Harmful algae in the planktonic food web of the Baltic Sea

This study deals with algal species occurring commonly in the Baltic Sea: haptophyte Prymnesium parvum, dinoflagellates Dinophysis acuminata, D. norvegica and D. rotundata, and cyanobacterium Nodularia spumigena. The hypotheses are connected to the toxicity of the species, to the factors determining toxicity, to the consequences of toxicity and to the transfer of toxins in the aquatic food web. Since the Baltic Sea is severely eutrophicated, the fast-growing haptophytes have potential in causing toxic blooms. In our studies, the toxicity (as haemolytic activity) of the haptophyte P. parvum was highest under phosphorus-limited conditions, but the cells were toxic also under nitrogen limitation and under nutrient-balanced growth conditions. The cellular nutrient ratios were tightly related to the toxicity. The stoichiometric flexibility for cellular phosphorus quota was higher than for nitrogen, and nitrogen limitation led to decreased biomass. Negative allelopathic effects on another algae (Rhodomonas salina) could be observed already at low P. parvum cell densities, whereas immediate lysis of R. salina cells occurred at P. parvum cell densities corresponding to natural blooms. Release of dissolved organic carbon from the R. salina cells was measured within 30 minutes, and an increase in bacterial number and biomass was measured within 23 h. Because of the allelopathic effect, formation of a P. parvum bloom may accelerate after a critical cell density is reached and the competing species are eliminated. A P. parvum bloom indirectly stimulates bacterial growth, and alters the functioning of the planktonic food web by increasing the carbon transfer through the microbial loop. Our results were the first reports on DSP toxins in Dinophysis cells in the Gulf of Finland and on PTX-2 in the Baltic Sea. Cellular toxin contents in Dinophysis spp. ranged from 0.2 to 149 pg DTX-1 cell-1 and from 1.6 to 19.9 pg PTX-2 cell-1 in the Gulf of Finland. D. norvegica was found mainly around the thermocline (max. 200 cells L-1), whereas D. acuminata was found in the whole mixed layer (max. 7 280 cells L-1). Toxins in the sediment trap corresponded to 1 % of DTX-1 and 0.01 % PTX-2 of the DSP pool in the suspended matter. This indicates that the majority of the DSP toxins does not enter the benthic community, but is either decomposed in the water column, or transferred to higher trophic levels in the planktonic food chain. We found that nodularin, produced by Nodularia spumigena, was transferred to the copepod Eurytemora affinis through three pathways: by grazing on filaments of small Nodularia, directly from the dissolved pool, and through the microbial food web by copepods grazing on ciliates, dinoflagellates and heterotrophic nanoflagellates. The estimated proportion of the microbial food web in nodularin transfer was 22-45 % and 71-76 % in our two experiments, respectively. This highlights the potential role of the microbial food web in the transfer of toxins in the planktonic food web.

Missing-In-Metastasis (MIM) regulates cell morphology by promoting plasma membrane and actin cytoskeleton dynamics

The cells of multicellular organisms have differentiated to carry out specific functions that are often accompanied by distinct cell morphology. The actin cytoskeleton is one of the key regulators of cell shape subsequently controlling multiple cellular events including cell migration, cell division, endo- and exocytosis. A large set of actin regulating proteins has evolved to achieve and tightly coordinate this wide range of functions. Some actin regulator proteins have so-called house keeping roles and are essential for all eukaryotic cells, but some have evolved to meet the requirements of more specialized cell-types found in higher organisms enabling complex functions of differentiated organs, such as liver, kidney and brain. Often processes mediated by the actin cytoskeleton, like formation of cellular protrusions during cell migration, are intimately linked to plasma membrane remodeling. Thus, a close cooperation between these two cellular compartments is necessary, yet not much is known about the underlying molecular mechanisms. This study focused on a vertebrate-specific protein called missing-in-metastasis (MIM), which was originally characterized as a metastasis suppressor of bladder cancer. We demonstrated that MIM regulates the dynamics of actin cytoskeleton via its WH2 domain, and is expressed in a cell-type specific manner. Interestingly, further examination showed that the IM-domain of MIM displays a novel membrane tubulation activity, which induces formation of filopodia in cells. Following studies demonstrated that this membrane deformation activity is crucial for cell protrusions driven by MIM. In mammals, there are five members of IM-domain protein family. Functions and expression patterns of these family members have remained poorly characterized. To understand the physiological functions of MIM, we generated MIM knockout mice. MIM-deficient mice display no apparent developmental defects, but instead suffer from progressive renal disease and increased susceptibility to tumors. This indicates that MIM plays a role in the maintenance of specific physiological functions associated with distinct cell morphologies. Taken together, these studies implicate MIM both in the regulation of the actin cytoskeleton and the plasma membrane. Our results thus suggest that members of MIM/IRSp53 protein family coordinate the actin cytoskeleton:plasma membrane interface to control cell and tissue morphogenesis in multicellular organisms.

