<p>Platelet-derived growth factor receptor α (PDGFRα) plays an important role during embryogenesis. After implantation, the patterns of expression of Pdgfrα and its ligand Pdgf-A undergo an "autocrine-paracrine transition", in that Pdgf-A becomes expressed in the ectoderm and epithelia, while Pdgfrα is expressed in the adjacent mesenchymal tissue. In human tumors, such as malignant glioma, both PDGF and PDGFRα are overexpressed within the same tissue, indicating that an autocrine PDGF loop is generated in the tumors. This thesis is focused on the <i>in vivo</i> functionality of the <i>PDGFR</i>α gene (<i>PDGFRA</i>) promoter, arid on the effect of autocrine PDGF-A stimulation in transgenic n-iice during embryogenesis. </p><p>To test the <i>in vivo</i> promoter function of a human PDGFRA 2.2 kb 5' flanking fragment, we generated transgenic mouse lines and found that the 2.2 kb fragment was able to promote lacZ reporter gene expression in most of the endogenous Pdgfra expressing tissues. Absence of expression and "ectopic" expression of the transgenic lacZ were also observed. To investigate the autocrine PDGF effect, we produced autocrine PDGF-As (A short-chain) transient transgenic embryos. These transgenic embryos carried a 6 kb mouse <i>Pdgfra</i> 5' flanking sequence linked to a human PDGF-As cDNA. The pattern of expression of the PDGF-As transgene mRNA was similar to that of lacZ. Some of the transgenic embryos exhibited severe abnormal phenotypes, such as midline fusion defects in the cephalic and craniofacial region and small body size, and these embryos die at mid-gestation stage. These findings indicate that a paracrine pattern of expression and the dosage of PDGF are important for sustaining normal embryo development, especially with regard to the middline fusion in craniofacial regions. </p><p>The possible signaling pathways that may be involved in regulating <i>Pdgfra</i> activity were also studied by comparison of patterns of mRNA expression of Gli, Ptc, and Paxl with that of Pdgfra. The results pointed to the possibility that the Shh signaling pathway may be involved in the regulation of <i>Pdgfra</i> expression for example during early bone and foregut development. The specific regulatory mechanisms may vary for different tissues.</p>

<p>Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer.</p><p>Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.</p>

<p>All-trans retinoic acid (ATRA), a biologically active metabolite of vitamin A, is a powerful inducer of terminal differentiation and growth arrest of several myeloid cell lines <i>in vitro</i>. Although the efficacy of ATRA as an anti-cancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), knowledge concerning the molecular mechanisms directing ATRA-induced differentiation and cell cycle arrest of myeloid cells is lacking. Our results show, for the first time, that the complex regulation of cell cycle proteins and myeloid-specific transcription factors induced by ATRA relies on functional Stat1. We found that Stat1 is activated by both tyrosine-701 and serine-727 phosphorylation upon ATRA-induced differentiation of the human monoblastic cell line U-937. Expression of phosphorylation deficient mutants of Stat1 (Stat1Y701F or Stat1S727A) inhibited both ATRA-induced differentiation and cell cycle arrest of U-937 cells, pointing to a requirement of active Stat1 in these processes. </p><p>Detailed analysis of the molecular mechanism of ATRA-induced cell cycle arrest and differentiation showed that the onset of cell cycle arrest was associated with a decrease in c-Myc and cyclin E levels and upregulation of p27^Kip1 and p21^WAF1/CIP1. This was followed by a rapid fall in cyclin A and B and a coordinate dephosphorylation of the retinoblastoma protein (pRb). The inhibition of ATRA-induced cell-cycle arrest by constitutive expression of Stat1Y701F or Stat1S727A was associated with impaired regulation of these cyclins and p27^Kip1, positioning Stat1 activation upstream of these events. To further understand the process of ATRA-induced differentiation, the regulation of myeloid-specific transcription factors was investigated during ATRA-treatment. Notably, ATRA-induced upregulation of Stat2, ICSBP and C/EBP-ε was selectively impaired in sublines expressing Stat1Y701F or Stat1S727A, suggesting an important function of these factors downstream Stat1. Taken together, the work in this thesis clearly demonstrates that Stat1 plays a key role in ATRA-induced terminal differentiation of myeloid cells, through regulation of cell cycle proteins and myeloid-specific transcription factors. </p>

