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.

The viral coat protein is regulated by HSP70 and HSP40 in Potato virus A infection

Plus-stranded (plus) RNA viruses multiply within a cellular environment as tightly integrated units and rely on the genetic information carried within their genomes for multiplication and, hence, persistence. The minimal genomes of plus RNA viruses are unable to encode the molecular machineries that are required for virus multiplication. This sets requisites for the virus, which must form compatible interactions with host components during multiplication to successfully utilize primary metabolites as building blocks or metabolic energy, and to divert the protein synthesis machinery for production of viral proteins. In fact, the emerging picture of a virus-infected cell displays tight integration with the virus, from simple host and virus protein interactions through to major changes in the physiological state of the host cell. This study set out to develop a method for the identification of host components, mainly host proteins, that interact with proteins of Potato virus A (PVA; Potyvirus) during infection. This goal was approached by developing affinity-tag based methods for the purification of viral proteins complexed with associated host proteins from infected plants. Using this method, host membrane-associated viral ribonucleoprotein (RNP) complexes were obtained, and several host and viral proteins could be identified as components of these complexes. One of the host proteins identified using this strategy was a member of the heat shock protein 70 (HSP70) family, and this protein was chosen for further analysis. To enable the analysis of viral gene expression, a second method was developed based on Agrobacterium-mediated virus genome delivery into plant cells, and detection of virally expressed Renilla luciferase (RLUC) as a quantitative measure of viral gene expression. Using this method, it was observed that down-regulation of HSP70 caused a PVA coat protein (CP)-mediated defect associated with replication. Further experimentation suggested that CP can inhibit viral gene expression and that a distinct translational activity coupled to replication, referred to as replication-associated translation (RAT), exists. Unlike translation of replication-deficient viral RNA, RAT was dependent on HSP70 and its co-chaperone CPIP. HSP70 and CPIP together regulated CP turnover by promoting its modification by ubiquitin. Based on these results, an HSP70 and CPIP-driven mechanism that functions to regulate CP during viral RNA replication and/or translation is proposed, possibly to prevent premature particle assembly caused by CP association with viral RNA.

Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.

Personporträttet som tidningsgenre : En närläsningsstudie med fokus på innehållsliga teman, berättarkonventioner och kön

The thesis The portrait interview as a newspaper genre. A qualitative close reading focussing on topical motifs, conventions of narration, and gender defines the portrait interview as a newspaper genre and analyses how the personalities in the portraits are constructed textually. The main body of material consists of 107 portrait interviews in two morning newspapers, Dagens Nyheter (published in Stockholm, Sweden) and Hufvudstadsbladet (published in Swedish in Helsinki, Finland), during two one-week periods (week 46/1999 and week 38/2002). There is also complementary material of 59 portraits from four magazines. The study is carried out within the research traditions of journalistic genre studies, gender and journalism, and critical text analysis. It is comprised of a qualitative close reading focussing on content (topical motifs or themes), conventions of narration, and gender. The methods used to carry out the study are qualitative close reading and quantitative content analysis. The analysis identifies the stylistic elements that differentiate the portrait genre from other journalistic genres, as well as from the autobiographical genre, and explores what opportunities and limitations these elements present for the inclusion of even more women protagonists in the portrait genre. The portrait interview is an exception from the critical mission of journalism in general, with its position as a genre of politeness. Since a typical characteristic of the portrait interview genre is that it pays tribute to the protagonist, the genre reveals the kind of personalities and lives that are seen as admirable in society. Four levels of portrait interview are defined: the prototype portrait, the pure portrait, the hybrid portrait and the marginal portrait. The prototype is a raw version of a portrait that fulfils the criteria but may be lacking in content and stylistics. The pure portrait does not lack these qualities and resembles an ideal portrait. The hybrid is a borderline case which relates to another genre or is a mixture between the portrait and some other genre, most commonly the news genre. The marginal portrait does not fulfil the criteria, and can therefore be seen as an inadequate portrait. For example, obituaries and caricatures are excluded if the protagonist s voice is never quoted. The analysis resulted in three factors that in part help to explain why the portrait interview genre has somewhat more female protagonists than journalistic news texts do in general. The four main reasons why women are presented somewhat more in the portrait genre than in other journalistic genres are: (i) women are shown as exceptions to the female norm when, for example, taking a typical male job or managing in positions where there are few women; (ii) women are shown as representing female themes ; (iii) use of the double bind as a story-generating factor; and (iv) the intimisation of journalism. The double bind usually builds up the narration on female ambiguity in the contradiction between private and public life, for example family and career, personal desire and work. The intimisation of journalism and the double bind give women protagonists somewhat more publicity also because of the tendency of portrait interviews to create conflicts within the protagonist, as an exception to journalism in general where conflicts are created or seen as existing between, for example, persons, groupings or parties. Women protagonists and their lives create an optimal narration of inner conflicts originating in the double bind as men are usually not seen as suffering from these conflicts. The analysis also resulted in gendered portrait norms: The feminine portrait norm and the masculine portrait norm or more concretely, professional life and family life as expectation and exception. Women are expected to be responsible parents and mediocre professionals, while men are expected to be professionals and in their free time engaging fathers. Key words: journalism, genre, portrait interview, gender, interview, newspaper, women s magazine.