Genomic approach to organ-specific growth control

Growth is a fundamental aspect of life cycle of all organisms. Body size varies highly in most animal groups, such as mammals. Moreover, growth of a multicellular organism is not uniform enlargement of size, but different body parts and organs grow to their characteristic sizes at different times. Currently very little is known about the molecular mechanisms governing this organ-specific growth. The genome sequencing projects have provided complete genomic DNA sequences of several species over the past decade. The amount of genomic sequence information, including sequence variants within species, is constantly increasing. Based on the universal genetic code, we can make sense of this sequence information as far as it codes proteins. However, less is known about the molecular mechanisms that control expression of genes, and about the variations in gene expression that underlie many pathological states in humans. This is caused in part by lack of information about the second genetic code that consists of the binding specificities of transcription factors and the combinatorial code by which transcription factor binding sites are assembled to form tissue-specific and/or ligand-regulated enhancer elements. This thesis presents a high-throughput assay for identification of transcription factor binding specificities, which were then used to measure the DNA binding profiles of transcription factors involved in growth control. We developed ‘enhancer element locator’, a computational tool, which can be used to predict functional enhancer elements. A genome-wide prediction of human and mouse enhancer elements generated a large database of enhancer elements. This database can be used to identify target genes of signaling pathways, and to predict activated transcription factors based on changes in gene expression. Predictions validated in transgenic mouse embryos revealed the presence of multiple tissue-specific enhancers in mouse c- and N-Myc genes, which has implications to organ specific growth control and tumor type specificity of oncogenes. Furthermore, we were able to locate a variation in a single nucleotide, which carries a susceptibility to colorectal cancer, to an enhancer element and propose a mechanism by which this SNP might be involved in generation of colorectal cancer.

The Gerbera cDNA Microarray: A Tool for Large-Scale Identification of Genes Involved in Flower Development

During the past ten years, large-scale transcript analysis using microarrays has become a powerful tool to identify and predict functions for new genes. It allows simultaneous monitoring of the expression of thousands of genes and has become a routinely used tool in laboratories worldwide. Microarray analysis will, together with other functional genomics tools, take us closer to understanding the functions of all genes in genomes of living organisms. Flower development is a genetically regulated process which has mostly been studied in the traditional model species Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida. The molecular mechanisms behind flower development in them are partly applicable in other plant systems. However, not all biological phenomena can be approached with just a few model systems. In order to understand and apply the knowledge to ecologically and economically important plants, other species also need to be studied. Sequencing of 17 000 ESTs from nine different cDNA libraries of the ornamental plant Gerbera hybrida made it possible to construct a cDNA microarray with 9000 probes. The probes of the microarray represent all different ESTs in the database. From the gerbera ESTs 20% were unique to gerbera while 373 were specific to the Asteraceae family of flowering plants. Gerbera has composite inflorescences with three different types of flowers that vary from each other morphologically. The marginal ray flowers are large, often pigmented and female, while the central disc flowers are smaller and more radially symmetrical perfect flowers. Intermediate trans flowers are similar to ray flowers but smaller in size. This feature together with the molecular tools applied to gerbera, make gerbera a unique system in comparison to the common model plants with only a single kind of flowers in their inflorescence. In the first part of this thesis, conditions for gerbera microarray analysis were optimised including experimental design, sample preparation and hybridization, as well as data analysis and verification. Moreover, in the first study, the flower and flower organ-specific genes were identified. After the reliability and reproducibility of the method were confirmed, the microarrays were utilized to investigate transcriptional differences between ray and disc flowers. This study revealed novel information about the morphological development as well as the transcriptional regulation of early stages of development in various flower types of gerbera. The most interesting finding was differential expression of MADS-box genes, suggesting the existence of flower type-specific regulatory complexes in the specification of different types of flowers. The gerbera microarray was further used to profile changes in expression during petal development. Gerbera ray flower petals are large, which makes them an ideal model to study organogenesis. Six different stages were compared and specifically analysed. Expression profiles of genes related to cell structure and growth implied that during stage two, cells divide, a process which is marked by expression of histones, cyclins and tubulins. Stage 4 was found to be a transition stage between cell division and expansion and by stage 6 cells had stopped division and instead underwent expansion. Interestingly, at the last analysed stage, stage 9, when cells did not grow any more, the highest number of upregulated genes was detected. The gerbera microarray is a fully-functioning tool for large-scale studies of flower development and correlation with real-time RT-PCR results show that it is also highly sensitive and reliable. Gene expression data presented here will be a source for gene expression mining or marker gene discovery in the future studies that will be performed in the Gerbera Laboratory. The publicly available data will also serve the plant research community world-wide.

