<p>It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease.</p><p>The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (<6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma. </p>

<p>Although the practice of releasing fish into the wild is common in the management and conservation of fish populations, the success of release programmes and the potential harmful genetic and ecological effects that may follow are rarely considered. This thesis focuses on genetic and ecological consequences of fish releases, exemplified by supportive breeding of brown trout (<i>Salmo trutta</i>) and translocation of European eel (<i>Anguilla anguilla</i>). Specific questions addressed include: What is the relative performance of hatchery produced fish released to support wild populations, and do released hatchery fish contribute to the natural productivity? What is the variation in reproductive success in the wild, and how does it affect the genetic consequences of a supportive breeding programme? Is there a spatial genetic structure in the European eel that must be considered in the management of this rapidly declining species?</p><p>Experiments conducted under natural and near-natural conditions in the River Dalälven, Sweden, suggest that hatchery produced trout can reproduce in the wild. In fact, when the pronounced variation between individual breeders was accounted for, there were no detectable differences between hatchery produced and wild born trout in reproductive success or offspring survival. These results were supported by molecular genetic data suggesting a pronounced gene flow from hatchery to wild trout in the river. Hatchery reared trout were, however, found to exhibit reduced survival rates immediately following release into the wild, an effect that was most likely due to phenotypic responses to the hatchery environment during ontogeny and a lack of experience of the wild.</p><p>In sharp contrast to recently published studies, the present genetic analyses of European eels sampled across the whole distribution range suggest no spatial genetic structure but a subtle temporal genetic heterogeneity within sampled locations. These results emphasise the need to consider temporal replication when assessing population structure of marine species.</p><p>The results obtained have general implications for the management and conservation of fish populations. First, supportive breeding of threatened salmonid populations might be successful, not only for boosting the census size and thereby reducing the short-term probability of extinction, but also for reducing the risks of inbreeding depression and loss of adaptive potential in future generations. However, the results also highlight the need to restore the natural productivity of a population under supportive breeding to avoid a potential reduction in fitness due to hatchery selection. Further, the lack of a detectable spatial genetic structure in the European eel suggests that the management strategy of translocating juvenile eels from locations were they are overabundant to other suitable freshwater habitats does not necessarily have to include genetic considerations with respect to the geographical origin of the translocated eels.</p>

<p>This thesis examined neurotoxic effects of nicotine exposure during a defined critical period of neonatal brain development in mice.</p><p>In our environment there are numerous hazardous contaminants that an individual can be exposed to during its entire lifetime. In many mammalian species the neonatal period is characterised by a rapid development of the brain. The present studies have identified a defined critical period during the neonatal brain development in mice, where exposure to low doses of nicotine causes permanent disturbances in the cholinergic nicotinic receptors and altered behaviour response to nicotine at adult age. This adult reaction to nicotine, a hypoactive response, was the opposite of that observed in control animals and animals exposed to nicotine before or after this period. Animals showing a hypoactive response to nicotine lacked nicotinic low affinity binding sites in the cerebral cortex. Furthermore, neonatal exposure to nicotine affected learning and memory in adult animals, an effect that was time-dependent. This thesis also showed that neonatal exposure to nicotine increased adult susceptibility to a repeated exposure of nicotine, manifested as an even more pronounced effect in spontaneous behaviour after challenging doses of nicotine. In these animals the nicotinic receptors in the cerebral cortex, assayed by a-bungarotoxin, was decreased. </p><p>Neonatal exposure to nicotine was also shown to increase adult susceptibility to the organophosphate paraoxon, a known cholinergic agent, and to the brominated flame retardant 2,2´,4,4´,5-pentabromodiphenyl ether, a novel environmental agent, at adult age. This was seen at doses that did not affect behaviour in control animals, and was manifested as deranged spontaneous behaviour and reduced habituation, aberrations that also worsened with age. </p><p>The results indicate that differences in adult susceptibility to environmental pollutants are not necessarily an inherited condition. Rather they may well be acquired by low dose exposure to toxic agents during early life.</p>

