Paradise Reconsidered : A Study of the Ophite Myth and Ritual and their Relationship to Sethianism

This thesis examines the mythology in and social reality behind a group of texts from the Nag Hammadi and related literature, to which certain leaders of the early church attached the label, Ophite, i.e., snake people. In the mythology, which essentially draws upon and rewrites the Genesis paradise story, the snake's advice to eat from the tree of knowledge is positive, the creator and his angels are demonic beasts and the true godhead is depicted as an androgynous heavenly projection of Adam and Eve. It will be argued that this unique mythology is attested in certain Coptic texts from the Nag Hammadi and Berlin 8502 Codices (On the Origin of the World, Hypostasis of the Archons, Apocryphon of John, Eugnostos, Sophia of Jesus Christ), as well as in reports by Irenaeus (Adversus Haereses 1.30), Origen (Contra Celsum 6.24-38) and Epiphanius (Panarion 26). It will also be argued that this so-called Ophite evidence is essential for a proper understanding of Sethian Gnosticism, often today considered one of the earliest forms of Gnosticism; there seems to have occurred a Sethianization of Ophite mythology. I propose that we replace the current Sethian Gnostic category by a new one that not only adds texts that draw upon the Ophite mythology alongside these Sethian texts, but also arranges the material in smaller typological units. I also propose we rename this remodelled and expanded Sethian corpus "Classic Gnostic." I have divided the thesis into four parts: (I) Introduction; (II) Myth and Innovation; (III) Ritual; and (IV) Conclusion. In Part I, the sources and previous research on Ophites and Sethians will be examined, and the new Classic Gnostic category will be introduced to provide a framework for the study of the Ophite evidence. Chapters in Part II explore key themes in the mythology of our texts, first by text comparison (to show that certain texts represent the Ophite mythology and that this mythology is different from Sethianism), and then by attempting to unveil social circumstances that may have given rise to such myths. Part III assesses heresiological claims of Ophite rituals, and Part IV is the conclusion.

Bending the Rules of Cell Protrusions : Molecular Mechanisms and Biological Roles of Inverse-BAR Proteins in Cell Morphogenesis

Plasma membrane adopts myriad of different shapes to carry out essential cellular processes such as nutrient uptake, immunological defence mechanisms and cell migration. Therefore, the details how different plasma membrane structures are made and remodelled are of the upmost importance. Bending of plasma membrane into different shapes requires substantial amount of force, which can be provided by the actin cytoskeleton, however, the molecules that regulate the interplay between the actin cytoskeleton and plasma membrane have remained elusive. Recent findings have placed new types of effectors at sites of plasma membrane remodelling, including BAR proteins, which can directly bind and deform plasma membrane into different shapes. In addition to their membrane-bending abilities, BAR proteins also harbor protein domains that intimately link them to the actin cytoskeleton. The ancient BAR domain fold has evolved into at least three structurally and functionally different sub-groups: the BAR, F-BAR and I-BAR domains. This thesis work describes the discovery and functional characterization of the Inverse-BAR domains (I-BARs). Using synthetic model membranes, we have shown that I-BAR domains bind and deform membranes into tubular structures through a binding-surface composed of positively charged amino acids. Importantly, the membrane-binding surface of I-BAR domains displays an inverse geometry to that of the BAR and F-BAR domains, and these structural differences explain why I-BAR domains induce cell protrusions whereas BAR and most F-BAR domains induce cell invaginations. In addition, our results indicate that the binding of I-BAR domains to membranes can alter the spatial organization of phosphoinositides within membranes. Intriguingly, we also found that some I-BAR domains can insert helical motifs into the membrane bilayer, which has important consequences for their membrane binding/bending functions. In mammals there are five I-BAR domain containing proteins. Cell biological studies on ABBA revealed that it is highly expressed in radial glial cells during the development of the central nervous system and plays an important role in the extension process of radial glia-like C6R cells by regulating lamellipodial dynamics through its I-BAR domain. To reveal the role of these proteins in the context of animals, we analyzed MIM knockout mice and found that MIM is required for proper renal functions in adult mice. MIM deficient mice displayed a severe urine concentration defect due to defective intercellular junctions of the kidney epithelia. Consistently, MIM localized to adherens junctions in cultured kidney epithelial cells, where it promoted actin assembly through its I-BAR andWH2 domains. In summary, this thesis describes the mechanism how I-BAR proteins deform membranes and provides information about the biological role of these proteins, which to our knowledge are the first proteins that have been shown to directly deform plasma membrane to make cell protrusions.