GDP-L-fucose: synthesis and role in inflammation

GDP-L-fucose: synthesis and role in inflammation The migration of leukocytes from intravascular locations to extravascular sites is essential to the immune responses. The initial attachment of leukocytes to the endothelium and the rolling step of the leukocyte extravasation cascade are mediated by selectins, a family of cell adhesion molecules on cell surfaces. Selectins are able to recognize glycoproteins and glycolipids containing the tetrasaccharide sialyl Lewis x (sLex, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc). Several glycosyltransferases are involved in the biosynthesis of sLex, fucosyltransferase VII (Fuc-TVII) being the last enzyme to modify the sLex structure. Fuc-TVII transfers L-fucose from GDP-L-fucose to sialylated N-acetyllactosamine. GDP-L-fucose is synthesized in the cytosol via two different metabolic pathways. The major, constitutively active de novo pathway involves conversion of GDP-α-D-mannose to GDP-β-L-fucose. In the alternative salvage pathway, L-fucokinase synthesizes from free fucose L-fucose-1-phosphate, which is further converted to GDP-L-fucose by GDP-L-fucose pyrophosphorylase. GDP-L-fucose is translocated from the cytosol to Golgi for fucosylation via the GDP-fucose transporter. This thesis involved the study of the synthesis of GDP-L-fucose via the salvage pathway: cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. The gene expression levels of these enzymes were found to be relatively high in various tissues; the mRNA levels were highest in brain, ovary and testis. This study also describes molecular cloning of rat fucosyltransferase VII (FUT7) and its expression as a functional enzyme. Gene expression levels of GDP-L-fucose synthesizing enzymes, GDP-fucose transporter and FUT7 were determined in inflamed tissues as well as cancer cells. Our results revealed a clear upregulation of the enzymes involved in the synthesis of GDP-L-fucose via de novo pathway, GDP-fucose transporter and FUT7 in inflamed tissues and in cancer cells. On the contrary, the GDP-L-fucose salvage pathway was found to be irrelevant in inflammation and in tumorigenesis. Furthermore, our results indicated the transcriptional coregulation of Golgi transporters involved in the synthesis of sulfo sLex, i.e. CMP-sialic acid, GDP-fucose and 3 phosphoadenosine 5 -phosphosulfate transporters, in inflammation.

Environmental effects of thermal and radioactive discharges from nuclear power plants in the boreal brackish-water conditions of the northern Baltic Sea

