This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus. Keywords: protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein

Climatic variation and change affect the dynamics of organisms and ecosystem processes. This thesis examines phytoplankton as a target variable to trace climatic impacts on Lake Erken (Sweden) with special emphasis on the spring bloom. A strong correlation between the timing of the spring bloom and the North Atlantic Oscillation (NAO) illustrates the link between atmospheric pressure variations and local biological processes. The predictive power increased by applying a recently established regional Scandinavian Circulation Index (SCI). Changes to an earlier timing of the spring bloom and elevated water temperature were induced by the global warming trend. The climate signal was still persistent in summer manifested by an enhanced summer phytoplankton biomass. Between spring and summer, the phytoplankton was mainly controlled by phosphorus limitation. The application of a new method to measure alkaline phosphatase activity revealed that P-limitation varied between species and among individual cells. Combining the above knowledge and literature data, the impact of the NAO on the timing of life history events, biomass and trophic cascade in aquatic and terrestrial ecosystems was quantitatively tested with a meta-analysis. In all environments, pronounced effects of the NAO were apparent, indicating the generality of climate effects found in different ecosystems. Finally, a regional climate model was applied, forcing a physical lake model from which future lake conditions were simulated. The simulation revealed a one-month shorter ice cover period with two years out of ten being completely ice free. Internal eutrophication is one of the expected consequences. In conclusion, the strong influences of global and regional climate are apparent in local physical, chemical and biological variables and will most probably also in future affect the structure and function of processes in lakes.

Persistent environmental pollutants were examined for their interaction with the glucocorticoid hormone system. The focus was placed on interference with the glucocorticoid synthesis and the glucocorticoid-signalling pathway in various in vitro test systems. Several aryl methyl sulphones competitively inhibited CYP11B1 activity in mouse adrenocortical Y1 cells. The DDT metabolite, 3-methylsulphonyl-2,2’-bis(4-chlorophenyl)-1,1’-dichloroethene (3-MeSO2-DDE) had a higher affinity to the enzyme than the endogenous substrate, 11-deoxycorticosterone. In fact, 3-MeSO2-DDE (Ki 1.6 μM) was almost as potent as the drug metyrapone (Ki 0.8 μM), a well-known inhibitor of the enzyme. 3-MeSO2-DDE inhibited CYP11B1 activity in human adrenocortical H295R carcinoma cells, and at higher concentrations the CYP21 activity. The human H295R cell line seems to be a useful test system for studies of enzyme activities and could be used to screen endocrine disrupting chemicals interfering with the glucocorticoid hormone synthesis. Several chiral PCB methyl sulphones and the fungicide tolylfluanid proved to be antagonists to the glucocorticoid receptor (GR) in rat hepatoma cells and/or Chinese hamster ovary cells stable transformed with a human GR and a responsive reporter vector. The 4-methylsulphonyl-2,3,6,2’,4’,5’-hexachlorobiphenyl (4-MeSO2-CB149) enantiomers had similar antagonistic effect on the GR. Co-exposure of substances led to additive inhibitory effects on glucocorticoid-regulated protein synthesis in rat hepatoma cells. In general, 4-substituted but not 3-substituted methylsulphonyl-PCBs interacted with the glucocorticoid hormone system. In the environment, humans and wildlife are constantly exposed to a wide range of chemicals. Considering the effects of these substances via mechanisms of actions described in this thesis, interference of xenobiotics with the glucocorticoid hormone system deserves further attention. In conclusion, environmental pollutants can interact with the glucocorticoid hormone system in vitro, yet the effects of the tested substances on this hormone system remain to be established in vivo.

Picophytoplankton were inferior competitors for inorganic phosphorus compared to heterotrophic bacteria. This may be due to the source of energy available for the heterotrophs, while cell-size was of minor importance. However, picophytoplankton were superior to large phytoplankton in the competition for nutrients at low concentrations. Biomass of picophytoplankton was low in brownwater lakes and high in clearwater lakes, compared to the biomass of heterotrophic bacteria. The results suggest that picophytoplankton are inferior to heterotrophic bacteria in the competition for inorganic nutrients in brownwater lakes, where the production of heterotrophic bacteria is subsidized by humic dissolved organic carbon (DOC) Relative to large phytoplankton, picophytoplankton were most important in lakes with intermediate water colour, despite the fact that the lowest nutrient concentrations were found in the clearwater lakes. Large phytoplankton in the clearwater lakes may be able to overcome nutrient competition with picophytoplankton by vertical migration. In conclusion, changes in nutrient content, light availability and concentrations of DOC affect the interactions of heterotrophic bacteria, picophytoplankton and large phytoplankton and are therefore important factors for the structure of the food web in the pelagic zones of lakes. Picophytoplankton (planktonic algae and cyanobacteria, < 2 µm) constitute an important component of pelagic food webs. They are linked to larger phytoplankton and heterotrophic bacteria through complex interactions including competition, commensalism and predation. In this thesis, field and laboratory studies on the competitive ability of picophytoplankton are reported.