Folding and Selective Exit of Reporter Proteins from the Yeast Endoplasmic Reticulum

The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditions the beta-lactamase portion unfolds for translocation. Cytosolic machinery must be responsible for the unfolding. The unfolding is a prerequisite for translocation through the Sec61 channel into the lumen of the ER, where the polypeptide is again folded into a bioactive and secretion-competent conformation. Lhs1p is a member of the Hsp70 family, which functions in the conformational repair of misfolded proteins in the yeast ER. It contains Hsp70 motifs, thus it has been thought to be an ATPase, like other Hsp70 members. In order to understand its activity, authentic Lhs1p and its recombinant forms expressed in E. coli, were purified. However, no ATPase activity of Lhs1p could be detected. Nor could physical interaction between Lhs1p and activators of the ER Hsp70 chaperone Kar2p, such as the J-domain proteins Sec63p, Scj1p, and Jem1p and the nucleotide exchange factor Sil1p, be demonstrated. The domain structure of Lhs1p was modelled, and found to consist of an ATPase-like domain, a domain resembling the peptide-binding domain (PBD) of Hsp70 proteins, and a C-terminal extension. Crosslinking experiments showed that Lhs1p and Kar2p interact. The interacting domains were the C-terminal extension of Lhs1p and the ATPase domain of Kar2p, and this interaction was independent of ATPase activity of Kar2p. A model is presented where the C-terminal part of Lhs1p forms a Bag-like 3 helices bundle that might serve in the nucleotide exchange function for Kar2p in translocation and folding of secretory proteins in the ER. Exit of secretory proteins in COPII-coated vesicles is believed to be dependent of retrograde transport from the Golgi to the ER in COPI-coated vesicles. It is thought that receptors escaping to the Golgi must be recycled back to the ER exit sites to recruit cargo proteins. We found that Hsp150 leaves the ER even in the absence of functional COPI-traffic from the Golgi to the ER. Thus, an alternative, COPI-independent ER exit pathway must exists, and Hsp150 is recruited to this route. The region containing the signature guiding Hsp150 to this alternative pathway was mapped.

Marking one-summer old whitefish with fluorescent pigment spraying method and results of whitefish stockings in the Gulf of Bothnia

Anadromous whitefish is one of the most important fish species in the Finnish coastal fisheries in the Gulf fo Bothnia. To compensate the lost reproduction due to river damming and to support the fisheries, several million one-summer old whitefish are released yearly into the Gulf of Bothnia. Since there are naturally reproducing whitefish in the Gulf as well, and the wild and stocked fish can not be separated in the catch, stocking impact can only be estimated by marking the stocked fish. Due to the small size and large number of released whitefish, the scattered fishery and large area where the whitefish migrate, most of the traditionally used fish marking methods were either unsuitable (e.g. Carlin-tags) or proved to be too expensive (e.g. coded wire tags). Fluorescent pigment spraying method offers a fast and cost-effective method to mass-mark young fish. However, the results are not always satisfactory due to low long-time retention of the marks in some species. The method has to be tested and proper marking conditions and methods determined for each species. This thesis is based on work that was accomplished while developing the fluorescent pigment spraying method for marking one-summer old whitefish fingerlings, and it draws together the results of mass-marking whitefish fingerlings that were released in the Gulf of Bothnia. Fluorescent pigment spraying method is suitable for one-summer old whitefish larger than 8 cm total length. The water temperature during the marking should not exceed 10o C. Suitable spraying pressure is 6 bars measured in the compressor outlet, and the distance of the spraying gun nozzle should be ca 20 cm from the fish. Under such conditions, the marking results in long-term retention of the mark with low or no mortality. The stress level of the fish (measured as muscle water content) rises during the marking procedure, but if the fish are allowed to recover after marking, the overall stress level remains within the limits observed in normal fish handling during the capture-loading-transport-stocking procedure. The marked whitefish fingerlings are released into the sea at larger size and later in the season than the wild whitefish. However, the stocked individuals migrate to the southern feeding grounds in a similar pattern to the wild ones. The catch produced by whitefish stocking in the Gulf of Bothnia varied between released fingerling groups, but was within the limits reported elsewhere in Finland. The releases in the southern Bothnian Bay resulted in a larger catch than those made in the northern Bothnian Bay. The size of the released fingerlings seemed to have some effect on survival of the fish during the first winter in the sea. However, when the different marking groups were compared, the mean fingerling size was not related to stocking success.

