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

Conceptual and statistical modelling of environmental effects in population dynamics

Population dynamics are generally viewed as the result of intrinsic (purely density dependent) and extrinsic (environmental) processes. Both components, and potential interactions between those two, have to be modelled in order to understand and predict dynamics of natural populations; a topic that is of great importance in population management and conservation. This thesis focuses on modelling environmental effects in population dynamics and how effects of potentially relevant environmental variables can be statistically identified and quantified from time series data. Chapter I presents some useful models of multiplicative environmental effects for unstructured density dependent populations. The presented models can be written as standard multiple regression models that are easy to fit to data. Chapters II IV constitute empirical studies that statistically model environmental effects on population dynamics of several migratory bird species with different life history characteristics and migration strategies. In Chapter II, spruce cone crops are found to have a strong positive effect on the population growth of the great spotted woodpecker (Dendrocopos major), while cone crops of pine another important food resource for the species do not effectively explain population growth. The study compares rate- and ratio-dependent effects of cone availability, using state-space models that distinguish between process and observation error in the time series data. Chapter III shows how drought, in combination with settling behaviour during migration, produces asymmetric spatially synchronous patterns of population dynamics in North American ducks (genus Anas). Chapter IV investigates the dynamics of a Finnish population of skylark (Alauda arvensis), and point out effects of rainfall and habitat quality on population growth. Because the skylark time series and some of the environmental variables included show strong positive autocorrelation, the statistical significances are calculated using a Monte Carlo method, where random autocorrelated time series are generated. Chapter V is a simulation-based study, showing that ignoring observation error in analyses of population time series data can bias the estimated effects and measures of uncertainty, if the environmental variables are autocorrelated. It is concluded that the use of state-space models is an effective way to reach more accurate results. In summary, there are several biological assumptions and methodological issues that can affect the inferential outcome when estimating environmental effects from time series data, and that therefore need special attention. The functional form of the environmental effects and potential interactions between environment and population density are important to deal with. Other issues that should be considered are assumptions about density dependent regulation, modelling potential observation error, and when needed, accounting for spatial and/or temporal autocorrelation.

VEGF-C and angiopoietins in lymphangiogenesis

Angiogenesis and lymphangiogenesis occur during development as the result of tightly coordinated signalling programs to generate two hierarchically organised vascular systems. All tissues and organs are dependent on a functional blood vasculature for oxygen and nutrients, whereas the lymphatic vasculature functions to collect excess tissue fluid, passing it through lymph nodes for immune surveillance, and returning it to the blood circulation. Effectors that control developmental angiogenesis and lymphangiogenesis are also involved in pathological settings, and therefore potential targets for therapy. Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) growth factors, signalling through endothelial VEGFR and Tie receptors, have been established as key regulators of angiogenic and lymphangiogenic processes in development and disease. In this study, we aimed to obtain a clearer understanding of the vascular effects of stimulation by VEGF-C, Ang1 and Ang2, all known to be involved in lymphangiogenesis. In cell culture models, we found that both intrinsic and microenvironmental regulatory mechanisms are involved in the regulation of endothelial cell phenotypes, and distinct responses to VEGF signalling are induced by specific receptor pathways in different endothelial cell types. Surprisingly, we also found that Ang1 induces sprouting lymphangiogenesis in vivo by a VEGFR-3 dependent mechanism, establishing Ang1 as a novel lymphangiogenic factor. Using inducible transgenic mouse models, we found that VEGF-C-induced lymphatic hyperplasia persisted independently of the growth factor, indicating that short pro-lymphangiogenic therapy could lead to lasting improvements in tissue oedema. While VEGF-C had blood vessel effects in embryos, no angiogenic side effects were observed in adult tissues. Furthermore, inducible transgenic expression of Ang2 during embryonic development confirmed Ang2 as an important regulator of lymphatic remodelling and mural cell contacts. The unexpected similarity of the lymphatic maturation defects caused by excess Ang2 to those observed in Ang2 deficient mice demonstrated that correct doses of Ang2 are crucial for the control of lymphatic development. Unlike Ang1, Ang2 did not induce lymphatic sprouting. Although Ang1 has been shown to be able to substitute for Ang2 during developmental lymphangiogenesis, their lymphatic effects are not identical. These findings further our understanding of the basic mechanisms of angiogenesis and lymphangiogenesis, important for the future development of targeted therapies for vascular diseases such as cancer, inflammation, lymphoedema and ischemia. VEGF-C and Ang1 especially emerged as promising candidates for pro-lymphangiogenic therapy.