Understanding the diversity of life is one of the main aims of evolutionary biology, and requires knowledge of the occurrence and causes of adaptive genetic differentiation among geographically distinct populations. Environmental stress caused by acidity may cause strong directional selection in natural populations, but is little explored from an evolutionary perspective. In this thesis, a series of laboratory experiments and field data was used to study evolutionary and ecological responses of amphibians to environmental acidity. Local adaptation to acid stress was studied in the moor frog (Rana arvalis).The results show that acid origin populations have higher acid stress tolerance during the embryonic stages than neutral origin populations, and that acid and neutral origin populations have diverged in embryonic and larval life-histories. The mechanisms underlying adaptive differentiation are partially mediated by maternal effects related to extra-embryonic membranes and egg size. Acid origin females invest in larger eggs and have a stronger egg size-fecundity trade-off than females from neutral areas, likely reflecting adaptive differentiation in maternal investment patterns. Potential carry-over effects of low pH, and the effects of UV-b/pH interaction were investigated in the common frog (R. temporaria). The results suggest that amphibian larvae are able to compensate for the negative effects of acidity experienced early in life, if conditions later turn beneficial. R. temporaria populations differed in their sensitivity to synergistic effects of low pH/UV-B, indicating variation in population responses to environmental stress. In conclusion, these results suggest rapid evolution in response to human induced environmental change, much of which may be mediated via adaptive maternal effects. Acidification may be a powerful selective force shaping life-history evolution.

The great difference in the level of chloroplast variation and its geographic structure among the three main species studied here demonstrates that forest species do not form a homogeneous group. Hazel shows a genetic structure similar to many other thermophilous species and this structure, in combination with fossil evidence, indicates that the post-glacial colonisation of most of Europe originated in a refugium in western France while the Balkan and Italy were colonised from a south-eastern refugium. In sallow and silver birch the chloroplast DNA variation and its structure does not fit with a scenario of glacial restriction to southern refugia and survival at intermediate latitudes is suggested for both species. The chloroplast DNA variation in silver birch suggests the presence of one western and one eastern European post-glacial colonisation route and limited contribution of southern populations in the colonisation of the rest of Europe. Unique haplotypes by the Ural Mountains indicates the possibility of a separate glacial origin of these populations. The study of chloroplast DNA in species closely related to sallow and silver birch indicate that extensive hybridisation and cytoplasmic gene flow occurs within both the Salix and Betula genera in Europe. The nuclear and chloroplast phylogenies of 14 Betula species were not in complete agreement with each other or with the classical division of the Betula genus into subgenera or sections. The phylogenetic structure implies that hybridisation has played a role in the evolution of the Betula genus. This thesis focuses on the chloroplast DNA variation in three forest tree genera: Corylus, Betula and Salix. Chloroplast PCR-RFLP is used to evaluate the post-glacial history of hazel, Corylus avellana, silver birch, Betula pendula and sallow, Salix caprea and to explore the possibility of introgression in the Salix and Betula genera. In addition, the chloroplast matK gene, its flanking regions and the nuclear ADH gene were used to study the phylogenetic relationships within the Betula genus.

Vanguerieae is a tribe in the mainly tropical angiosperm family Rubiaceae. The tribe currently comprises around 600 species in 27 genera, with the major part of the species distributed in Africa south of the Sahara. The phylogeny of the tribe was investigated using both molecular and morphological data. ITS sequences of the nuclear ribosomal DNA are used in all of the included papers, and in three papers also in combination with sequences from the plastid trnT-F region and/or the rpl16 intron. Thirty morphological characters were also investigated and used in combination with molecular data. Several strongly supported groups are found, but eight of the currently recognized genera are also revealed as para- or polyphyletic, i.e., Canthium, Fadogia, Lagynias, Pachystigma, Pyrostria, Rytigynia, Tapiphyllum, and Vangueria. A new classification based on the phylogenetic analyses is suggested and a review of the morphology of Vanguerieae with a focus on taxonomically informative characters is also included. The morphologically similar Ancylanthos, Lagynias, Pachystigma, and Tapiphyllum together with a species of Rytigynia, R. fuscosetulosa, are suggested for transfer to Vangueria. All dioecious species of Vanguerieae are found to form a strongly supported group. A study of the group is performed in which all dioecious genera and also twelve undescribed species from Madagascar are included. It is concluded that the whole group should be included in Pyrostria, which necessitates transfers of Canthium subgenus Bullockia and Scyphochlamys. Canthium subgenus Afrocanthium is found to have a position isolated from the type species of the genus and it is suggested that the subgenus is given generic rank as Afrocanthium. A comparison of nuclear and chloroplast phylogenies shows that several species in a subgroup of the tribe have incongruent positions, and possible explanations for the incongruencies are discussed.