<p>Since the beginning of mycology, studies of species concept in fungi have been mainly based on morphology, partially due to the history of mycology as part of botany. Current advances in biochemical and molecular research have provided mycologists with powerful tools that can be used for delineation of fungal taxa. Recently, an integrated approach to fungal taxonomy involving both morphological and molecular traits has found a wide application for identification of species, especially in anamorphic (mitosporic) fungi.</p><p>In this thesis, I have tried to use this approach for identification of species units in two rather unrelated groups of organisms. One of the case studies concerned <i>Colletotrichum acutatum</i> – a worldwide economically important plant pathogenic anamorphic fungus, which is exhibiting a high level of variation in both morphological and molecular features. This fungus has been intensively studied during the past decades, and several attempts have been made to find reliable markers to separate it from other closely related species of <i>Colletotrichum</i>. The second case studied in this thesis was <i>Mycelia Sterilia</i> – an artificial group of fungi, which are deficient in production of spores of any kind, therefore lacking the main morphological feature used for assigning them to any certain fungal taxon below class level. Due to this peculiarity, <i>Mycelia Sterilia</i> have usually been neglected, and currently there is no working species concept applicable to these fungi. </p><p>In this work, I have tried to clarify the relationships among <i>C. acutatum</i> and several other anamorphic (<i>C. gloeosporioides</i> and <i>C. fructigenum</i>) and teleomorphic (<i>Glomerella acutata</i>, <i>G. cingulata</i> and <i>G. miyabeana</i>) taxa that are closely related to each other. For this purpose, examination of morphological traits was employed in combination with comparison of DNA sequencing data from three loci and subsequent phylogenetic analysis. As a result, re-description of <i>C. acutatum</i> and separation of (at least) two new species was proposed.</p><p>For studies of <i>Mycelia Sterilia</i>, a large collection of sterile strains was screened in search for biologically interesting organisms. One novel pathogen has been found, and two plant growth promoting strains with antifungal properties were selected. Attempt for tentative identification of those fungi was made based on their morphological, physiological and molecular features. Sequencing of several genes and spacers of the ribosomal DNA array revealed that the plant pathogenic strain is closely related to the teleomorphic basidiomycete genus <i>Campanella</i>, and plant growth-promoting isolates were identified as belonging to the anamorphic ascomycete genus <i>Phoma</i>. However, assigning the sterile strains to any existing species was not possible.</p><p>The main conclusion of the thesis is that species in anamorphic fungi should be defined based on a combination of morphological and molecular methods, both equally important, involving as many aspects of fungal biology as is possible at our current state of knowledge. </p>

<p>A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin <i>O</i>-deethylase (EROD) was developed in rainbow trout (<i>Oncorhynchus mykiss</i>) and applied to Atlantic salmon (<i>Salmo salar</i>), Arctic charr (<i>Salvelinus alpinus</i>), Atlantic cod (<i>Gadus morhua</i>), saithe (<i>Pollachius virens</i>), and spotted wolffish (<i>Anarhichas minor</i>). Exposure to waterborne β-naphthoflavone (βNF; 10^-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10^-9 M) and the textile dye indigo (10^-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity.</p><p>A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne ³H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10^-7 M) and indigo (10^-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10^-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae.</p><p>My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.</p>

<p>Speciation is the process in which reproductive barriers evolve between populations. In this thesis I examine how sexual signals contribute to the maintenance, reinforcement or breakdown of reproductive barriers.</p><p>Male pied flycatchers (<i>Ficedula hypoleuca</i>) and collared flycatchers (<i>F. albicollis</i>) differ in song and plumage traits. However, where the two species coexist, several pied flycatchers sing a song resembling the collared flycatcher (mixed song). Mixed song is not caused by introgression from the collared flycatcher but is due to heterospecific copying. Mixed song provokes aggressive behaviour in collared flycatcher males and leads to heterospecific pairing and maladaptive hybridization. </p><p>The species differences in song were found to be larger in an old than a young hybrid zone. This was due to a reduction in the frequency of mixed song in the pied flycatcher and a divergence in the song of the collared flycatcher. Apparently, mixed song causes maladaptive hybridization, which over time leads to reinforcement of reproductive barriers by a song divergence.</p><p>Previous studies have shown that a character displacement in male plumage traits reinforces species barriers. Hence both plumage and song divergence reduce the incidence of hybridization. The evolution of male plumage traits has been so rapid, or selection has been so strong that rapidly evolving molecular markers are unable to trace it.</p><p>Hybrid females mate with a male of the same species as their father. Previous studies have shown that females use male plumage traits controlled by genes linked to the sex chromosomes (the Z) in species recognition. An association between preference and a sex-linked trait through the paternal line may render reinforcement of reproductive barriers more likely.</p><p>In conclusion, sexual signals are affected by species interactions that cause breakdown or reinforcement of reproductive barriers.</p>

<p>In the freshwater copepod, <i>Macrocyclops albidus,</i> food availability, rearing conditions and tapeworm infection clearly affected various life-history traits and their trade-offs. I found that low food availability clearly constrained resource allocations to several life-history (often phenotypically plastic) traits, whereas high food availability either allowed for adjustments in resource allocation patterns or allowed resources to be allocated to several traits without apparent trade-offs. </p><p>Both male and female copepods allocated resources according to food availability; developing more slowly and achieving smaller adult body size when food was scarce. When food availability was low females were constrained and produced fewer eggs (in total and per clutch), and started reproduction later than females with more food available. Males under low food availability allocated relatively more to spermatophore size (current reproduction) with decreasing body size. In contrast, when food availability was high males allocated resources to body size as well as spermatophore size. Overall, at maturity, copepods of both sexes were more similar in size than in age, suggesting that large body size was more important for fitness than fast development. </p><p>In nature the prevalence of copepods infected with cestode tapeworms was found to be low (0-3%). Female copepods, experimentally infected with the cestode <i>Schistocephalus solidus</i>, showed lower overall fecundity, especially when food availability was low. However, infected females produced a larger proportion of their life-time egg production early in life than non-infected females. This might be an adaptation to reduce future fitness costs of infection. Females grown under bad rearing conditions, but with high food availability, produced their first clutch earlier than females grown under good rearing conditions, indicating an adjustment in timing of reproduction. These findings contribute to our fundamental evolutionary understanding of how environmental conditions interact with life-history traits.</p>