Molecular studies on LIM-domain protein CRP1

Various intrinsic and external factors are constantly attacking the cells causing damage to DNA and to other cellular structures. Cells in turn have evolved with different kinds of mechanisms to protect against the attacks and to repair the damage. Ultraviolet radiation (UVR) is one of the major environmental genotoxic carcinogens that causes inflammation, mutations, immunosuppression, accelerated aging of the skin and skin cancers. Epidermis is the outermost layer of the skin consisting mostly of keratinocytes, whose primary function is to protect the skin against e.g. UV radiation. LIM domain proteins are a group of proteins involved in regulation of cell growth, damage signalling, cell fate determination and signal transduction. Despite their two zinc fingers, LIM domains do not bind to DNA, but rather mediate protein-protein interactions and function as modular protein binding interfaces. We initially identified CSRP1 as UVR-regulated transcript by using expression profiling. Here we have further studied the regulation and function of CRP1, a representative of cysteine rich protein- family consisting of two LIM domains. We find that CRP1 is increased by UVR in primary human keratinocytes and in normal human skin fibroblasts. Ectopic expression of CRP1 protected the cells against UVR and provided a survival advantage, whereas silencing of CRP1 rendered the cells more photosensitive. Actinic keratosis is a premalignant lesion of skin caused by excess exposure to sunlight and sunburn, which may lead to formation of squamous cell carcinoma. The expression of CRP1 was increased in basal keratinocytes of Actinic keratosis patient specimens suggesting that CRP1 may be increased by constant exposure to UVR and may provide survival advantage for the cells also in vivo. In squamous cell carcinoma, CRP1 was only expressed in the fibroblasts surrounding the tumour. Moreover, we found that ectopic expression of CRP1 suppresses cell proliferation. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine that regulates several functions in cell including growth, apoptosis and differentiation, and plays important roles in pathological disorders like cancer and fibrosis. We found that TGFbeta-signalling pathway regulates CRP1 at protein, but not at transcriptional level. The increase was mediated both through Smad and non-Smad signalling pathways involving MAPK/p38. Furthermore, we found that TGFbeta-mediated increase in CRP1 was associated with myofibroblast differentiation, and that CRP1 was significantly more expressed in idiopathic pulmonary fibrosis as compared to normal lung specimens. Since cell contractility is a distinct feature of myofibroblasts, and CRP1 is associated with actin cytoskeleton, we studied the role of CRP1 in cell contractility. CRP1 was found to localize to stress fibres that mediate contractility and to mediate myofibroblast contraction. These studies identify CRP1 as a stress responsive and cytokine regulated cytoskeletal protein that participates in pathological processes involved in fibrotic diseases and cancer.

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.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Other Side of This Life : Death, Value, and Social Being in Thomas Pynchon's Fiction

This dissertation analyzes the interrelationship between death, the conditions of (wo)man s social being, and the notion of value as it emerges in the fiction of the American novelist Thomas Pynchon (1937 ). Pynchon s present work includes six novels V. (1963), The Crying of Lot 49 (1966), Gravity s Rainbow (1973), Vineland (1990), Mason & Dixon (1997), Against the Day (2006) and several short stories. Death constitues a central thematic in Pynchon s work, and it emerges through recurrent questions of mortality, suicide, mass destruction, sacrifice, afterlife, entropy, the relationship between the animate and the inanimate, and the limits of representation. In Pynchon, death is never a mere biological given (or event); it is always determined within a certain historical, cultural, and ideological context. Throughout his work, Pynchon questions the strict ontological separation of life and death by showing the relationship between this separation and social power. Conceptual divisions also reflect the relationship between society and its others, and death becomes that through which lines of social demarcation are articulated. Determined as a conceptual and social "other side", death in Pynchon forms a challenge to modern culture, and makes an unexpected return: the dead return to haunt the living, the inanimate and the animate fuse, and technoscientific attempts at overcoming and controlling death result in its re-emergence in mass destruction and ecological damage. The questioning of the ontological line also affects the structuration of Pynchon's prose, where the recurrent narrated and narrative desire to reach the limits of representation is openly associated with death. Textualized, death appears in Pynchon's writing as a sudden rupture within the textual functioning, when the "other side", that is, the bare materiality of the signifier is foregrounded. In this study, Pynchon s cultural criticism and his poetics come together, and I analyze the subversive role of death in his fiction through Jean Baudrillard s genealogy of the modern notion of death from L échange symbolique et la mort (1976). Baudrillard sees an intrinsic bond between the social repression of death in modernity and the emergence of modern political economy, and in his analysis economy and language appear as parallel systems for generating value (exchange value/ sign-value). For Baudrillard, the modern notion of death as negativity in relation to the positivity of life, and the fact that death cannot be given a proper meaning, betray an antagonistic relation between death and the notion of value. As a mode of negativity (that is, non-value), death becomes a moment of rupture in relation to value-based thinking in short, rationalism. Through this rupture emerges a form of thinking Baudrillard labels the symbolic, characterized by ambivalence and the subversion of conceptual opposites.