This thesis integrates the fields of sexual selection, parental investment and sex role theory by investigating mutual mate choice and mate competition in the sex role reversed deep snouted pipefish Syngnathus typhle (Pisces: Syngnathidae) through a series of laboratory experiments. In S. typhle, the female transfers her eggs to the male's brood pouch where they are nourished and oxygenated for about a month, when the male gives birth to the independent fry. Mate choice was found to be adaptive. Both sexes benefited from mating with preferred partners in terms of increased offspring viability and got larger, or faster growing, offspring when mating with large fish. Females were also shown to prefer males with thicker brood pouches. Thus, females, the more competitive sex, had multiple preferences. Both male and female choice behaviour was found to be flexible and influenced by available information on partner quality. In addition, males, but not females, copied the mate choice of consexuals. Both sexes were found to take their own quality in relation to surrounding competitors into account when deciding whether to display to potential partners. Male-male competition was found to influence both the mate choice of males and, potentially, overrule the mate choice of females. Males did not compete as intensely as females, nor did they use their sexual ornament in this context as females do. Rather, the ornament was used in interactions with females, and males that displayed more received more eggs. The findings in this thesis emphasise the importance of not viewing mate choice and competition as opposite behaviours, but rather to apply a dynamic approach in mate choice studies, integrating choice and competition in both sex

Krüppel-related zinc finger proteins constitute the largest single class of transcription factors within the human genome. Members of this protein family have the ability to either activate or repress transcription depending on the presence of specific activator or repressor domains within the protein. Approximately one third of the Krüppel-related zinc finger proteins contain an evolutionarily well-conserved repressor domain termed the KRAB domain. This domain acts as a potent repressor of transcription by interacting with the co-repressor protein, TIF1β. TIF1β then, in turn, recruits HP1 proteins, HDACs and probably other proteins involved in gene silencing. In order to identify novel KRAB-containing zinc finger proteins, one mouse monocytic cDNA library and two testis cDNA libraries were screened for novel members of this multigene family. Six novel KRAB-ZNF cDNAs, four mouse and two human, were isolated. The corresponding proteins were all shown to contain N-terminally located KRAB domains as well as varying numbers of C-terminally located zinc finger motifs. An extensive comparative sequence analysis of the KRAB domains of these proteins together with KRAB domains from a large number of previously identified KRAB-ZNF proteins resulted in a clear subdivision into three different subfamilies, A+B, A+b and A. Later, we also isolated a fourth KRAB box, which is present downstream of the KRAB A box in a few proteins of the KRAB A family. This module was named KRAB C. Potential functional differences between these different subfamilies were investigated. In line with previous observations, the KRAB A box was shown to repress transcription, an activity which was enhanced by the presence of the KRAB B box. However, addition of neither the KRAB b box nor the KRAB C box had any effect on repression. Moreover, all KRAB A motifs had the ability to bind TIF1β, and this binding was increased both by the presence of the KRAB B box and by the KRAB C box. The KRAB b box, however, did not seem to contribute to TIF1β-binding. One of the novel human cDNAs, HKr19, was found to be a member of the large ZNF91 family of KRAB zinc finger genes. Interestingly, the expression of HKr19 and a number of other closely related genes were restricted to lymphoid cells, indicating that these genes may be involved in regulating lineage commitment. The effect of HKr19 on cell viability was investigated by transfection into human embryonic kidney cells (HEK 293). The results indicated that HKr19, or its zinc finger domain in isolation, were toxic to these cells when expressed at high levels. The MZF6D protein, on the other hand, showed a testis-specific expression. In situ hybridization analysis located this expression to meiotic germ cells, suggesting a role for this protein in spermatogenesis. Further, the evolutionary perspectives of this large gene family were addressed, and its enormous expansion throughout evolution probably includes numerous duplication events. The results from two extensive sequence analyses give clues to how the repetitive nature of the ZNF motif has given rise to both internal duplications of single motifs as well as duplications of entire genes resulting in gene clusters.

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. 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.