Immune response to insulin and changes in the gut immune system in children with or at risk for type 1 diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing β-cells in the pancreatic islets. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against β-cell molecules. Insulin is the only β-cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive T-cells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the HLA gene region but the environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow's milk which both affect the immune system through the gut. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow's milk during early infancy and later spreads to include human insulin. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother's breast milk on the development of antibodies to dietary insulin in the child was examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAA led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells with insulin. The insulin stimulation induced increased expression of regulatory T-cell markers, such as Foxp3, in those patients treated with insulin than in patients examined before initiating insulin treatment. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that insulin concentration in mother's breast milk correlates inversely with the levels of bovine insulin-specific antibodies in those infants who were exposed to cow's milk proteins in their diet, suggesting that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother's breast milk was increased. This finding may indicate that in those children prone to β-cell autoimmunity, breast milk insulin does not promote tolerance to insulin. In the small intestinal biopsies the presence of several immunological markers were quantified with the RT-PCR. From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D.

Actin Dynamics in Muscle Cells

In every cell, actin is a key component involved in migration, cytokinesis, endocytosis and generation of contraction. In non-muscle cells, actin filaments are very dynamic and regulated by an array of proteins that interact with actin filaments and/or monomeric actin. Interestingly, in non-muscle cells the barbed ends of the filaments are the predominant assembly place, whereas in muscle cells actin dynamics was reported to predominate at the pointed ends of thin filaments. The actin-based thin filament pointed (slow growing) ends extend towards the middle of the sarcomere's M-line where they interact with the thick filaments to generate contraction. The actin filaments in muscle cells are organized into a nearly crystalline array and are believed to be significantly less dynamic than the ones in other cell types. However, the exact mechanisms of the sarcomere assembly and turnover are largely unknown. Interestingly, although sarcomeric actin structures are believed to be relatively non-dynamic, many proteins promoting actin dynamics are expressed also in muscle cells (e.g ADF/cofilin, cyclase-associated protein and twinfilin). Thus, it is possible that the muscle-specific isoforms of these proteins promote actin dynamics differently from their non-muscle counterparts, or that actin filaments in muscle cells are more dynamic than previously thought. To study protein dynamics in live muscle cells, I used primary cell cultures of rat cardiomyocytes. My studies revealed that a subset of actin filaments in cardiomyocyte sarcomeres displays rapid turnover. Importantly, I discovered that the turnover of actin filaments depends on contractility of the cardiomyocytes and that the contractility-induced actin dynamics plays an important role in sarcomere maturation. Together with previous studies those findings suggest that sarcomeres undergo two types of actin dynamics: (1) contractility-dependent turnover of whole filaments and (2) regulatory pointed end monomer exchange to maintain correct thin filament length. Studies involving an actin polymerization inhibitor suggest that the dynamic actin filament pool identified here is composed of filaments that do not contribute to contractility. Additionally, I provided evidence that ADF/cofilins, together with myosin-induced contractility, are required to disassemble non-productive filaments in developing cardiomyocytes. In addition, during these studies we learned that isoforms of actin monomer binding protein twinfilin, Twf-1 and Twf-2a localise to myofibrils in cardiomyocytes and may thus contribute to actin dynamics in myofibrils. Finally, in collaboration with Roberto Dominguez s laboratory we characterized a new actin nucleator in muscle cells - leiomodin (Lmod). Lmod localises towards actin filament pointed ends and its depletion by siRNA leads to severe sarcomere abnormalities in cardiomyocytes. The actin filament nucleation activity of Lmod is enhanced by interactions with tropomyosin. We also revealed that Lmod expression correlates with the maturation of myofibrils, and that it associates with sarcomeres only at relatively late stages of myofibrillogenesis. Thus, Lmod is unlikely to play an important role in myofibril formation, but rather might be involved in the second step of the filament arrangement and/or maintenance through its ability to promote tropomyosin-induced actin filament nucleation occurring at the filament pointed ends. The results of these studies provide valuable new information about the molecular mechanisms underlying muscle sarcomere assembly and turnover. These data offer important clues to understanding certain physiological and pathological behaviours of muscle cells. Better understanding of the processes occurring in muscles might help to find strategies for determining, diagnosis, prognosis and therapy in heart and skeletal muscles diseases.

Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

The role of MADS and TCP transcription factors in Gerbera hybrida flower development

Angiosperms represent a huge diversity in floral structures. Thus, they provide an attractive target for comparative developmental genetics studies. Research on flower development has focused on few main model plants, and studies on these species have revealed the importance of transcription factors, such as MADS-box and TCP genes, for regulating the floral form. The MADS-box genes determine floral organ identities, whereas the TCP genes are known to regulate flower shape and the number of floral organs. In this study, I have concentrated on these two gene families and their role in regulating flower development in Gerbera hybrida, a species belonging to the large sunflower family (Asteraceae). The Gerbera inflorescence is comprised of hundreds of tightly clustered flowers that differ in their size, shape and function according to their position in the inflorescence. The presence of distinct flower types tells Gerbera apart from the common model species that bear only single kinds of flowers in their inflorescences. The marginally located ray flowers have large bilaterally symmetrical petals and non-functional stamens. The centrally located disc flowers are smaller, have less pronounced bilateral symmetry and carry functional stamens. Early stages of flower development were studied in Gerbera to understand the differentiation of flower types better. After morphological analysis, we compared gene expression between ray and disc flowers to reveal transcriptional differences in flower types. Interestingly, MADS-box genes showed differential expression, suggesting that they might take part in defining flower types by forming flower-type-specific regulatory complexes. Functional analysis of a CYCLOIDEA-like TCP gene GhCYC2 provided evidence that TCP transcription factors are involved in flower type differentiation in Gerbera. The expression of GhCYC2 is ray-flower-specific at early stages of development and activated only later in disc flowers. Overexpression of GhCYC2 in transgenic Gerbera-lines causes disc flowers to obtain ray-flower-like characters, such as elongated petals and disrupted stamen development. The expression pattern and transgenic phenotypes further suggest that GhCYC2 may shape ray flowers by promoting organ fusion. Cooperation of GhCYC2 with other Gerbera CYC-like TCP genes is most likely needed for proper flower type specification, and by this means for shaping the elaborate inflorescence structure. Gerbera flower development was also approached by characterizing B class MADS-box genes, which in the main model plants are known regulators of petal and stamen identity. The four Gerbera B class genes were phylogenetically grouped into three clades; GGLO1 into the PI/GLO clade, GDEF2 and GDEF3 into the euAP3 clade and GDEF1 into the TM6 clade. Putative orthologs for GDEF2 and GDEF3 were identified in other Asteraceae species, which suggests that they appeared through an Asteraceae-specific duplication. Functional analyses indicated that GGLO1 and GDEF2 perform conventional B-function as they determine petal and stamen identities. Our studies on GDEF1 represent the first functional analysis of a TM6-like gene outside the Solanaceae lineage and provide further evidence for the role of TM6 clade members in specifying stamen development. Overall, the Gerbera B class genes showed both commonalities and diversifications with the conventional B-function described in the main model plants.