During recent decades, thermal and radioactive discharges from nuclear power plants into the aquatic environment have become the subject of lively debate as an ecological concern. The target of this thesis was to summarize the large quantity of results obtained in extensive monitoring programmes and studies carried out in recipient sea areas off the Finnish nuclear power plants at Loviisa and Olkiluoto during more than four decades. The Loviisa NPP is located on the coast of the Gulf of Finland and Olkiluoto NPP on that of the Bothnian Sea. The state of the Gulf of Finland is clearly more eutrophic; the nutrient concentrations in the surface water are about 1½ 2 times higher at Loviisa than at Olkiluoto, and the total phosphorus concentrations still increased in both areas (even doubled at Loviisa) between the early 1970s and 2000. Thus, it is a challenge to distinguish the local effects of thermal discharges from the general eutrophication process of the Gulf of Finland. The salinity is generally low in the brackish-water conditions of the northern Baltic Sea, being however about 1 higher at Olkiluoto than at Loviisa (the salinity of surface water varying at the latter from near to 0 in early spring to 4 6 in late autumn). Thus, many marine and fresh-water organisms live in the Loviisa area close to their limit of existence, which makes the biota sensitive to any additional stress. The characteristics of the discharge areas of the two sites differ from each other in many respects: the discharge area at Loviisa is a semi-enclosed bay in the inner archipelago, where the exchange of water is limited, while the discharge area at Olkiluoto is more open, and the exchange of water with the open Bothnian Sea is more effective. The effects of the cooling water discharged from the power plants on the temperatures in the sea were most obvious in winter. The formation of a permanent ice cover in the discharge areas has been delayed in early winter, and the break-up of the ice occurs earlier in spring. The prolonging of the growing season and the disturbance of the overwintering time, in conditions where the biota has adjusted to a distinct rest period in winter, have been the most significant biological effects of the thermal pollution. The soft-bottom macrofauna at Loviisa has deteriorated to the point of almost total extinction at many sampling stations during the past 40 years. A similar decline has been reported for the whole eastern Gulf of Finland. However, the local eutrophication process seems to have contributed into the decline of the zoobenthos in the discharge area at Loviisa. Thermal discharges have increased the production of organic matter, which again has led to more organic bottom deposits. These have in turn increased the tendency of the isolated deeps to a depletion of oxygen, and this has further caused strong remobilization of phosphorus from the bottom sediments. Phytoplankton primary production and primary production capacity doubled in the whole area between the late 1960s and the late 1990s, but started to decrease a little at the beginning of this century. The focus of the production shifted from spring to mid- and late summer. The general rise in the level of primary production was mainly due to the increase in nutrient concentrations over the whole Gulf of Finland, but the thermal discharge contributed to a stronger increase of production in the discharge area compared to that in the intake area. The eutrophication of littoral vegetation in the discharge area has been the most obvious, unambiguous and significant biological effect of the heated water. Myriophyllum spicatum, Potamogeton perfoliatus and Potamogeton pectinatus, and vigorous growths of numerous filamentous algae as their epiphytes have strongly increased in the vicinity of the cooling water outlet, where they have formed dense populations in the littoral zone in late summer. However, the strongest increase of phytobenthos has extended only to a distance of about 1 km from the outlet, i.e., the changes in vegetation have been largest in those areas that remain ice-free in winter. Similar trends were also discernible at Olkiluoto, but to a clearly smaller extent, which was due to the definitely weaker level of background eutrophy and nutrient concentrations in the Bothnian Sea, and the differing local hydrographical and biological factors prevailing in the Olkiluoto area. The level of primary production has also increased at Olkiluoto, but has remained at a clearly lower level than at Loviisa. In spite of the analogous changes observed in the macrozoobenthos, the benthic fauna has remained strong and diversified in the Olkiluoto area. Small amounts of local discharge nuclides were regularly detected in environmental samples taken from the discharge areas: tritium in seawater samples, and activation products, such as 60Co, 58Co, 54Mn, 110mAg, 51Cr, in suspended particulate matter, bottom sediments and in several indicator organisms (e.g., periphyton and Fucus vesiculosus) that effectively accumulate radioactive substances from the medium. The tritium discharges and the consequent detection frequency and concentrations of tritium in seawater were higher at Loviisa, but the concentrations of the activation products were higher at Olkiluoto, where traces of local discharge nuclides were also observed over a clearly wider area, due to the better exchange of water than at Loviisa, where local discharge nuclides were only detected outside Hästholmsfjärden Bay quite rarely and in smaller amounts. At the farthest, an insignificant trace amount (0.2 Bq kg-1 d.w.) of 60Co originating from Olkiluoto was detected in Fucus at a distance of 137 km from the power plant. Discharge nuclides from the local nuclear power plants were almost exclusively detected at the lower trophic levels of the ecosystems. Traces of local discharge nuclides were very seldom detected in fish, and even then only in very low quantities. As a consequence of the reduced discharges, the concentrations of local discharge nuclides in the environment have decreased noticeably in recent years at both Loviisa and Olkiluoto. Although the concentrations in environmental samples, and above all, the discharge data, are presented as seemingly large numbers, the radiation doses caused by them to the population and to the biota are very low, practically insignificant. The effects of the thermal discharges have been more significant, at least to the wildlife in the discharge areas of the cooling water, although the area of impact has been relatively small. The results show that the nutrient level and the exchange of water in the discharge area of a nuclear power plant are of crucial importance.

