The reproductive organs of conifers and angiosperms differ in morphology in several fundamental respects. The conifer Norway spruce (Picea abies) form pollen and seed cones from separate meristems whereas angiosperms bear bipartite flowers with sepals and petals surrounding two inner whorls of stamens and carpels. Despite these differences in morphology this thesis present data to suggest that reproductive development in conifers and angiosperms is regulated by a similar molecular mechanism. This implies an evolutionary conservation of the major mechanism for reproductive development since the origin of seed plants. Flower organ identity in angiosperms is determined by regulatory genes belonging to the MADS-box gene family of transcription factors. This thesis presents the cloning and characterisation of four novel MADS-box genes from Norway spruce. Three of these genes DAL11, DAL12 and DAL13 are most closely related to angiosperm B function genes i.e. genes required for petal and stamen development. DAL11, 12 and 13 all are specifically active in developing pollen cones, with different temporal and spatial expression pattern. Functional analysis in transgenic Arabidopsis and yeast suggest that the reproductive aspect of the B-function is conserved between conifers and angiosperms. The results also suggest that the B-function in conifers is separated into one shoot identity and one organ identity determinant. A fourth gene presented; DAL10, is specifically expressed in vegetative parts of pollen- and seed cones. Phylogenetically DAL10 is not closely related to any of the known angiosperm clades, but rather forms a separate clade with other gymnosperm genes, suggesting a gymnosperm specific function. We suggest that the DAL10 activity reflects a function in the determination of the reproductive shoot.

Prophenoloxidase (proPO) is a key enzyme for generation of melanin and is activated by the proPO activating enzyme (ppA) to its active form, PO. The active ppA was purified and cloned from crayfish hemocytes and it is a typical serine proteinase containing a clip, a proline-rich, and a glycine-rich domain. A recombinant protein containing the clip-domain, with homology to horseshoe crab big defensin and mammalian â-defensin, had antibacterial activity in vitro against gram-positive bacteria. The proPO activating system (proPO system) is triggered by lipopolysaccharides (LPS) or â-1,3-glucans. An LPS and â-1,3-glucan binding protein (LGBP) was characterized from crayfish hemocytes. The results of an LGBP antibody inhibition assay suggest that LGBP is directly involved in the proPO system. The primary structure of a crayfish masquerade-like (mas) protein has homology to serine proteinases except for a substitution within the catalytic triad, which renders it without proteinase activity. The crayfish mas-like protein has also binding activity to various gram-negative bacteria and yeast. When the mas-like protein binds to microorganisms, it is processed by a proteolytic enzyme. The mas-like protein exhibited cell adhesion and opsonic activities suggesting that it plays a role in defense against parasites.

There are 42 members of the homeodomain-leucine zipper (HDZip) family of transcription factors in Arabidopsis thaliana. This thesis focuses on the functional analysis of one member of this family, ATHB16, and on the biochemical properties of HDZip proteins. To assess the function of the ATHB16 gene, the expression of ATHB16 was altered in transgenic Arabidopsis plants by using sense and antisense RNA constructs under the control of the 35S promoter. The reciprocal phenotypic effects associated with elevated and reduced levels of ATHB16 expression suggested that, in wild-type plants, ATHB16 acts as a mediator of blue and red light effects on the regulation of plant growth and the timing of the floral transition. In wild-type Arabidopsis, expression of ATHB16 is high in leaves, intermediate in adult roots and inflorescences, and low in stems and siliques. The expression of ATHB16 in the root is markedly increased in response to exogenous abscisic acid (ABA) treatment, but is reduced in the ABA response mutants abil and abi2, suggesting that ATHB16 may be involved in ABA signal transduction. This hypothesis was corroborated by observations of alterations in sensitivity to ABA inhibition of root growth in seedlings of a T-DNA insertion mutant of ATHB16 and of transgenic plants with elevated ATHB16 levels. HDZip proteins bind DNA as dimers. DNA-binding studies showed that different HDZip proteins interact with very similar target sequences in vitro and that they selectively form heterodimers with each other. For example, it was demonstrated that ATHB16 can heterodimerize with ATHB6 and ATHB7 in yeast and with ATHB5 in vitro, suggesting that ATHB16 may interact with other HDZip proteins in Arabidopsis. This interaction may have functional significance, since it may provide a mechanism for the plant to integrate different input signals, like light of different spectral qualities and water availability in the regulation of its growth.

This thesis focuses on reproduction in a simultaneous hermaphrodite with internal fertilization; the nudibranch Aeolidiella glauca. Unlike most other nudibranchs, where copulation is the rule, A. glauca was found to transfer sperm via external spermatophores that were attached to the partner's back. Despite elaborate courtship the actual spermatophore transfer, which always involved two animals only, was of short duration. In most matings (88%) spermatophores were reciprocally exchanged. A. glauca was further found to be very promiscuous. During mating and sperm transfer the receiver exerts considerable control over sperm, and manipulative behaviours designed to increase the donor's reproductive success are thus likely to have evolved. An example of such manipulative behaviour may be A. glauca's<b> </b>unique spermatophore avoidance behaviour. I found that slugs carrying a sign of previous mating activity, i.e. a spermatophore, were discriminated against in a situation where mate choice was possible. The presence of spermatophores was further found to reduce slugs' ability to interrupt matings, and displace other slugs. Body size, however, had no direct effect on displacement in A. glauca as small slugs interrupted matings as successfully as large ones. Furthermore, pair formation and mating were found to be random with respect to size. This was true also for pairs formed in the field. In addition to mate choice hermaphrodites may increase their reproductive success by differential sex allocation. I tested whether differing mate encounter rates had any effects on allocation to male and female function in A. glauca. Slugs with more mating opportunities mated more, and had higher proportional spermatophore production that others. As predicted they also laid significantly fewer eggs than slugs presented with partners less often.