Phytoplankton ecology of subarctic lakes in Finnish Lapland

Climate is warming and it is especially seen in arctic areas, where the warming trend is expected to be greatest. Arctic freshwater ecosystems, which are a very characteristic feature of the arctic landscape, are especially sensitive to climate change. They could be used as early warning systems, but more information about the ecosystem functioning and responses are needed for proper interpretation of the observations. Phytoplankton species and assemblages could be especially suitable for climate-related studies, since they have short generation times and react rapidly to changes in the environment. In addition, phytoplankton provides a good tool for lake classifications, since different species have different requirements and tolerance ranges for various environmental factors. The use of biological indicators is especially useful in arctic areas, were many of the chemical factors commonly fall under the detection limit and therefore do not provide much information about the environment. This work brings new information about species distribution and dynamics of arctic freshwater phytoplankton in relation to environmental factors. The phytoplankton of lakes in Finnish Lapland and other European high-altitude or high-latitude areas were compared. Most lakes were oligotrophic and dominated by flagellated species belonging to chrysophytes, cryptophytes and dinoflagellates. In Finnish Lapland cryptophytes were of less importance, whereas desmids had high species richness in many of the lakes. In Pan-European scale, geographical and catchment-related factors were explaining most of the differences in species distributions between different districts, whereas lake water chemistry (especially conductivity, SiO2 and pH) was most important regionally. Seasonal and interannual variation of phytoplankton was studied in subarctic Lake Saanajärvi. Characteristic phytoplankton species in this oligotrophic, dimictic lake belonged mainly to chrysophytes and diatoms. The maximum phytoplankton biomass in Lake Saanajärvi occurs during autumn, while spring biomass is very low. During years with heavy snow cover the lake suffers from pH drop caused by melt waters, but the effects of this acid pulse are restricted to surface layers and last for a relatively short period. In addition to some chemical parameters (mainly Ca and nutrients), length of the mixing cycle and physical factors such as lake water temperature and thermal stability of water column had major impact on phytoplankton dynamics. During a year with long and strong thermal stability, the phytoplankton community developed towards an equilibrium state, with heavy dominance of only a few taxa for a longer period of time. During a year with higher windiness and less thermal stability, the species composition was more diverse and species with different functional strategies were able to occur simultaneously. The results of this work indicate that although arctic lakes in general share many common features concerning their catchment and water chemistry, large differences in biological features can be found even in a relatively small area. Most likely the lakes with very different algal flora do not respond in a similar way to differences in the environmental factors, and more information about specific arctic lake types is needed. The results also show considerable year to year differences in phytoplankton species distribution and dynamics, and these changes are most likely linked to climatic factors.