<p>A series of novel methods for detection of known sequence variants in DNA, in particular single nucleotide polymorphism, using padlock probes and rolling circle replication are presented. DNA probes that can be circularized – padlock probes – are ideal for rolling circle replication. Circularized, but not unreacted probes, can generate powerful signal amplification by allowing the reacted probes to template a rolling circle replication (RCR) reaction. However, when hybridized and ligated to a target DNA molecule with no nearby ends, the probes are bound to the target sequence, inhibiting the RCR reaction is. This problem can be solved by generating a branched DNA probe with two 3’ arms such that the probes may be circularized while leaving the second 3’ arm as a primer for the RCR reaction. We describe how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5’ end but two distinct 3’ ends that extend from the 2’ and 3’ carbons of an internal nucleotide. An even stronger approach to circumvent the topological problem that can inhibit RCR is to restriction digest the template downstream of the padlock recognition site. By using Phi 29 DNA polymerase with efficient 3’ exonuclease and strand displacement activity, the template strand can then be used to prime the RCR reaction. The amplified molecule is contiguous with the target DNA, generating an anchored localized signal. The kinetics of the reaction was investigated by following the reaction in real-time using molecular beacon probes. Localized RCR signal were obtained on DNA arrays, allowing detection of as little as 104-105 spotted molecules, of either single- or double-stranded M13 DNA, in a model experiment. We have also established a serial rolling circle amplification procedure. By converting rolling circle products to a second and even third generation of padlock probes the signal was amplified thousand-fold per generation. This procedure provides sufficient sensitivity for detection of single-copy gene sequences in 50 ng of human genomic DNA, and large numbers of probes were amplified in parallel with excellent quantitative resolution.</p>

<p>Angiogenesis can be defined as the formation of new blood vessels from pre-existing ones. Angiogenesis is required for development and maintenance of our vascular system and thus of fundamental importance to our existence. The endothelial cells that line the inside of the vessels de-differentiate, migrate, proliferate and re-differentiate during angiogenesis. Angiogenesis is tightly regulated, controlled by several angiogenic factors of various classes that promote angiogenesis but also by anti-angiogenic factors that counteract the effect of the pro-angiogenic factors. We have examined three factors involved in angiogenesis regulation, Vascular endotelial growth factor (VEGFR) -3, the matrix protein laminin-1 and the collagen XVIII derived fragment endostatin. </p><p>Five tyrosine phosphorylation sites in the cytoplasmic tail of VEGFR-3 were identified by phosphopeptide mapping (PPM). The data was confirmed by PPM using point-mutated receptors generated by site-directed mutagenesis.</p><p>Laminin-1 was found to promote angiogenesis in the chicken chorioallantoic membrane assay and in a synergistic fashion together with suboptimal levels of fibroblast growth factor 2 (FGF-2) in embryoid bodies. Laminin-1 also promoted endothelial tubular morphogenesis in vitro, and upregulated the expression of the endothelial differentiation marker Jagged-1. </p><p>Endostatin was shown to affect endothelial FGF-2-induced cell survival and morphogenesis. This was a result of direct binding to endothelial cells and induction of tyrosine phosphorylation of many proteins including the adaptor protein Shb. The apoptotic and morphogenic responses induced by endostatin was shown to be dependent on Shb. Further, endostatin inhibited endothelial migration and affected molecules implicated in migration. In particular, FGF-2 induced actin reorganization, and β-catenin regulation was modulated by endostatin. </p>

<p>Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The <i>VMD2</i> gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD.</p><p>In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a <i>C. elegans</i> protein family, expressed in retina, was identified as the <i>VMD2</i> gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the <i>VMD2</i> gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. <i>VMD2</i> expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation.</p><p>Analysis of mouse <i>Vmd2</i> and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. <i>Vmd2</i> expression levels were highest around birth.</p>

<p>The phylogeography of a wide ranging temperate species, the adder, <i>Vipera berus</i>, was investigated using several genetic tools, with special emphasis on the post-glacial colonisation pattern of Fennoscandia. The area was colonised from two directions by adder populations representing different glacial refugia. The two populations meet in three places and the main contact zone is situated in Northern Finland. The two other contact zones are the result of dispersal across the Baltic Sea to the Umeå archepelago and South-Western Finland. Asymmetrically distributed nuclear genetic variation compared to mitochondrial DNA in the northern contact zone suggests a skewed gene flow from the east to the west across the zone. This pattern might reflect differences in dispersal among sexes and lineages, or may be accounted for by a selective advantage for nuclear variation of eastern origin among Fennoscandian adders.</p><p>The phylogeographic pattern for adders across the entire species range was addressed by sequencing part of the mitochondrial genome and scoring microsatellite markers. The adder can be divided into three major genetic groups. One group is confined to the Balkan peninsula harbouring the distribution range of <i>V. b. bosniensis</i>. A second, well differentiated group is restricted to the Southern Alps. These two areas have probably served as refugia for adders during a number of ice ages for the adders. The third group is distributed across the remainder of the species’ range, from extreme Western Europe to Pacific Russia and can be further divided into one ancestral group inhabiting the Carpathians refugial area, and three more recent groups inhabiting areas west, north and east of the Alps. The adder provides an example of a species where the Mediterranean areas are housing endemic populations, rather than the sources for post-glacial continental colonisation. Continent-wide colonisation has instead occurred from up to three cryptic northern refugia. </p>

