<p>This thesis attempts to improve our understanding of the role of population size for the process of speciation. First, the effect of population size on speciation is studied using several meta-analyses of published laboratory experiments. Second, the effect of population size on behaviour is studied using a laboratory population of the blowfly <i>Lucilia sericata</i>. Third, the effect of population size on morphological and genetic variation is studied using wings and microsatellites from wild populations of <i>L. illustris</i> as well as experimentally bottlenecked populations of <i>L. sericata</i>. The meta-analyses showed that the result of many previous laboratory experiments on sympatric and parapatric speciation may have been biased by too small population sizes. Reduced interbreeding was less likely to develop in small populations where the selection against hybridisation often seemed to have been opposed by inbreeding depression or loss of genetic variation. In allopatric speciation experiments, no general consistent effect of population size was observed. There was no support for speciation through founder events. In fact, significant assortative mating was only found in vicariance experiments where derived populations was tested against each other. Population size influenced reproductive behaviour in <i>L. sericata</i>. There was a positive effect of increasing number of males on egg-laying but only as long as the female was in the company of at least one other female. Female mate choice and a positive effect of number of eggs on larval survival are suggested to be the underlying factors. No historic bottlenecks could be detected in the fly populations, but strong genetic indications suggest a fine grained genetic population structure of wild <i>Lucilia</i> flies. Bottlenecks had unpredictable effects on wing morphology as well as on genetic variation and fitness in a laboratory stock of <i>L. sericata</i>. Thus a bottlenecked population will not necessarily have a higher chance of evolving morphological novelties than one which has not undergone a bottleneck. However, among many bottlenecked populations there is a good chance that in at least one of them the conditions will be conducive to morphological change and evolution. In this statistical sense, thus, strong population fluctuations may enhance the probability of speciation events.</p>

<p>Understanding the processes that shape the spatial distribution of genetic variation within species is central to the evolutionary study of diversification and demography. Neutral genetic variation reflects past demographic events as well as current demographic characteristics of populations, and the correct interpretation of genetic data requires that the relative impact of these forces can be identified. Details of breeding systems can affect the genetic structure through effects on effective migration rate or on effective population size. Restrictions in recombination rate lead to associations between neutral marker genes and genes under natural selection. Although the effects on genetic structure can be substantial, the process will often be difficult to tell apart from stochastic effects of history or genetic drift, which may suggest erroneous conclusions about demography.</p><p>In cyclically parthenogenetic freshwater invertebrates, which alternate between sexual and asexual reproduction, demographic fluctuations and reliance on diapausing eggs for dispersal enhances neutral genetic differentiation as well as effects of selection on associated genes. Although genetic founder effects are expected to be profound and long-lasting in these species, genetic hitch-hiking may reduce initial strong differentiation rapidly if better adapted genes are introduced by mutation or immigration. Fluctuating environmental conditions have been suggested to generate rapid shifts in the frequencies of clones during the asexual phase. In the presence of egg banks resting in sediments, genetic diversity is stabilised and the importance of migration for differentiation is reduced.</p><p>Studies of unstable and young populations of cyclically parthenogenetic <i>Daphnia pulex</i> showed substantial variation for important fitness traits, within as well as between populations, despite hypothesised recent founder effects. Neutral markers indicated genetic equilibrium, but changes in clonal composition during asexuality disrupted the genetic structure in a manner compatible with local adaptation and exclusion of immigrants. This illustrates that the forces affecting sexual progeny may be markedly different from those shaping the structure among asexual individuals.</p>

<p>Genomic imprinting marks a subset of autosomal loci expressed in parent of origin-dependent monoallelic expression in a non-Mendelian fashion. To restore totipotency and to reset the imprint according to the sex of the individual, the mark must be erased during germline development. The imprinted <i>Igf2-H19</i> loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the <i>H19 </i>is silenced when paternally inherited, the <i>Igf2</i> is silenced when maternally inherited. </p><p>The differentially methylated 5'-flank of <i>H19</i> gene, termed imprinting control region (ICR), shown to display a unique chromatin organisation harbours hypersensitive sites in linker regions flanked by positioned nucleosomes on the maternal allele. This unique chromatin conformation functions as a methylation-sensitive and unidirectional chromatin insulator, which later was found to depend on the chromatin insulator protein CTCF. </p><p>The <i>H19</i> ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the <i>H19</i> ICR and the promoter of the reporter gene required for this effect was 1.2 ± 0.3kb which can be compared to the 1.9 kb distance between the endogenous <i>H19 </i>ICR and <i>H19</i> promoter. Results suggest that the <i>H19</i> ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal <i>cis</i>-regulatory elements to avoid adverse effects on neighbouring promoters. </p><p>Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived <i>H19</i> ICR and associated with factors that interact with the CTCF target sites. CTCF is itself poly(ADP-ribosy)lated and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide relieves the insulator function of the <i>H19</i> ICR. </p><p>Designed zinc finger proteins were applied to examine if epigenetic marks provided an obstacle for targeted activation and silencing. The zinc finger protein ZFP809 with activator/repressor domain able to efficiently activate/silence the <i>IGF2</i> target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal <i>IGF2</i> and paternal <i>H19</i> allele, respectively. Results suggested that imprinted genes are not normally exposed to strong <i>cis</i>-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions.</p>