Phylogenetics, taxon delimitation and phylogenetic diversity

Throughout the history of Linnean taxonomy, species have been described with varying degrees of justification. Many descriptions have been based on only a few ambiguous morphological characters. Moreover, species have been considered natural, well-defined units whereas higher taxa have been treated as disparate, non-existent creations. In the present thesis a few such cases were studied in detail. Often the species-level descriptions were based on only a few specimens and the variation previously thought to be interspecific was found to be intraspecific. In some cases morphological characters were sufficient to resolve the evolutionary relationships between the taxa, but generally more resolution was gained by the addition of molecular evidence. However, both morphological and molecular data were found to be deceptive in some cases. The DNA sequences of morphologically similar specimens were found to differ distinctly in some cases, whereas in other closely related species the morphology of specimens with identical DNA sequences differed substantially. This study counsels caution when evolutionary relationships are being studied utilizing only one source of evidence or a very limited number of characters (e.g. barcoding). Moreover, it emphasizes the importance of high quality data as well as the utilization of proper methods when making scientific inferences. Properly conducted analyses produce robust results that can be utilized in numerous interesting ways. The present thesis considered two such extensions of systematics. A novel hypothesis on the origin of bioluminescence in Elateriformia beetles is presented, tying it to the development of the clicking mechanism in the ancestors of these animals. An entirely different type of extension of systematics is the proposed high value of the white sand forests in maintaining the diversity of beetles in the Peruvian Amazon. White sand forests are under growing pressure from human activities that lead to deforestation. They were found to harbor an extremely diverse beetle fauna and many taxa were specialists living only in this unique habitat. In comparison to the predominant clay soil forests, considerably more elateroid beetles belonging to all studied taxonomic levels (species, genus, tribus, and subfamily) were collected in white sand forests. This evolutionary diversity is hypothesized to be due to a combination of factors: (1) the forest structure, which favors the fungus-plant interactions important for the elateroid beetles, (2) the old age of the forest type favoring survival of many evolutionary lineages and (3) the widespread distribution and fragmentation of the forests in the Miocene, favoring speciation.

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.

Cancer incidence, mortality, and pregnancy outcome among women treated for cervical intraepithelial neoplasia

Cervical cancer develops through precursor lesions, i.e. cervical intraepithelialneoplasms (CIN). These can be detected and treated before progression to invasive cancer. The major risk factor for developing cervical cancer or CIN is persistent or recurrent infection with high-risk human papilloma virus (hrHPV). Other associated risk factors include low socioeconomic status, smoking, sexually transmitted infections, and high number of sexual partners, and these risk factors can predispose to some other cancers, excess mortality, and reproductive health complications as well. The aim was to study long-term cancer incidence, mortality, and reproductive health outcomes among women treated for CIN. Based on the results, we could evaluate the efficacy and safety of CIN treatment practices and estimate the role of the risk factors of CIN patients for cancer incidence, mortality, and reproductive health. We collected a cohort of 7 599 women treated for CIN at Helsinki University Central Hospital from 1974 to 2001. Information about their cancer incidence, cause of death, birth of children and other reproductive endpoints, and socio-economic status were gathered through registerlinkages to the Finnish Cancer Registry, Finnish Population Registry, and Statistics Finland. Depending on the endpoints in question, the women treated were compared to the general population, to themselves, or to an age- and municipality-matched reference cohort. Cervical cancer incidence was increased after treatment of CIN for at least 20 years, regardless of the grade of histology at treatment. Compared to all of the colposcopically guided methods, cold knife conization (CKC) was the least effective method of treatment in terms of later CIN 3 or cervical cancer incidence. In addition to cervical cancer, incidence of other HPV-related anogenital cancers was increased among those treated, as was the incidence of lung cancer and other smoking-related cancers. Mortality from cervical cancer among the women treated was not statistically significantly elevated, and after adjustment for socio-economic status, the hazard ratio (HR) was 1.0. In fact, the excess mortality among those treated was mainly due to increased mortality from other cancers, especially from lung cancer. In terms of post-treatment fertility, the CIN treatments seem to be safe: The women had more deliveries, and their incidence of pregnancy was similar before and after treatment. Incidence of extra-uterine pregnancies and induced abortions was elevated among the treated both before and after treatment. Thus this elevation did not occur because they were treated rather to a great extent was due to the other known risk factors these women had in excess, i.e. sexually transmitted infections. The purpose of any cancer preventive activity is to reduce cancer incidence and mortality. In Finland, cervical cancer is a rare disease and death from it even rarer, mostly due to the effective screening program. Despite this, the women treated are at increased risk for cancer; not just for cervical cancer. They must be followed up carefully and for a long period of time; general health education, especially cessation of smoking, is crucial in the management process, as well as interventions towards proper use of birth control such as condoms.