Kritisk-händelse-baserad metodutveckling

Denna artikel diskuterar den metodutveckling inom marknadsföring som baseras på den allmänt och mycket frekvent använda kritisk-händelse metod som grundar sig på Flanagen (1954). Genom att forskningen inom nämnda område alltmer handlar om kundrelationer är det naturligt att även metoder som används anpassas till detta. Det finns emellertid flere frågetecken när man fokuserar på kritiska händelser och kundrelationer. Ett sådant är konsekvensen av den kritiska händelsen. Utan att kartlägga en verklig konsekvens av kritiska händelser som inträffat (verkligt beteende) är det emellertid svårt att utveckla metoder för framtida och kommande beteende. Därför diskuteras dessa frågor här i avsikt att föra utvecklingen mot en applicering av kritisk-händelse metoder i pågående kundrelationer.

Conditionally One of 'Us' : A Study of Print Media, Minorities and Positioning Practices

This study deals with how ethnic minorities and immigrants are portrayed in the Finnish print media. The study also asks how media users of various ethnocultural backgrounds make sense of these mediated stories. A more general objective is to elucidate negotiations of belonging and positioning practices in an increasingly complex society. The empirical part of the study is based on content analysis and qualitative close reading of 1,782 articles in five newspapers (Hufvudstadsbladet, Vasabladet, Helsingin Sanomat, Iltalehti and Ilta-Sanomat) during various research periods between 1999 and 2007. Four case studies on print media content are followed up by a focus group study involving 33 newspaper readers of Bosnian, Somalian, Russian, and 'native' Finnish backgrounds. The study draws from different academic and intellectual traditions; mainly media and communication studies, sociology and social psychology. The main theoretical framework employed is positioning theory, as developed by Rom Harré and others. Building on this perspective, situational self-positioning, positioning by others, and media positioning are seen as central practices in the negotiation of belonging. In support of contemporary developments in social sciences, some of these negotiations are seen as occurring in a network type of communicative space. In this space, the media form one of the most powerful institutions in constructing, distributing and legitimising values and ideas of who belongs to 'us', and who does not. The notion of positioning always involves an exclusionary potential. This thesis joins scholars who assert that in order to understand inclusionary and exclusionary mechanisms, the theoretical starting point must be a recognition of a decent and non-humiliating society. When key insights are distilled from the five empirical cases and related to the main theories, one of the major arguments put forward is that the media were first and foremost concerned with a minority actor's rightful or unlawful belonging to the Finnish welfare system. However, in some cases persistent stereotypes concerning some immigrant groups' motivation to work, pay taxes and therefore contribute are so strong that a general idea of individualism is forgotten in favour of racialised and stagnated views. Discussants of immigrant background also claim that the positions provided for minority actors in the media are not easy to identify with; categories are too narrow, journalists are biased, the reporting is simplifying and carries labelling potential. Hence, although the will for the communicative space to be more diverse and inclusive exists — and has also in many cases been articulated in charters, acts and codes — the positioning of ethnic minorities and immigrants differs significantly from the ideal.

Representation av kvinnor och män – Hur prioriterar riksdagsledamöter frågor i den finska riksdagen? En kvantitativ studie av motionerna i riksdagen 2007

Pro gradu uppsatsen diskuterar betydelsen av representation för grupper i samhället genom att diskutera kvinnors representation. Kvinnor är en grupp i samhället som länge varit underrepresenterad, men som under de senaste 100 åren fått en betydligt bättre position i den offentliga beslutfattningen. För att ge bakgrund till frågeställningen beskrivs kvinnors deltagande i den finska riksdagen. Begreppen deskriptiv, substantiell och symbolisk representation diskuteras. Undersökningar av kvinnors representation både deskriptivt och substantiellt på parlamentsnivå presenteras. Forskningsfrågan i uppsatsen består av fyra frågeställningar om hur motioner görs i riksdagen och hur kön kan identifieras i motionerna. Frågeställningarna bygger på den teoretiska diskussionen och meningen med frågeställningar är att styra analysen av motionerna. I teoriavsnittet behandlas kvinnors representation och den vetenskapliga diskussionen om sambandet mellan substantiell och deskriptiv representation. Diskussionen utgår från Hanna Pitkins olika kategorier av representation. I den teoretiska diskussionen behandlas även begreppen den kritiska massan, närvarons politik och kritiska aktörer. I stycket diskuteras även vilka frågeställningar, som är relevanta att undersöka när man diskuterar kvinnors representation i samhället. När representationen av kvinnor diskuteras är det viktigt att minnas att kvinnor inte utgör en homogen grupp och att även män kan representera kvinnor innehållsmässigt. Efter den teoretiska diskussionen presenteras metoden för undersökningen. Som metod används kvantitativa statistiska metoder för att kunna undersöka ett större material, som kan ge en bättre helhetsbild av hur kvinnor representeras i motionerna. Materialet för undersökningen är motioner som är gjorda i den finska riksdagen 2007. Resultaten i undersökningen visar att motionerna används av både opposition och regering och att det finns en möjlighet för samarbete över partigränser. Andra faktorer, som påverkade hur aktivt man gjorde motioner var bland annat riksdagsledamöternas ålder och den valregion ledamöterna var från. När man såg speciellt på motionärernas kön framkom det skillnader i kvinnors och mäns motionsbeteende. Överlag gör män mera motioner än kvinnor i den finska riksdagen. Kvinnor har än idag en större tendens att göra motioner om socialpolitik och speciellt familje- och barnpolitik än vad män har. Männen igen väljer att göra motioner, som traditionellt har setts som starkt manliga, så som trafik och allmänna utrymmen. Resultaten visade också att kvinnors intressen inte är vanligt som motivering för motioner. Man fann också att riksdagsledamöters koppling till kvinnoorganisationer hade en betydelse när det gällde sannolikheten att göra motioner som motiverades med kvinnors intressen i samhället. Uppsatsen diskuterar orsaker till resultaten och ser att det finns orsaker att fortsätta diskutera sambandet mellan deskriptiv och substantiell representation.