<p>An overall coordination between the expressions of genes is required for the proper development of an individual. Although most genes are expressed from both the constituent alleles of the genome, a small subset of autosomal genes are preferentially expressed from only one of the parental alleles, a phenomenon known as genomic imprinting. </p><p>The imprinted <i>H19</i> and <i>Igf2</i> genes are considered paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of sequence located upstream of <i>H19</i>, known as the imprinting control region (ICR). This region shows differential methylation, with hypermethylation specifically on the paternal allele. On the maternal allele this region acts as an insulator and harbours maternal specific hypersensitive sites. </p><p>The hypersensitive sites were identified as the result of association of the vertebrate insulator protein CTCF with the region. This association was investigated in both an <i>in vitro</i> episomal system and in an <i>in vivo</i> mouse model system by mutating the CTCF target sites at the <i>H19</i> ICR. The importance of CTCF for the insulator property of the region was confirmed in both instances. In the mouse model, the disruption of the binding was also observed to affect the methylation profile of the ICR, which ultimately resulted in the de-repression of the maternal <i>Igf2</i> allele.</p><p>The relevance of multiple CTCF target sites in higher vertebrates for the proper insulator function was investigated using another knock-in mouse model with mutation at a single CTCF target site in the <i>H19</i> ICR. The investigation confirmed the cooperation between the target sites for the establishment of a functional insulator on the maternal allele. Target sites in the ICR were also analysed for their differential binding affinity for the CTCF protein.</p><p>The utilisation of the CTCF target sites was examined in different human tumours and cell lines. Methylation analysis conveyed a lack of correlation between the loss of insulator function and methylation status of the ICR with the loss of imprinting (LOI) of <i>IGF2</i>. Investigations also identified a novel mechanism, which neutralised the chromatin insulator function of the <i>H19</i> ICR without affecting its chromatin conformation. This principle might also help in explaining the loss of <i>IGF2</i> imprinting observed in some instances.</p><p>In conclusion, this thesis confirms the importance of CTCF in the formation of an epigenetically regulated chromatin insulator at the ICR, which in turn controls the expression pattern of <i>H19</i> and <i>Igf2</i>. The studies also confirm the role of CTCF in the maintenance of the methylation profile of the region. Investigations into the loss of <i>IGF2 </i>imprinting in human cancer indicate the involvement of other novel mechanisms besides CTCF in the regulation of insulator function.</p>

<p>A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting.</p><p>The imprinted state of the <i>H19</i> and <i>Igf2</i> genes is controlled by a short stretch of sequences upstream of <i>H19</i> known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited <i>Igf2</i> allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the <i>H19</i> ICR, which plays an important role in maintaining the imprinting status of <i>H19</i> and <i>Igf2</i>, was shown to lose the insulator property upon CpG methylation.</p><p>Another ICR in the <i>Kcnq1</i> locus regulates long-range repression of <i>p57Kip2</i> and <i>Kcnq1</i> on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing <i>H19</i> and <i>Igf2</i>, on the distal end of mouse chromosome 7. Similarly to the <i>H19</i> ICR, the <i>Kcnq1</i> ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions.</p><p>The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the <i>H19</i> ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF <i>in vivo</i> occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding. </p><p>In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.</p>