Semi-analytical models for remote sensing of water quality parameters need to be parameterized with specific inherent optical properties. In this thesis, data on specific inherent optical properties of Swedish lakes and coastal waters is presented. Also, the problems of measuring in situ spectral backscattering are addressed. It is shown how measured specific inherent optical properties are used to parameterize semi-analytical bio-optical models. The models are then used to produce large synthetic data sets based on the distribution of water quality parameters, and from these data sets, band ratio or single band ratio algorithms for remote estimation of water quality parameters are constructed. A similar model was also used to calculate under water PAR from measured water quality parameters. The specific inherent optical properties of Swedish lakes and coastal waters are very similar to earlier reported data from the oceanic environment. However, different relations of the water quality parameters will affect the inherent optical properties absorption and backscattering. The absorption spectra are dominated by yellow substance with terrestrial origin. Phytoplankton absorption is low, and account in general only for about 10 % of the total absorption in regions where phytoplankton pigments are active. The spectral backscattering is dominated by suspended particulate inorganic matter. Phytoplankton backscattering is almost negligible, except in cases where the phytoplankton community is dominated by highly scattering cyanobacteria. Experiences from remote sensing campaigns and modeling shows that remote chlorophyll estimation is most effective at longer wavelengths, where the absorption of yellow substance is low. However, modeling also predicts that large uncertainties have to be expected in the estimation of chlorophyll, both from variation in the specific phytoplankton absorption and from influences of other optically active water quality parameters.

For a complete picture of the lake ecosystem, it is necessary to understand the mechanisms regulating biomass and nutrient status (nutrient limitation, optimal supply, or surplus) of benthic algae, which are important primary producers and a food resource for grazers. This thesis gives an overview of the natural variation of benthic algae at different scales of space and time and on different substrates, and unravels some of the underlying factors. Algal nutrient status was assessed using the C:N:P (carbon:nitrogen:phosphorus) ratio of the entire natural benthic community. A review, observations, and experiments confirmed that a C:N:P ratio of about 158:18:1 (molar basis) represented an optimal nutrient supply, and that substantially higher C:N, N:P, or C:P ratios reflected algal growth limitation caused by an N or P nutrient deficiency. Horizontal variation of benthic algal biomass and nutrient status was patchy, of similar amount for all investigated distances, substrates, and lakes, and constituted a dominant proportion of the total variation. For example, patches of nutrient limited algae were found within only 10 m distance from patches with a nutrient surplus. Thus, horizontal variation should not be neglected when sampling benthic algae in lakes. Field observations suggested an impact of wind, nutrients, and grazers on the horizontal variation. Light and nutrients might have caused the observed vertical and temporal variation. Field experiments confirmed a simultaneous control of benthic algal biomass by nutrients and grazing, mediated by light and temperature. Grazing effects were larger than nutrient effects, but the comparison of natural communities in lakes of different trophy suggested that benthic algal biomass was controlled by nutrients in the long run. An important nutrient supply was animal excretions, causing a low C:N:P ratio of epizoon on zebra mussels, and algal communities associated with macrograzers. A field experiment revealed that 15N circulated one week longer in epizoon associated with a sessile caddisfly than in surrounding epilithon. In conclusion, the regulation of benthic algal biomass and nutrient status in lakes is complex, and benthic animals should be looked at not only as grazers, but also as a nutrient source.

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 Lucilia sericata. Third, the effect of population size on morphological and genetic variation is studied using wings and microsatellites from wild populations of L. illustris as well as experimentally bottlenecked populations of L. sericata. 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 L. sericata. 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 Lucilia flies. Bottlenecks had unpredictable effects on wing morphology as well as on genetic variation and fitness in a laboratory stock of L. sericata. 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.

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. 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. Studies of unstable and young populations of cyclically parthenogenetic Daphnia pulex 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.

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 Igf2-H19 loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the H19 is silenced when paternally inherited, the Igf2 is silenced when maternally inherited. The differentially methylated 5'-flank of H19 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. The H19 ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the H19 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 H19 ICR and H19 promoter. Results suggest that the H19 ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal cis-regulatory elements to avoid adverse effects on neighbouring promoters. Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived H19 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 H19 ICR. 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 IGF2 target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal IGF2 and paternal H19 allele, respectively. Results suggested that imprinted genes are not normally exposed to strong cis-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions.

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. The imprinted H19 and Igf2 genes are considered paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of sequence located upstream of H19, 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. 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 in vitro episomal system and in an in vivo mouse model system by mutating the CTCF target sites at the H19 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 Igf2 allele. 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 H19 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. 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 IGF2. Investigations also identified a novel mechanism, which neutralised the chromatin insulator function of the H19 ICR without affecting its chromatin conformation. This principle might also help in explaining the loss of IGF2 imprinting observed in some instances. 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 H19 and Igf2. The studies also confirm the role of CTCF in the maintenance of the methylation profile of the region. Investigations into the loss of IGF2 imprinting in human cancer indicate the involvement of other novel mechanisms besides CTCF in the regulation of insulator function.