education seemed to utterly increase the teachers' readiness to use alternative teaching methods. The most important conclusions were the following: In order to integrate EPR software as a natural tool in teaching planning and documenting health care, it is crucial that the teachers have sufficient basic skills in computing and that more teachers have personal experience of using EPR software. In order for computer supported teaching to become accepted it is necessary to arrange with extensive further education for the teachers presently working, and for that further education to succeed it should be backed up locally among other things by sufficient support in matters concerning computer supported teaching. The attitudes towards computing showed significant gender differences. Based on the findings it is suggested that basic skills in computing should also include an awareness of data safety in relation to work in different kinds of computer networks, and that projects of this kind should be built up around a proper project organisation with sufficient resources. Suggestions concerning curricular development and further education are also presented. Conclusions concerning the research method were that reminders have a better effect, and that respondents tend to answer open-ended questions more verbosely in electronically distributed online surveys compared to traditional surveys. A method of utilising randomized passwords to guarantee respondent anonymity while maintaining sample control is presented. Keywords: computer-assisted learning, computer-assisted instruction, health care, social work, vocational education, computerized patient record, online survey

Systematic literature review: Research on Supported Playgroups

Overview This review of research conducted with supported playgroups was prepared for the Queensland Department of Education, Training and Employment (DETE). The report provides a synthesis of the research on the effectiveness of supported playgroups to improve child, parent, and community outcomes and to identify key features of supported playgroups that support effective outcomes. Supported playgroups are community-based services that provide a low intensity parenting intervention, through regular group sessions for parent-child dyads. Supported playgroups target vulnerable families who may benefit from parenting support. Supported playgroups have common goals to enhance children’s early learning and parental wellbeing. Method A search strategy was devised to identify research studies, nationally and internationally, that involved parent-child group programs for families with young children, delivered under the leadership of an employed facilitator. Academic databases and other data sources were explored for studies conducted in the period from 2004 to 2014. Summary descriptions of the research studies were developed; assessment of research methodologies was made; research evidence on the effectiveness of supported playgroups to improve child, parent, and community outcomes was identified; and comparative analyses of the implementation features of supported playgroups were completed. Findings The search strategy identified 34 research publications, reporting on 29 different programs. Twenty-six of the studies report on research conducted in Australia and eight reported on research conducted in other countries, including the United Kingdom, Canada, and the United States. Three clusters of playgroups were identified: Category 1 - Standard supported playgroups; Category 2 - Mobile playgroups; Category 3 – Supported playgroups with specific interventions. The research studies identified encompassed experimental and non-experimental research designs. The studies of standard supported playgroups and mobile playgroups were most often qualitative studies and modest in scale, in terms of the number of research participants. Experimental and quasi-experimental research designs characterised the studies identified in the category of supported playgroups with specific interventions. Overall, the research studies that were categorised as supported playgroups with specific interventions provided stronger evidence for effectiveness to improve parental behaviour in ways that are known to support children’s early developmental competence. Qualitative studies, including case studies and ethnographic research, documented important features of program delivery, such as the importance of facilitators’ interpersonal skills to positive experiences for families in the playgroups; as well as the important opportunities that the playgroups afforded to vulnerable families to reduce social isolation. Conclusions The potential for supported playgroups to improve a broad range of learning and psychosocial outcomes for children and parents was suggested by many of the research studies. However, the nature of the research designs employed means that it is not possible to conclude that there is strong evidence of the impact of supported playgroups on child, parent, and community outcomes. The qualitative studies did provide rich descriptions about the implementation processes of playgroups and also captured the variability in the delivery of the playgroups in terms of who participated, local contextual factors that impacted on the playgroup experiences, and the nature of the experiences of parents within the playgroups. Research methodologies need to be employed that address the limitations of the studies to date. This would provide more defensible evidence that supported playgroups have an impact over time on outcomes for children, families, and communities. Overall, this area of research remains relatively under-evaluated in terms of rigorous research designs. The identified research studies point to some promising research directions but do not yet enable strong claims to be made about the effectiveness of the standard playgroup or mobile playgroup models to impact on parenting outcomes. Data collected from interview and survey methodologies clearly identifies that supported playgroups are highly acceptable to families. Given the popularity of supported playgroups to engage families across diverse communities, and the reported high levels of satisfaction and benefits identified within many of the research studies, it is clear that the provision of supported playgroups is fulfilling an important community need by providing support to parents with young children. However, there is a need to strengthen the evidence base that supported playgroups are an effective early parenting intervention that improves outcomes for children, parents, and communities.

Characterization of the mitochondrial active-site serine protein LACTB: filaments in the mitochondrial intermembrane space

Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases.

Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Adult-type hypolactasia: Genotype-phenotype correlation

Adult-type hypolactasia (primary lactose malabsorption, lactase non-persistence) is the most common enzyme deficiency worldwide, and manifests with symptoms of lactose intolerance such as abdominal pain, gas formation and diarrhea. In humans with adult-type hypolactasia, lactase activity is high at birth, but declines during childhood to about one-tenth of the activity at birth. In 2002, a one base polymorphism C/T-13910, located 14 kilobases from the starting codon of the lactase-phlorizin hydrolase (LPH) gene was observed to be associated with the persistence of lactase activity. The T-13910 allele (C/T-13910 and T/T-13910 genotypes) associates with persistence of lactase activity throughout life, whereas the C/C-13910 genotype associates with adult-type hypolactasia. In this thesis work, the timing and mechanism of decline of lactase enzyme activity during development was studied using the C/T-13910 polymorphism as a molecular marker. We observed an excellent correlation between low lactase activity and the C/C-13910 genotype in all subjects > 12 years of age, irrespective their ethnicity. In children of African origin, the lactase activity declined somewhat earlier than among Finnish children. Furthermore, we observed an increasing imbalance in the relative lactase mRNA expression from the C-13910 and T-13910 alleles in Finnish children beginning from five years of age. The genetic test for adult-type hypolactasia showed a sensitivity of 93% and a specificity of 100% in the Finnish children and adolescents > 12 years of age. The relation of milk consumption and the milk-related abdominal complaints to the C/T-13910 genotypes associated with lactase persistence/non-persistence was studied by a questionnaire-based approach in > 2100 Finns. Both Finnish children and adults with the C/C-13910 genotype consumed significantly less dairy products compared to those with the C/T-13910 and T/T-13910 genotypes. Flatulence was the only of the abdominal symptoms of lactose intolerance that subjects with the C/C-13910 genotype reported significantly more often than those with the C/T-13910 and T/T-13910 genotypes. A minor proportion (<10%) of subjects with the C/C-13910 genotype, nevertheless, reported drinking milk without any symptoms afterwards. There was no association between cow's milk allergy starting as a newborn and adult-type hypolactasia. In an association study an increased risk of colorectal cancer was observed among those with molecular diagnosis of adult-type hypolactasia. It warrants further studies to clarify whether the increased risk observed in the Finnish population is associated with lactose or decreased intake of dairy products in these subjects.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

Campylobacter Infections of Domestic Origin in Finland

Campylobacter jejuni and C. coli are the leading causes of human bacterial gastroenteritis in developed countries. Most human Campylobacter infections are sporadic and a seasonal peak in the distribution of infections can be seen in the summer months in several countries, including Finland. A variety of risk factors for Campylobacter infections have been identified; handling and eating poultry, drinking unpasteurized milk, contact with domestic animals, and travelling abroad. However, the relative importance of the different risk factors in sporadic cases of Campylobacter infection remains unknown. In most cases, the infection is self-limiting and no specific treatment is required. Campylobacter enteritis can cause a wide range of complications, including reactive arthritis (ReA) that is reported in 1-5% of the cases. Seven clinical microbiology laboratories serving different geographical areas of Finland, participated in this multi-centre study, conducted during a seasonal peak in 2002. In a matched case-control study, domestically-acquired sporadic Campylobacter infections from three geographical areas were collected. The final study comprised 100 cases and 137 controls. Risk factors for sporadic domestically-acquired Campylobacter infections were identified on the basis of a questionnaire; swimming in natural waters was found to be a novel risk factor for Campylobacter infection. Other independent risk factors were tasting or eating raw or undercooked meat and drinking untreated water from a dug well. The role of bacterial strain and host characteristics are not fully understood in Campylobacter infections. Exposure factors, demographical characteristics, and the serotype of the Campylobacter isolate may affect the severity of the enteritis. This cross-sectional study comprised 114 patients with C. jejuni enteritis, diagnosed in three clinical microbiology laboratories; most of the patients had participated in the previous case-control study. Swimming was associated with age ≤ 5 years and serotype Pen 6,7 was found significantly more often among patients reporting swimming. The geographical distribution among serotypes varied; serotype Pen 4-complex appeared more often in patients from urban areas and serotype Pen 21 among patients from more rural areas. Thus, risk factors and sources of infection for C. jejuni infection may vary among individuals depending on age and geographical location. The in vitro susceptibilities of C. jejuni and C. coli strains isolated from patients infected abroad (85 strains) or domestically (393 strains) revealed that susceptibility to erythromycin is still high, even among isolates of foreign origin. However, the novel antimicrobial agent telithromycin did not offer any advantage over erythromycin; isolates with high minimal inhibitory concentrations (MICs) for erythromycin also showed reduced susceptibility to telithromycin. Reduced susceptibility to fluoroquinolones was detected almost exclusively among isolates of foreign origin and half of these isolates with high MICs for fluoroquinolones also showed elevated MICs for doxycycline. Questionnaires concerning complications associated with C. jejuni enteritis were sent to patients two months after becoming ill; 201 patients from seven different geographical areas were included in the study. Musculoskeletal complications after C. jejuni infection were commonly reported by patients (39%). The incidence of classical ReA was 4% and that of Achilles enthesopathy and/or heel pain 9%. Other C. jejuni-associated reactive joint symptoms were commonly reported, however, due to their milder nature seldom seen and diagnosed by a physician. The severity of the enteritis may predict further complications; stomach ache during enteritis was connected to the development of later joint pain. Early antimicrobial treatment, within two days from the start of symptoms, shortened the duration of diarrhoea by two days but did not prevent later musculoskeletal complications. Campylobacter is an important human enteropathogen and causes a significant burden of illness. As the incidence of Campylobacter infections is high, the importance of the infection and the occurrence of complications will increase. This stresses the importance of understanding the risk factors for acquiring Campylobacter infection and how bacterial strain and host characteristics may affect the risk for infection. The role of antimicrobial treatment for acute Campylobacter enteritis seems to be marginal and should be used restrictively.