<p>This thesis examines seasonal, diel and vertical distribution patterns of activity of host-seeking <i>Ixodes ricinus</i> (L.) ticks at three localities in south-central Sweden. In addition, by examining the prevalence of infection in ticks with Lyme borreliosis (LB) spirochetes, <i>Borrelia burgdorferi</i> s.l, information for estimating relative LB risk in humans and the effect of control measures directed against this tick vector is provided.</p><p>The seasonal activity pattern of <i>I. ricinus</i> was, in general, bimodal with peaks of activity in May-June and August-September. Tick densities were generally high at Torö and low at Kungshamn-Morga. The greatest variation in tick density occurred at the sample site level, which indicated a patchy distribution of ticks. The diel activity of adult <i>I. ricinus</i> sampled at Bogesund showed a distinct nocturnal activity peak while nymphal ticks exhibited no particular diel variation. At the meadow site, there was a strong negative association between activity of each tick stage and ambient air temperature, and larval ticks also showed a nocturnal activity peak. <i>I. ricinus</i> of all stages were present in the vegetation up to at least 140 cm above ground level. At Torö, host-seeking larvae were found at significantly lower levels (below 20 cm) in the vegetation compared to nymphs and adults (50-59 and 60-79 cm, respectively). Vegetation structure is likely to be the main factor governing tick vertical distribution at this locality. The northern limit of the geographical distribution of <i>I. ricinus</i> in Sweden corresponds with the southern boundary of the taiga zone, as well as with several other climatic or vegetational isoclines primarily associated with the vegetation period.</p><p>The prevalence rates of <i>Borrelia</i> spirochetes, recorded by phase-contrast microscopy in host-seeking <i>I. ricinus</i>, were 0% in larvae, 5.8-13.1% in nymphs and 14.5-28.6% in adult ticks. The human LB risk, estimated by the number of <i>Borrelia</i>-infected nymphs per hectare, was greater at Torö than at Kungshamn-Morga and greater in woodland than in open areas. The risk also possessed a bimodal seasonal pattern similar to that of subadult host-seeking activity. Controlling the number of infected nymphs through de-ticking of reservoir hosts seems not to be an effective control measure in Sweden due to the ubiquitous availability of alternative reservoir hosts.</p>

<p>Peat mosses (<i>Sphagnum</i>) are widely distributed and are a major component of mire vegetation and peat throughout the boreal and temperate regions. Most boreal <i>Sphagnum</i> species regularly produce sporophytes, but the ecological role of the spore has been questioned. This study shows that the spores can form a spore bank and have the ability to germinate and contribute to moss establishment whenever suitable conditions occur. The results suggest that spore production is important for explaining the wide distribution and omnipresence of <i>Sphagnum</i> in nutrient-poor wetlands. The results further imply that initial recruitment from spores predominates in <i>Sphagnum</i> after disturbance or formation of suitable habitats.</p><p> A series of experiments showed that addition of phosphorus-containing substrates, such as fresh plant litter or moose dung, resulted in spore establishment on bare, moist peat. A field experiment indicated establishment rates of about 1% of sown, germinable spores on peat with added substrates. Plant litter on moist soil, without a closed cover of bryophytes, is an important safe site for the establishment of <i>Sphagnum</i> spores. The results fit the observed pattern of colonisation by <i>Sphagnum</i> beneath <i>Eriophorum vaginatum</i> tussocks in mires severely disturbed by peat extraction. Successful long-distance dispersal was indicated by the occurrence of several regionally new or rare <i>Sphagnum</i> species in disturbed mires.</p><p>Spore number per sporophyte ranged among <i>Sphagnum</i> species from 18 500 to 240 000, with a trade-off between spore number and spore size. Annual spore production was estimated at 15 million spores per square metre on two investigated mires. Sporophyte production showed a large interannual variation. Sporophyte production was positively related to the amount of precipitation the preceding summer. This was probably because a high water level promoted gametangium formation. Spore dispersal occurred in July and August. The earlier timing of spore dispersal in the more drought-sensitive, hollow-inhabiting sphagna should reduce the risk of sporophytes drying out prematurely during summer droughts.</p><p>Spores kept refrigerated up to 13 years retained high germinability. A field experiment showed that <i>Sphagnum</i> can form a persistent spore bank, with a potential longevity of several decades.</p>