<p>Mitochondrial DNA (mtDNA) has been a potent tool in studies of the evolution of modern humans, human migrations and the dynamics of human populations over time. The popularity of this cytoplasmic genome has largely been due to its clonal inheritance (in Man) allowing the tracing of a direct genetic line. In addition, a comparatively high rate of nucleotide substitution facilitates phylogenetic resolution among relatively closely related individuals of the same species.</p><p>In this thesis, a statistically supported phylogeny based on complete mitochondrial genome sequences is presented which, for the first time, unambiguously places the root of modern human mitochondrial lineages in Africa in the last 200 thousand years. This conclusion provides strong support for the “recent African origin” hypothesis. Also, the complete genome data underline the problematic nature of traditional approaches to analyses of mitochondrial phylogenies.</p><p>The dispersal of anatomically modern humans from the African continent is examined through single nucleotide polymorphism (SNP) and sequence data. These data imply an expansion from Africa about 57 thousand years ago and a subsequent population dispersal into Asia. The dispersal coincides with a major population division that may be the result of multiple migratory routes to East Asia.</p><p>Also investigated is the question of a common origin for the indigenous peoples of Australia and New Guinea. Previous studies have been equivocal on this question with some presenting evidence for a common genetic origin and other proposing separate histories. Our data reveals an ancient genetic link between Australian Aborigines and the peoples of the New Guinea highlands.</p>

<p>The aim of this thesis is to study phylogeny, species-wide phylogeography and genetic diversity in <i>Formica</i> ants across Eurasia in connection with the history of biotic responses to Quaternary environmental changes.</p><p>The mitochondrial DNA phylogeny of Palaearctic <i>Formica</i> species supported the subgeneric grouping based on morphological similarity. The exception was that <i>F. uralensis</i> formed a separate phylogenetic group. The mitochondrial DNA phylogeny of the <i>F. rufa </i>group showed the division into three major phylogenetic groups: one with the species <i>F. polyctena</i> and <i>F. rufa</i>, one with <i>F. aquilonia</i>, <i>F. lugubris</i> and <i>F. paralugubris</i>, and the third one with <i>F. pratensis</i>.</p><p>West-east phylogeographic divisions were found in <i>F. pratensis</i> suggesting post-glacial colonization of western Europe and a wide area from Sweden to the Baikal Lake from separate forest refugia. In contrast, no phylogeographic divisions were detected in either <i>F. lugubris </i>or<i> F. exsecta</i>. Contraction of the distribution range to a single refugial area during the late Pleistocene and the following population expansion could offer a general explanation for the lack of phylogeographic structure across most of Eurasia in these species.</p><p>Sympatrically distributed and ecologically similar species <i>F. uralensis </i>and<i> F. candida</i> showed clear difference in the phylogeographic structure that reflected difference in their vicariant history. Whereas no phylogeographic divisions were detected in <i>F. uralensis</i> across Europe, <i>F. candida</i> showed a well-supported phylogeographic division between the western, the central and the southern group.</p><p>In socially polymorphic <i>F. cinerea</i>, the overall level of intrapopulation microsatellite diversity was relatively high and differentiation among populations was low, indicating recent historical connections. The lack of correspondence between genetic affinities and geographic locations of studied populations did not provide any evidence for differentiating between alternative hypotheses concerning the directions and sources of postglacial colonization of Fennoscandia.</p>

<p>Sex chromosomes are useful in elucidating the evolutionary factors affecting diversity and divergence. In particular, Y chromosome analyses may complement studies using mitochondrial DNA for inferring sex-specific population genetic processes.</p><p>Y chromosome studies have been scarce due to limited access to genetic markers and the dynamic evolution of Y. Conserved Y-specific primers that could amplify a diverse set of mammalian species were developed from comparison of gametologous X and Y sequences. Y-specific sequence, generally more than one kb, was amplified for all 20 species examined.</p><p>Intraspecific diversity on mammalian Y was found to be reduced even when male-biased mutation rate and effective population size were corrected for. A number of factors can cause this low variation on Y of which selection on a haploid chromosome seems most important.</p><p>The field vole (<i>Microtus agrestis</i>), a common and well-studied small mammal in Eurasia, was examined for X and Y variability. Earlier studies on mtDNA had shown that the field vole is separated in two distinct lineages in Europe. The X and Y chromosome sequences confirmed the deep split and suggested that the two lineages of field vole should be reclassified as two separate species.</p><p>Two distinct Y chromosome haplogroups were found in modern European cattle, distributed among breeds according to a north-south gradient. Ancient DNA analysis of European aurochsen showed the northern haplogroup to be the most common, possibly indicating local hybridization between domestic cows and wild aurochs bulls in Europe.</p>