Role of cell cycle regulators in development of the inner ear

The inner ear originates from an ectodermal thickening called the otic placode. The otic placode invaginates and closes to an otic vesicle, the otocyst. The otocyst epithelium undergoes morphogenetic changes and cell differentiation, leading to the formation of the labyrinth-like mature inner ear. Epithelial-mesenchymal interactions control inner ear morphogenesis, but the modes and molecules are largely unresolved. The expressions of negative cell cycle regulators in the epithelium of the early-developing inner ear have also not been elucidated. The mature inner ear comprises the hearing (cochlea) and balance (vestibular) organs that contain the nonsensory and sensory cells. In mammals, the inner ear sensory cells, called hair cells, exit the cell cycle during embryogenesis and are mitotically quiescent during late-embryonic differentiation stages and postnatally. The mechanisms that maintain this hair cell quiescense are largely unresolved. In this work I examined 1) the epithelial-mesenchymal interactions involved in inner ear morphogenesis, 2) expression of negative cell cycle regulators in the epithelium of the early developing inner ear and 3) the molecular mechanisms that maintain the postmitotic state of inner ear sensory cells. We observed that during otocyst stages, epithelial fibroblast growth factor 9 (Fgf9) communicates with the surrounding mesenchyme, where its receptors are expressed. Fgf9 inactivation leads to reduced proliferation of the surrounding vestibular mesenchyme and to the absence of semicircular canals. Semicircular canal development is blocked, since fusion plates do not form. These results show that the mesenchyme directs fusion plate formation and give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of proliferation. We show that the members of the Cip/Kip family of CKIs (p21Cip1, p27Kip1 and p57Kip2) are expressed in the early-developing inner ear. Our expression data suggest that CKIs divide the otic epithelium into proliferative and nonproliferative compartments that may underlie shaping of the otocyst. At later stages, CKIs regulate proliferation of the vestibular appendages, and this may regulate their continual growth. In addition to restricting proliferation, CKIs may play a role in regional differentiation of various epithelial cells. Differentiating and adult inner ear hair cells are postmitotic and do not proliferate in response to serum or mitogenic growth factors. In our study, we show that this is the result of the activity of negative cell cycle regulators. Based on expression profiles, we first focused on the retinoblastoma (Rb) gene, which functions downstream of the CKIs. Analysis of the inner ear phenotype of Rb mutant mice show, that the retinoblastoma protein regulates the postmitotic state of hair cells. Rb inactivation leads to hyperplasia of vestibular and cochlear sensory epithelia that is a result of abnormal cell cycle entry of differentiated hair cells and of delayed cell cycle exit of the hair cell precursor cells. In addition, we show that p21Cip1 and p19Ink4d cooperate in maintaining the postmitotic state of postnatal auditory hair cells. Whereas inactivation of p19Ink4d alone leads to low-level S-phase entry (Chen et al., 2003) and p21Cip1 null mutant mice have a normal inner ear phenotype, codeletion of p19Ink4d and p21Cip1 triggers high-level S-phase entry of auditory hair cells during early postnatal life, which leads to supernumerary hair cells. The ectopic hair cells undergo apoptosis in all of the mutant mice studied, DNA damage being the immediate cause of this death. These findings demonstrate that the maintenance of the postmitotic state of hair cells is regulated by Rb and several CKIs, and that these cell cycle regulators are critical for the lifelong survival of hair cells. These data have implications for the future design of therapies to induce hair cell regrowth.

Coastal environmental gradients – Key to reproduction habitat mapping of freshwater fish in the Baltic Sea