<p>This dissertation is primarily focused on the <i>Buellia</i> species with pluriseptate spores, often referred to the section or genus <i>Diplotomma</i>, and secondarily on the Physciaceae. An ultrastructural study at family level gives new information on spore characters. Variations in the composition and structure of the spore wall layers and septa are described.</p><p>A phylogenetic analysis of the Physciaceae, based on morphological and chemical characters, is presented. In the resulting trees the species with pluriseptate spores appear in a poorly resolved clade together with other crustose species with 1-septate spores. A subsequent analysis, excluding foliose and fruticose species, shows that the species with pluriseptate spores do not form a monophyletic group, and that only a few are grouped together with the type species of <i>Diplotomma</i>. Due to low resolution, poor branch support, and uncertainty concerning the typification of <i>Buellia</i> all species treated are retained in <i>Buellia</i>.</p><p>Altogether 35 species with pluriseptate spores are treated, including ten new to science, viz. <i>B. aeruginosa</i>, <i>B. morsina</i>, <i>B. muriformis</i>, <i>B. oidaliella</i>, <i>B. pallido-marginata</i>, <i>B. romoletia</i>, <i>B. rubroreagens</i>, <i>B. terricola</i>, <i>B. tombadorensis</i>, and <i>B. tri-septata</i>. Historical outlines are presented, and information on life strategy, morphology, chemistry, ecology, and distribution is given. All species are similar and macroscopically not easily distinguished, having crustose thalli and blackish apothecia, but there is a great variation in microscopic characters, secondary chemistry, substrate preferences, habitat ecology, and distribution range. Several names are typified and many synonyms are listed. References are given to all species referred to <i>Diplotomma</i> in literature.</p>

<p>Mammalian pregnancy is an immunological paradox. The foetus, which expresses both paternal and maternal cell-surface molecules, has to be protected from rejection by the maternal immune system. At the same time, the mother has to have an efficient immune defence and must provide her offspring with antibodies.</p><p>The first part of this thesis investigates some of the mechanisms involved in the foetal avoidance of maternal rejection reactions. Placental absence of MHC class II expression, as well as a bias for Th2-cytokines at the maternal-foetal interface are suggested to be important for foetal survival. The results showed that placental MHC class II expression cannot be induced <i>in vivo</i>. Transfections of trophoblast cells with MHC class II genes, however, resulted in detectable MHC class II cell-surface expression, indicating that a post-transcriptional block does not exist in these cells.</p><p>By using IL-4- and IL-10-double deficient mice, it was shown that neither maternal nor foetal expression of these cytokines were crucial for completion of allogeneic pregnancy.</p><p>In the second part of the thesis, the effect of transmission of immunoglobulin G (IgG) from the mother to the offspring was studied. It was observed that viable maternal Ig-secreting cells occasionally infiltrated the B cell-deficient offspring and remained functional for long periods. In this study "green fluorescent mice" were used as a tool. Furthermore, neonatal ingestion of wild type milk increased the survival of adoptively transferred B-lineage cells in B cell-deficient mice, suggesting that suckling of IgG-containing milk could be used to facilitate B cell-reconstitution in B cell-deficient mice. Finally, results from studies on normal mice showed that absence of maternal IgG-transmission during their neonatal development resulted in elevated serum-IgG production, as well as enhanced immune reactions upon immunisations in adult life. This showed that maternal IgG can have long-term immunoregulatory effects in the offspring.</p>

<p>This thesis shows that socially subordinate Arctic charr (<i>Salvelinus alpinus</i>) display elevated brain serotonergic (5-HT) and norepinephric activity along with a chronic activation of the hypothalamic-pituitary-interrenal (HPI) axis, including elevated plasma concentrations of á-MSH. Furthermore, subordinate fish showed an inhibition of aggressive behaviour and darker body coloration, skin darkness being positively correlated with plasma á-MSH. Fish kept on dark background, and thus being darker in body colour, were less aggressive than conspecifics interacting on white background, supporting the hypothesis that skin darkening could signal social submission. The 5-HT_1A-receptor agonist 8-OH-DPAT stimulated HPI axis activity in non-stressed fish, but if administrated to stressed fish it inhibited HPI axis activity, suggesting that 5-HT_1A receptors may act as both post- and pre-synaptic receptors. 8-OH-DPAT also induced skin darkening in both non-stressed and stressed fish. Stimulation of brain dopaminergic activity by L-dopa treatment counteracted the stress-induced inhibition of aggressive behaviour, and stress related effects on brain 5-HT activity and plasma levels of cortisol. In conclusion, social subordination in Arctic charr results in skin darkening and an inhibition of aggressive behaviour. Stress-induced effects, that could be mediated by elevated brain 5-HT activity, and serve as a way of signalling social position and coping with stress.</p>