Stanniocalcin-1 in cell stress and differentiation

Stanniocalcin-1 (STC-1) is a 56 kD homodimeric protein which was originally identified in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. STC-1 was considered unique to fish until the cloning of cDNA for human STC-1 in 1995 and mouse Stc-1 in 1996. STC-1 is conserved through evolution with human and salmon STC-1 sharing 60% identity and 80% similarity. The surprisingly high homology between mammalian and fish STC-1 and the protective actions of STC-1 in terminally differentiated neurons, originally reported by my colleagues, prompted me to further study the role of STC-1 in cell stress and differentiation. One purpose was to determine whether there is an inter-relationship between terminally differentiated cells and STC-1 expression. The study revealed an accumulation of STC-1 in mature megakaryocytes and adipocytes, i.e. postmitotic cells with limited or lost proliferative capacity. Still proliferating uninduced cells were negative for STC-1 mRNA and protein, whereas differentiating cells accumulated STC-1 in their cytoplasm. Interestingly, in liposarcomas the grade inversely correlated with STC-1 expression. Another aim was to study how STC-1 gene expression is regulated. Given that IL-6 is a cytokine with neuroprotective actions, by unknown mechanisms, we examined whether IL-6 regulates STC-1 gene expression. Treatment of human neural Paju cells with IL-6 induced a dose-dependent upregulation of STC-1 mRNA levels. This induction of STC-1 expression by IL-6 occurred mainly through the MAPK signaling pathway. Furthermore, I studied the role of IL-6-mediated STC-1 expression as a mechanism of cytoprotection conferred by hypoxic preconditioning (HOPC) in brain and heart. My findings show that Stc-1 was upregulated in brain after hypoxia treatment. In the brain of IL-6 deficient mice, however, no upregulation of Stc-1 expression was evident. After induced brain injury the STC-1 response in brains of IL-6 transgenic mice, with IL-6 overexpression in astroglial cells, was stronger than in brains of WT mice. These results indicate that IL-6-mediated expression of STC-1 is one molecular mechanism of HOPC-induced tolerance to brain ischemia. The protection conferred by HOPC in heart occurs during a bimodal time course comprising early and delayed preconditioning. Interestingly, my results showed that the expression of Stc-1 in heart was upregulated in a biphasic manner during HOPC. IL-6 deficient mice did not, however, show a similar biphasic manner of Stc-1 upregulation as did WT mice. Instead, only an early upregulation of Stc-1 expression was evident. The results suggest that the upregulation of Stc-1 during the delayed preconditioning is IL-6-dependent. The upregulated expression of Stc-1 during the early preconditioning, however, is only partly IL-6-dependent and possibly also directly mediated by HIF-1. These findings suggest that STC-1 is a pro-survival protein for terminally differentiated cells and that STC-1 expression may in fact be regulated by stress. In addition, I show that STC-1 gene upregulation, mediated in part by IL-6, is a new mechanism of protection conferred by HOPC in brain and heart. Because of its importance for fundamental biological processes, such as differentiation and cytoprotection, STC-1 may have therapeutic implications for management of stroke, neurodegenerative diseases, cancer, and obesity.