Tactile processing in human somatosensory and auditory cortices

Tactile sensation plays an important role in everyday life. While the somatosensory system has been studied extensively, the majority of information has come from studies using animal models. Recent development of high-resolution anatomical and functional imaging techniques has enabled the non-invasive study of human somatosensory cortex and thalamus. This thesis provides new insights into the functional organization of the human brain areas involved in tactile processing using magnetoencephalography (MEG) and functional magnetic resonance imaging (fMRI). The thesis also demonstrates certain optimizations of MEG and fMRI methods. Tactile digit stimulation elicited stimulus-specific responses in a number of brain areas. Contralateral activation was observed in somatosensory thalamus (Study II), primary somatosensory cortex (SI; I, III, IV), and post-auditory belt area (III). Bilateral activation was observed in secondary somatosensory cortex (SII; II, III, IV). Ipsilateral activation was found in the post-central gyrus (area 2 of SI cortex; IV). In addition, phasic deactivation was observed within ipsilateral SI cortex and bilateral primary motor cortex (IV). Detailed investigation of the tactile responses demonstrated that the arrangement of distal-proximal finger representations in area 3b of SI in humans is similar to that found in monkeys (I). An optimized MEG approach was sufficient to resolve such fine detail in functional organization. The SII region appeared to contain double representations for fingers and toes (II). The detection of activations in the SII region and thalamus improved at the individual and group levels when cardiac-gated fMRI was used (II). Better detection of body part representations at the individual level is an important improvement, because identification of individual representations is crucial for studying brain plasticity in somatosensory areas. The posterior auditory belt area demonstrated responses to both auditory and tactile stimuli (III), implicating this area as a physiological substrate for the auditory-tactile interaction observed in earlier psychophysical studies. Comparison of different smoothing parameters (III) demonstrated that proper evaluation of co-activation should be based on individual subject analysis with minimal or no smoothing. Tactile input consistently influenced area 3b of the human ipsilateral SI cortex (IV). The observed phasic negative fMRI response is proposed to result from interhemispheric inhibition via trans-callosal connections. This thesis contributes to a growing body of human data suggesting that processing of tactile stimuli involves multiple brain areas, with different spatial patterns of cortical activation for different stimuli.

Basement membrane and provisional matrix components (collagen type IV, laminins and von Willebrand factor) in rheumatoid arthritis and Sjögren s syndrome

The aim of this study was twofold- Firstly, to determine the composition of the type IV collagen which are the major components of the basement membrane (BM), in the synovial lining of the rheumatoid arthritis (RA) patient and in the BM in the labial salivary gland of the Sjögrens syndrome (SS) patient. Secondly, this thesis aimed to investigate the role of the BM component laminin α4 and laminin α5 in the migration of neutrophils from the blood vessels thorough the synovial lining layer into synovial fluid and the presence of vWF in the microvasculature of labial salivary gland in SS. Our studies showed that certain α chains type IV collagen are low in RA compared to control synovial linings, while laminin α5 exhibited a pattern of low expression regions at the synovial lining interface towards the joint cavity and fluid. Also, high numbers of macrophage-like lining cells containing MMP-9 were found in the lining. MMP-9 was also found in the synovial fluid. Collagen α1/2 (IV) mRNA was found to be present in high amount compared to the other α(IV) chains and also showed intense labelling in immunohistochemical staining in normal and SS patients. In healthy glands α5(IV) and α6(IV) chains were found to be continuous around ducts but discontinuous around acini. The α5(IV) and α6(IV) mRNAs were present in LSG explants and HSG cell line, while in SS these chains seemed to be absent or appear only in patches around the ductal BM and tended to be absent around acini in immunohistochemical staining, indicating that their synthesis and/or degradation seemed to be locally regulated around acinar cells. The provisional matrix component vWF serves as a marker of vascular damage. Microvasculature in SS showed signs of focal damage which in turn might impair arteriolar feeding, capillary transudation and venular drainage of blood. However, capillary density was not decreased but rather increased, perhaps as a result of angiogenesis compensatory to microvascular damage. Microvascular involvement of LSG may contribute to the pathogenesis of this syndrome. This twofold approach allows us to understand the intricate relation between the ECM components and the immunopathological changes that occur during the pathogenesis of these inflammatory rheumatic disease processes. Also notably this study highlights the importance of maintaining a healthy ECM to prevent the progression or possibly allow reversal of the disease to a considerable level. Furthermore, it can be speculated that a healthy BM could quarantine the inflamed region or in case of cancer cells barricade the movement of malignant cells thereby preventing further spread to the surrounding areas. This understanding can be further applied to design appropriate drugs which act specifically to maintain a proper BM/BM like intercellular matrix composition.