Habitat requirements of fish are most strict during the early life stages, and the quality and quantity of reproduction habitats lays the basis for fish production. A considerable number of fish species in the northern Baltic Sea reproduce in the shallow coastal areas, which are also the most heavily exploited parts of the brackish marine area. However, the coastal fish reproduction habitats in the northern Baltic Sea are poorly known. The studies presented in this thesis focused on the influence of environmental conditions on the distribution of coastal reproduction habitats of freshwater fish. They were conducted in vegetated littoral zone along an exposure and salinity gradient extending from the innermost bays to the outer archipelago on the south-western and southern coasts of Finland, in the northern Baltic Sea. Special emphasis was placed on reed-covered Phragmites australis shores, which form a dominant vegetation type in several coastal archipelago areas. The main aims of this research were to (1) develop and test new survey and mapping methods, (2) investigate the environmental requirements that govern the reproduction of freshwater fish in the coastal area and (3) survey, map and model the distribution of the reproduction habitats of pike (Esox lucius) and roach (Rutilus rutilus). The white plate and scoop method with a standardized sampling time and effort was demonstrated to be a functional method for sampling the early life stages of fish in dense vegetation and shallow water. Reed-covered shores were shown to form especially important reproduction habitats for several freshwater fish species, such as pike, roach, other cyprinids and burbot, in the northern Baltic Sea. The reproduction habitats of pike were limited to sheltered reed- and moss-covered shores of the inner and middle archipelago, where suitable zooplankton prey were available and the influence of the open sea was low. The reproduction habitats of roach were even more limited and roach reproduction was successful only in the very sheltered reed-covered shores of the innermost bay areas, where salinity remained low (< 4‰) during the spawning season due to freshwater inflow. After identifying the critical factors restricting the reproduction of pike and roach, the spatial distribution of their reproduction habitats was successfully mapped and modelled along the environmental gradients using only a few environmental predictor variables. Reproduction habitat maps are a valuable tool promoting the sustainable use and management of exploited coastal areas and helping to maintain the sustainability of fish populations. However, the large environmental gradients and the extensiveness of the archipelago zone in the northern Baltic Sea demand an especially high spatial resolution of the coastal predictor variables. Therefore, the current lack of accurate large-scale, high-resolution spatial data gathered at exactly the right time is a considerable limitation for predictive modelling of shallow coastal waters.

Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.

Effects of oxygen provision on the physiology of baker's yeast Saccharomyces cerevisiae

The availability of oxygen has a major effect on all organisms. The yeast Saccharomyces cerevisiae is able to adapt its metabolism for growth in different conditions of oxygen provision, and to grow even under complete lack of oxygen. Although the physiology of S. cerevisiae has mainly been studied under fully aerobic and anaerobic conditions, less is known of metabolism under oxygen-limited conditions and of the adaptation to changing conditions of oxygen provision. This study compared the physiology of S. cerevisiae in conditions of five levels of oxygen provision (0, 0.5, 1.0, 2.8 and 20.9% O2 in feed gas) by using measurements on metabolite, transcriptome and proteome levels. On the transcriptional level, the main differences were observed between the three level groups, 0, 0.5 2.8 and 20.9% O2 which led to fully fermentative, respiro-fermentative and fully respiratory modes of metabolism, respectively. However, proteome analysis suggested post-transcriptional regulation at the level of 0.5 O2. The analysis of metabolite and transcript levels of central carbon metabolism also suggested post-transcriptional regulation especially in glycolysis. Further, a global upregulation of genes related to respiratory pathways was observed in the oxygen-limited conditions and the same trend was seen in the proteome analysis and in the activities of enzymes of the TCA cycle. The responses of intracellular metabolites related to central carbon metabolism and transcriptional responses to change in oxygen availability were studied. As a response to sudden oxygen depletion, concentrations of the metabolites of central carbon metabolism responded faster than the corresponding levels of gene expression. In general, the genome-wide transcriptional responses to oxygen depletion were highly similar when two different initial conditions of oxygen provision (20.9 and 1.0% O2) were compared. The genes related to growth and cell proliferation were transiently downregulated whereas the genes related to protein degradation and phosphate uptake were transiently upregulated. In the cultures initially receiving 1.0% O2, a transient upregulation of genes related to fatty acid oxidation, peroxisomal biogenesis, response to oxidative stress and pentose phosphate pathway was observed. Additionally, this work analysed the effect of oxygen on transcription of genes belonging to the hexose transporter gene family. Although the specific glucose uptake rate was highest in fully anaerobic conditions, none of the hxt genes showed highest expression in anaerobic conditions. However, the expression of genes encoding the moderately low affinity transporters decreased with the decreasing oxygen level. Thus it was concluded that there is a relative increase in high affinity transport in anaerobic conditions supporting the high uptake rate.