Is a Majority Enough? Psychological Well-being and Its Relation to Academic and Prosocial Motivation, Self-regulation and Achievement at school

This cross-sectional study analyzed psychological well-being at school using the Self-Determination theory as a theoretical frame-work. The study explored basic psychological needs fulfillment (BPNS), academic (SRQ-A), prosocial self-regulation (SRQ-P) and motivation, and their relationship with achievement in general, special and selective education (N=786, 444 boys, 345 girls, mean age 12 yrs 8 mths). Motivation starts behavior which becomes guided by self-regulation. The perceived locus of control (PLOC) affects how self-determined this behavior will be; in other words, to what extent it is autonomously regulated. In order learn and thus to be able to accept external goals, a student has to feel emotionally safe and have sufficient ego-flexibility—all of which builds on satisfied psychological needs. In this study those conditions were explored. In addition to traditional methods Self-organizing maps (SOM), was used in order to cluster the students according to their well-being, self-regulation, motivation and achievement scores. The main impacts of this research were: a presentation of the theory based alternative of studying psychological well-being at school and usage of both the variable and person-oriented approach. In this Finnish sample the results showed that the majority of students felt well, but the well-being varied by group. Overall about for 11–15% the basic needs were deprived depending on the educational group. Age and educational group were the most effective factors; gender was important in relation to prosocial identified behavior. Although the person-oriented SOM-approach, was in a large extent confirming what was no-ticed by using comparison of the variables: the SEN groups had lower levels of basic needs fulfillment and less autonomous self-regulation, interesting deviations of that rule appeared. Some of the SEL- and GEN-group members ended up in the more unfavorable SOM-clusters, and not all SEN-group members belonged to the poorest clusters (although not to the best either). This evidence refines the well-being and self-regulation picture, and may re-direct intervention plans, and turn our focus also on students who might otherwise remain unnoticed. On the other hand, these results imply simultaneously that in special education groups the average is not the whole truth. On the basis of theoretical and empirical considerations an intervention model was sug-gested. The aim of the model was to shift amotivation or external motivation in a more intrinsic direction. According to the theoretical and empirical evidence this can be achieved first by studying the self-concept a student has, and then trying to affect both inner and environmental factors—including a consideration of the basic psychological needs. Keywords: academic self-regulation, prosocial self-regulation, basic psychological needs, moti-vation, achievement

Cellular and network mechanisms generating spontaneous population events in the immature rat hippocampus