Molecular characterization of mitochondrial ataxias : with emphasis on MIRAS and IOSCA

Uusi hermoston rappeumasairaus MIRAS: Suomessa kantajia joka 125. väestöstä Tässä väitöskirjatyössä on kuvattu uusi peittyvästi periytyvä hermoston rappeumasairaus, MIRAS (mitochondrial recessive ataxia syndrome), ja sen geenitausta. Tauti osoittautui tutkimuksessamme Suomen yleisimmäksi perinnölliseksi ataksiasairaudeksi. Tutkimuksessa on tutkittu perinnöllisiä aivosairauksia, joissa yhtenä oireena on ataksia (kävelyn epävarmuus, tasapainovaikeus ja liikkeiden haparointi), sekä lukuisia muita aivojen toimintahäiriöstä johtuvia oireita. Seuloessamme suomalaisilta ataksiapotilailta MIRAS-geenivirhettä, 27 potilasta sai diagnoosin aikaisemmin tuntemattomalle, etenevälle ataksiasairaudelleen. Tutkimuksen tuloksena kyseisen geenivirheen DNA-diagnostiikka on otettu käyttöön suomalaisissa ja eurooppalaisissa laboratorioissa, ja toista sataa potilasta ympäri maailman on saanut diagnoosin. Suomen väestössä joka 125. kantaa MIRAS geenivirhettä, mutta taudin saa vain, jos perii geenivirheen molemmilta vanhemmiltaan. MIRAS on taudinkuvaltaan vaihteleva, mutta vaikea etenevä neurologinen sairaus. Useilla potilailla esiintyvät oireet ovat ataksia, puheen puuromaisuus (dysartria), ääreishermorappeuma (neuropatia), pakkoliikkeet, psykiatriset oireet sekä vaikea epilepsia. Erityisen tärkeää MIRAS-taudin tunnistaminen on siihen liittyvän epilepsian hoitopäätöksessä: valproaatti-lääkitys voi aiheuttaa MIRAS-potilaille vaikean maksavaurion. Väitöskirjatyön tuloksena selvisi, että kaikki suomalaiset, norjalaiset, belgialaiset, englantilaiset, australialaiset ja uusi-seelantilaiset MIRAS potilaat olivat kaukaista sukua samalle, tuhansia vuosia sitten eläneelle eurooppalaiselle esivanhemmalle. Ataksiasairauksien tautimekanismeja selvitimme tutkimalla MIRAS-ataksiaa ja sitä muistuttavaa IOSCA sairautta (infantile onset spinocerebellar ataxia), jonka aiheuttaa peittyvästi periytyvä geenivirhe Twinkle-geenissä. Tutkimuksessa löydettiin myös uusi, Twinkle-geenin geenivirheestä johtuva taudinkuva: vaikea-asteinen, varhaisella iällä alkava aivosairaus, jossa on lisäksi viitteitä maksasairaudesta. Löysimme potilaiden aivoista muutoksia mitokondrioiden eli solun voimalaitosten perimän määrässä. Nämä tulokset antavat arvokasta lisätietoa ataksiasairauksien taustalla olevista muutoksista, joiden ymmärtäminen on välttämätön edellytys hoitomahdollisuuksien tutkimiselle tulevaisuudessa.