Polymorphic low penetrance genes and breast cancer : The role of genes involved in metabolism of xenobiotics, estrogens and reactive oxygen species

Various endogenous and exogenous factors have been reported to increase the risk of breast cancer. Many of those are related to prolonged lifetime exposure to estrogens. Furthermore, a positive family history of breast cancer and certain benign breast diseases are known to increase the risk of breast cancer. The role of lifestyle factors, such as use of alcohol and smoking has been an area of intensive study. Alcohol has been found to increase the risk of breast cancer, whereas the role of smoking has remained obscure. A multitude of enzymes are involved in the metabolism of estrogens and xenobiotics including the carcinogens found in tobacco smoke. Many of the metabolic enzymes exhibit genetic polymorphisms that can lead to inter-individual differences in their abilities to modify hazardous substrates. Therefore, in presence of a given chemical exposure, one subgroup of women may be more susceptible to breast carcinogenesis, since they carry unfavourable forms of the polymorphic genes involved in the metabolism of the chemical. In this work, polymorphic genes encoding for cytochrome P450 (CYP) 1A1 and 1B1, N-acetyl transferase 2 (NAT2), sulfotransferase 1A1 (SULT1A1), manganese superoxide dismutase (MnSOD) and vitamin D receptor (VDR) were investigated in relation to breast cancer susceptibility in a Finnish population. CYP1A1, CYP1B1 and SULT1A1 are involved in the metabolism of both estrogens and xenobiotics, whereas NAT2 is involved only in the latter. MnSOD is an antioxidant enzyme protecting cells from oxidative damage. VDR, in turn, mediates the effects of the active form of vitamin D (1,25(OH)2D3, calcitriol) on maintenance of calcium homeostasis and it has anti-proliferative effects in many cancer cells. A 1.3-fold (95% CIs 1.01-1.73) increased risk of breast cancer was seen among women who carried the NAT2 slow acetylator genotype and a 1.5-fold (95% CI 1.1-2.0) risk was found in women with a MnSOD variant A allele containing genotypes compared to women with the NAT2 rapid acetylator genotype or to those with the MnSOD VV genotype, respectively. Instead, women with the VDR a allele containing genotypes were found to be at a decreased risk for breast cancer (OR 0.73; 95% CI 0.54-0.98) compared to women with the AA genotype. No significant overall associations were found between SULT1A1 or CYP genotypes and breast cancer risk, whereas a combination of the CYP1B1 432Val allele containing genotypes with the NAT2 slow acetylator genotypes posed a 1.5-fold (95% CI 1.03-2.24) increased risk. Moreover, NAT2 slow acetylator genotype was found to be confined to women with an advanced stage of breast cancer (stages III and IV). Further evidence for the association of xenobiotic metabolising genes with breast cancer risk was found when active smoking was taken into account. Women who smoked less than 10 cigarettes/day and carried at least one CYP1B1 432Val variant allele, were at 3.1-fold (95% CI 1.32-7.12) risk of breast cancer compared to women who smoked the same amount but did not carry the variant allele. Furthermore, the risk was significantly increased with increasing number of the CYP1B1 432Val alleles (p for trend 0.005). In addition, women who smoked less than 5 pack-years and carried the NAT2 slow acetylator genotype were at a 2.6-fold (95% CI 1.01-6.48) increased risk of breast cancer compared to women who smoked the same amount but carried the NAT2 rapid acetylator genotype. Furthermore, the combination of the CYP1B1 432Val allele and the NAT2 slow acetylator genotype increased the risk of breast cancer by 2.5-fold (95% CI 1.11-5.45) among ever smokers. Instead, the MnSOD A allele was found to be a risk factor among postmenopausal long-term smokers (>15 years of smoking) (OR 5.1; 95% CI 1.4-18.4) or among postmenopausal women who had smoked more than 10 cigarettes/day (OR 5.5; 95% CI 1.3-23.4) compared to women who had similar smoking habits but carried the MnSOD V/V genotype. Similarly, within subgroups of postmenopausal women who were using oral contraceptives, hormone replacement therapy or alcohol, women carrying the MnSOD A allele genotypes seemed to be at increased risk of breast cancer compared to women with the MnSOD V/V genotype. A positive family history of breast cancer and high parity were shown to be inversely associated with breast cancer risk among women carrying the VDR ApaI a allele or among premenopausal women carrying the SULT1A1*2 allele, respectively.