<p>Estimating substitution frequencies at sites that influence (Ka) and do not influence (Ks) the amino acid sequence is important for understanding the dynamics of molecular sequence evolution and the selective pressures that have shaped genetic variation. </p><p>The aim of this work was to gain a deeper understanding of the driving forces of substitution frequency variation in human pathogens. <i>Rickettsia prowazekii</i>, the causative agent of epidemic typhus and <i>Helicobacter pylori</i>, which has been implicated in gastric diseases were used as model systems. A specific focus was on the evolution of orphan genes in <i>Rickettsia</i>. Additionally, adaptive sequence evolution and factors influencing protein evolutionary rates in <i>H. pylori</i> were studied.</p><p>The comparative sequence analyses of orphan genes using Typhus Group (TG) and Spotted Fever Group (SFG) <i>Rickettsia</i>, indicate that orphan genes in the SFG correspond to pseudogenes in the TG and that pseudogenes in the SFG correspond to extensively degraded gene remnants in the TG. The analysis also showed that ancestral gene sequences could be reconstructed from extant gene remnants of closely related species. The studies of split genes in <i>R. conorii</i> indicate that many of the small fragmented ORFs are probably pseudogenes. Analysis of the <i>H. pylori </i>carbamoyl phosphate synthetase provided an opportunity to understand natural selection acting on a protein undergoing adaptive evolution. Factors such as network properties, protein-protein interactions, gene essentiality and chromosomal position on protein evolutionary rates in <i>H. pylori</i> were studied, of which antigenicity and gene location were identified as the strongest factors. </p><p>In conclusion, high Ka/Ks ratios in human pathogens may reflect either adaptive sequence evolution or gene deterioration. Distinguishing between the two is an important task in molecular evolution and also of great relevance for medical microbiology and functional genomics research.</p>

<p>Animals show a great variety in physiological and behavioural responses to stressors. These responses are often bimodally distributed within populations and show consistency on an individual level over time and across situations, which in terrestrial vertebrates have been identified as proactive and reactive stress coping strategies. Proactive animals show lower cortisol responses, higher sympathetic activation and brain serotonergic activity compared to reactive animals. Behaviourally, proactive animals are more aggressive, more active in avoiding stressors, they form routines and show fewer cases of conditioned immobility compared to reactive animals. Our aim has been to reveal if such stress coping strategies exist in fish. Our results show that rainbow trout with high (HR) or low (LR) cortisol responses to stressors differs in sympathetic activation and brain serotonin turnover in the same manner as proactive and reactive mammals. HR fish showed less locomotor activity when reared in large groups (30 individuals) compared to LR fish. When reared in isolation there were no differences between HR and LR fish when exposed to stressors within a familiar environment. The adaption of a proactive coping style among reactive coping individuals when they are challenged within a familiar environment has previously been shown to be distinction between proactive and reactive coping mammals. However, when they were transferred to unfamiliar environments a behavioural difference between the two lines was observed indicating different stress coping strategies akin to those described in mammals. Finally, we observed a consistency over time in the cortisol response of an unselected line of rainbow trout. Fish from this line also demonstrated a correlation between behavioural responses to different stressors. However, there was no apparent connection between these behavioural responses and the cortisol response. Overall, the results of this thesis have strengthened the hypothesis that different stress coping strategies exist in teleost fish.</p>

<p>A central goal of evolutionary biology is to understand the processes responsible for morphological, genetic and behavioural differentiation between sexes and among geographically distinct populations. Perhaps the most significant processes are genetic drift, natural selection, phenotypic plasticity and sexual selection. The main aim of this thesis was to investigate differentiation among individuals and populations of the sex-role reversed pipefish (<i>Syngnathus typhle</i>) and, consequently, determine which processes may be responsible for emerging patterns. This unique species is characterised by males predominately choosing amongst displaying females.</p><p>In this thesis I revealed, on a microgeographic scale, morphological differentiation without genetic divergence among populations. Interestingly, females differed in size whereas the males did not. For females in this sex-role reversed species, the costs of expressing a plastic phenotype may be outweighed by the potential gains from greater survivorship, higher fecundity or increased mating success. Thus, females gain the ability to make themselves as conspicuous and attractive to males as possible in the specific environment they are living. Moreover, behavioural experiments, which focussed on describing “personalities”, reproductive investment strategies, and mate-sampling tactics, also indicated that males as well as females had the behavioural plasticity required to adjust to the environment in which they live. To this end, using video playbacks as experimental stimuli may be especially rewarding in this species.</p><p>Overall, the studies in this thesis acknowledge the ability of species to fine-tune their phenotype to maximise fitness and, therefore, highlight the importance of considering patterns of differentiation in an environment-specific context. </p>