Distinct endogenous network events, generated independently of sensory input, are a general feature of various structures of the immature central nervous system. In the immature hippocampus, these type of events are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings in vitro. GABA, the major inhibitory neurotransmitter of the adult brain, has a depolarizing action in immature neurons, and GDPs have been proposed to be driven by GABAergic transmission. Moreover, GDPs have been thought to reflect an early pattern that disappears during development in parallel with the maturation of hyperpolarizing GABAergic inhibition. However, the adult hippocampus in vivo also generates endogenous network events known as sharp (positive) waves (SPWs), which reflect synchronous discharges of CA3 pyramidal neurons and are thought to be involved in cognitive functions. In this thesis, mechanisms of GDP generation were studied with intra- and extracellular recordings in the neonatal rat hippocampus in vitro and in vivo. Immature CA3 pyramidal neurons were found to generate intrinsic bursts of spikes and to act as cellular pacemakers for GDP activity whereas depolarizing GABAergic signalling was found to have a temporally non-patterned facilitatory role in the generation of the network events. Furthermore, the data indicate that the intrinsic bursts of neonatal CA3 pyramidal neurons and, consequently, GDPs are driven by a persistent Na+ current and terminated by a slow Ca2+-dependent K+ current. Gramicidin-perforated patch recordings showed that the depolarizing driving force for GABAA receptor-mediated actions is provided by Cl- uptake via the Na-K-C1 cotransporter, NKCC1, in the immature CA3 pyramids. A specific blocker of NKCC1, bumetanide, inhibited SPWs and GDPs in the neonatal rat hippocampus in vivo and in vitro, respectively. Finally, pharmacological blockade of the GABA transporter-1 prolonged the decay of the large GDP-associated GABA transients but not of single postsynaptic GABAA receptor-mediated currents. As a whole the data in this thesis indicate that the mechanism of GDP generation, based on the interconnected network of bursting CA3 pyramidal neurons, is similar to that involved in adult SPW activity. Hence, GDPs do not reflect a network pattern that disappears during development but they are the in vitro counterpart of neonatal SPWs.

Biochemical and structural properties of Potato virus A VPg

The potato virus A (PVA) genome linked protein (VPg) is a multifunctional protein that takes part in vital infection cycle events such as replication and movement of the virus from cell to cell. VPg is attached to the 5´ end of the genome and is carried in the tip structure of the filamentous virus particle. VPg is also the last protein to be cleaved from the polyprotein. VPg interacts with several viral and host proteins and is phosphorylated at several positions. These features indicate a central role in virus epidemiology and a requirement for an efficient but flexible mechanism for switching between different functions. -- This study examines some of the key VPg functions in more detail. Mutations in the positively charged region from Ala38 to Lys44 affected the NTP binding, uridylylation, and in vitro translation inhibition activities of VPg, whereas in vivo translation inhibition was not affected. Some of the data generated in this study implicated the structural flexibility of the protein in functional activities. VPg lacks a rigid structure, which could allow it to adapt conformationally to different functions as needed. A major finding of this study is that PVA VPg belongs to the class of ´intrinsically disordered proteins´ (IDPs). IDPs are a novel protein class that has helped to explain the observed lack of structure. The existence of IDPs clearly shows that proteins can be functional and adapt a native fold without a rigid structure. Evidence for the intrinsic disorder of VPg was provided by CD spectroscopy, NMR, fluorescence spectroscopy, bioinformatic analysis, and limited proteolytic digestion. The structure of VPg resembles that of a molten globule-type protein and has a hydrophobic core domain. Approximately 50% of the protein is disordered and an α-helical stabilization of these regions has been hypothesized. Surprisingly, VPg structure was stabilized in the presence of anionic lipid vesicles. The stabilization was accompanied by a change in VPg structure and major morphological modifications of the vesicles, including a pronounced increase in the size and appearance of pore or plaque like formations on the vesicle surface. The most likely scenario seems to be an α-helical stabilization of VPg which induces formation of a pore or channel-like structure on the vesicle surface. The size increase is probably due to fusion or swelling of the vesicles. The latter hypothesis is supported by the evident disruption of the vesicles after prolonged incubation with VPg. A model describing the results is presented and discussed in relation to other known properties of the protein.

Modeling ecological and evolutionary processes in spatially structured populations

Many species inhabit fragmented landscapes, resulting either from anthropogenic or from natural processes. The ecological and evolutionary dynamics of spatially structured populations are affected by a complex interplay between endogenous and exogenous factors. The metapopulation approach, simplifying the landscape to a discrete set of patches of breeding habitat surrounded by unsuitable matrix, has become a widely applied paradigm for the study of species inhabiting highly fragmented landscapes. In this thesis, I focus on the construction of biologically realistic models and their parameterization with empirical data, with the general objective of understanding how the interactions between individuals and their spatially structured environment affect ecological and evolutionary processes in fragmented landscapes. I study two hierarchically structured model systems, which are the Glanville fritillary butterfly in the Åland Islands, and a system of two interacting aphid species in the Tvärminne archipelago, both being located in South-Western Finland. The interesting and challenging feature of both study systems is that the population dynamics occur over multiple spatial scales that are linked by various processes. My main emphasis is in the development of mathematical and statistical methodologies. For the Glanville fritillary case study, I first build a Bayesian framework for the estimation of death rates and capture probabilities from mark-recapture data, with the novelty of accounting for variation among individuals in capture probabilities and survival. I then characterize the dispersal phase of the butterflies by deriving a mathematical approximation of a diffusion-based movement model applied to a network of patches. I use the movement model as a building block to construct an individual-based evolutionary model for the Glanville fritillary butterfly metapopulation. I parameterize the evolutionary model using a pattern-oriented approach, and use it to study how the landscape structure affects the evolution of dispersal. For the aphid case study, I develop a Bayesian model of hierarchical multi-scale metapopulation dynamics, where the observed extinction and colonization rates are decomposed into intrinsic rates operating specifically at each spatial scale. In summary, I show how analytical approaches, hierarchical Bayesian methods and individual-based simulations can be used individually or in combination to tackle complex problems from many different viewpoints. In particular, hierarchical Bayesian methods provide a useful tool for decomposing ecological complexity into more tractable components.

Phylogeography and Population Genetics of the Moor frog (Rana arvalis Nilsson) in Northern Europe

The ongoing climate change along with increasing levels of pollutants, diseases, habitat loss and fragmentation constitute global threats to the persistence of many populations, species and ecosystems. However, for the long-term persistence of local populations, one of the biggest threats is the intrinsic loss of genetic variation. In order to adapt to changes in the environment, organisms must have a sufficient supply of heritable variation in traits important for their fitness. With a loss of genetic variation, the risk of extinction will increase. For conservational practices, one should therefore understand the processes that shape the genetic population structure and also the broader (historical) phylogenetic patterning of the species in focus. In this thesis, microsatellite markers were applied to study genetic diversity and population differentiation of the protected moor frog (Rana arvalis) in Fennoscandia from both historical (evolutionary) and applied (conservation) perspectives. The results demonstrate that R. arvalis populations are highly structured over rather short geographic distances. Moreover, the results suggest that R. arvalis recolonized Fennoscandia from two directions after the last ice age. This has had implications for the genetic structuring and population differentiation, especially in the northernmost parts where the two lineages have met. Compared to more southern populations, the genetic variation decreases and the interpopulation differentiation increases dramatically towards north. This could be an outcome of serial population bottlenecking along the recolonization route. Also, current isolation and small population sizes increase the effect of drift, thus reinforcing the observed pattern. The same pattern can also be seen in island populations. However, though R. arvalis on the island of Gotland has lost most of its neutral genetic variability, our results indicate that the levels of additive genetic variation have remained high. This conforms to the conjecture that though neutral markers are widely used in conservation purposes, they may be quite uninformative about the levels of genetic variation in ecologically important traits. Finally, the evolutionary impact of the typical amphibian mating behaviour on genetic diversity was investigated. Given the short time available for larval development, it is important that mating takes place as early as possible. The genetic data and earlier capture-recapture data suggest that R. arvalis gather at mating grounds they are familiar with. However, by forming leks in random to relatedness, and having multiple paternities in single clutches, the risk of inbreeding may be minimized in this otherwise highly philopatric species.

Functional studies of purified transmembrane proteases, omptins, of Yersinia pestis and Salmonella enterica.

Surface proteolysis is important in migration of cells through tissue barriers. In the case of prokaryotes, surface proteolysis has been associated with invasiveness of pathogenic bacteria from the primary infection site into circulation and secondary infection sites in the host. This study addressed surface proteases of two important bacterial pathogens, Yersinia pestis which is the causative agent of the lethal systemic zoonosis, plague, and Salmonella enterica serovar Typhimurium which is an oral-faecal pathogen that annually causes millions of cases of gastoenteritis that may develop to septicaemia. Both bacterial species express an ortholog of the omptin family of transmembrane β-barrel, outer membrane proteases/adhesins. This thesis work addressed the functions of isolated plasminogen activator Pla of Y. pestis and the PgtE omptin of S. enterica. Pla and PgtE were isolated as His6-fusion proteins in denaturing conditions from recombinant Escherichia coli and activated by adding lipopolysaccharide (LPS). The structural features in LPS that enhance plasminogen activation by His6-Pla were determined, and it was found that the lack of O-specifi c chain, the presence of outer core oligosaccharide, the presence of phosphates in lipid A, as well as a low level of acylation in lipid A influence the enhancement of Pla activity by LPS. A conserved lipid A phosphate binding motif in Pla and PgtE was found important for the enhancement of enzymatic activity by LPS. The results help to explain the biological signifi cance of the genetic loss of the O-specifi c chain biosynthesis in Y. pestis as well as the variations in LPS structure upon entry of Y. pestis into the human host. Expression of Pla in Y. pestis is associated with adhesiveness to lamin of basement membranes. Here, isolated and LPS-activated His6-Pla was coated onto fluorescent microparticles. The coating conferred specifi c adhesiveness of the particles to laminin and reconstituted basement membrane, thus confi rming the intrinsic adhesive characteristics of the Pla protein. The adhesiveness is thought to direct plasmin proteolysis at tissue barriers, thus increasing tissue damage and bacterial spread. Gelatinase activity has not been previously reported in enteric bacteria. Expression of PgtE in S. enterica was associated with cleavage of porcine skin gelatin, denaturated human type I collagen, as well as DQ-gelatin. Purifi ed His6-PgtE also degraded porcine skin gelatin and human type I gelatin but did not react with DQ-gelatin, indicating that minor differences are seen in proteolysis by isolated and cell-bound PgtE. Pla was less effective in gelatin degradation. The novel gelatinase activity in S. enterica is likely to enhance bacterial dissemination during infection.

Should I stay or should I go? : Reproductive and dispersal strategies of site-specific liverwort species

The area of intensively managed forests, in which required conditions for several liverwort species are seldom found, has expanded over the forest landscape during the last century. Liverworts are very sensitive to habitat changes, because they demand continuously moist microclimate. Consequently, about third of the forest liverworts have been classified as threatened or near threatened in Finland. The general objective of this thesis is to increase knowledge of the reproductive and dispersal strategies of the substrate-specific forest bryophytes. A further aim was to develop recommendations for conservation measures for species inhabiting unstable and stable habitats in forest landscape. Both population ecological and genetic methods have been applied in the research. Anastrophyllum hellerianum inhabits spatially and temporally limited substrate patches, decaying logs, which can be considered as unstable habitats. The results show that asexual reproduction by gemmae is the dominant mode of reproduction, whereas sexual reproduction is considerably infrequent. Unlike previously assumed, not only spores but also the asexual propagules may contribute to long-distance dispersal. The combination of occasional spore production and practically continuous, massive gemma production facilitates dispersal both on a local scale and over long distances, and it compensates for the great propagule losses that take place preceding successful establishment at suitable sites. However, establishment probability of spores may be restricted because of environmental and biological limitations linked to the low success of sexual reproduction. Long-lasting dry seasons are likely to result in a low success of sexual reproduction and decreased release rate of gemmae from the shoots, and consequent fluctuations in population sizes. In the long term, the substratum limitation is likely to restrict population sizes and cause local extinctions, especially in small-sized remnant populations. Contrastingly, larger forest fragments with more natural disturbance dynamics, to which the species is adapted, are pivotal to species survival. Trichocolea tomentella occupies stable spring and mesic habitats in woodland. The relatively small populations are increasingly fragmented with a high risk for extinction for extrinsic reasons. The results show that T. tomentella mainly invests in population persistence by effective clonal growth via forming independent ramets and in competitive ability, and considerably less in sexuality and dispersal potential. The populations possess relatively high levels of genetic diversity regardless of population size and of degree of isolation. Thus, the small-sized populations inhabiting stable habitats should not be neglected when establishing conservation strategies for the species and when considering the habitat protection of small spring sites. Restricted dispersal capacity, also on a relatively small spatial scale, is likely to prevent successful (re-)colonization in the potential habitat patches of recovering forest landscapes. By contrast, random short-range dispersal of detached vegetative fragments within populations at suitable habitat seems to be frequent. Thus, the restoration actions of spring and streamside habitats close to the populations of T. tomentella may contribute to population expansion. That, in turn, decreases the harmful effects of environmental stochasticity.