Chiral Synthons from Monoterpenes.1 Stereoselective Syntheses of (-)5S, 6S, 9R-6-Isopropyl-9-Methyl-2-Oxaspiro[4.4]Nonan-3-One and (-) 3aR, 6R, 7aR-3a-Methyl-6-Isopropenylhexahydrobenzofuran-2-One

Highly stereoselective syntheses of two C-12 chiral synthons 3 and 9, mentioned in the title, starting from the monoterpenes R-1 imonene and R-carvone, using radical cyc 1 sation as key reaction, are described.

Ecological communities in variable environments : dynamics and diversity under coloured environmental stochasticity

While environmental variation is an ubiquitous phenomenon in the natural world which has for long been appreciated by the scientific community recent changes in global climatic conditions have begun to raise consciousness about the economical, political and sociological ramifications of global climate change. Climate warming has already resulted in documented changes in ecosystem functioning, with direct repercussions on ecosystem services. While predicting the influence of ecosystem changes on vital ecosystem services can be extremely difficult, knowledge of the organisation of ecological interactions within natural communities can help us better understand climate driven changes in ecosystems. The role of environmental variation as an agent mediating population extinctions is likely to become increasingly important in the future. In previous studies population extinction risk in stochastic environmental conditions has been tied to an interaction between population density dependence and the temporal autocorrelation of environmental fluctuations. When populations interact with each other, forming ecological communities, the response of such species assemblages to environmental stochasticity can depend, e.g., on trophic structure in the food web and the similarity in species-specific responses to environmental conditions. The results presented in this thesis indicate that variation in the correlation structure between species-specific environmental responses (environmental correlation) can have important qualitative and quantitative effects on community persistence and biomass stability in autocorrelated (coloured) environments. In addition, reddened environmental stochasticity and ecological drift processes (such as demographic stochasticity and dispersal limitation) have important implications for patterns in species relative abundances and community dynamics over time and space. Our understanding of patterns in biodiversity at local and global scale can be enhanced by considering the relevance of different drift processes for community organisation and dynamics. Although the results laid out in this thesis are based on mathematical simulation models, they can be valuable in planning effective empirical studies as well as in interpreting existing empirical results. Most of the metrics considered here are directly applicable to empirical data.

The ecology of the mountain hare (Lepus timidus L.) in managed boreal forests: habitat associations at different forest scales

Aims of this thesis This study is part of a larger hare project in Finland, which provides answers to basic ecological questions regarding the mountain hare. This study of the ecology of the mountain hare focuses in particular on different levels of managed boreal forest. The feeding habits and intensity of mountain hares in winter are explored, and the connections between mountain hares versus the forest structure are also studied (e.g. habitat use and the importance of different forest layers for hares). The use of the environment by hares at the landscape level was examined (forest patch structures), and the home ranges of mountain hares were studied. Finally, the productivity and survival rate of mountain hare populations were also studied (discussion e.g. predator effects on hare populations). Conclusions Feeding intensity seemed to be highest in the spring-winter, when home ranges were also largest. Favourable food species are covered by snow in winter and the mobility of hares is highest during late winter. A shortage of suitable food species may be problematic for hares, especially during the winter period. In this study mountain hares preferred a dense shrub layer at local level and deciduous and mixed tree forest over coniferous forest at the landscape level. Food and shelter are vital for hares and the preference for particular habitats may also affect the population dynamics of the mountain hare. It would be possible to improve the quality of food and shelter or at least prevent the most negative habitat changes through forest management. At a local level it is also possible to add supplementary food for hares through the winter period. The intensive clearing of young sapling stands and especially the removal of deciduous shrubs and trees reduces the quality of habitats for the mountain hare. Mountain hares primarily live in forest habitat and it is possible that changes in the forest structure play a crucial role in mountain hare habitat preference. Ecological knowledge of the mountain hare is vital to create habitat structure more suitable for the species. More deciduous trees should be saved in managing forests and the mechanical clearing of the shrub layer should be done carefully.

Glutamatergic Modulation of Cue-Induced Drug-Seeking Behavior in the Rat

The characteristics of drug addiction include compulsive drug use despite negative consequences and re-occurring relapses, returns to drug use after a period of abstinence. Therefore, relapse prevention is one of the major challenges for the treatment of drug addiction. There are three main factors capable of inducing craving for drugs and triggering relapse long after cessation of drug use and dissipation of physical withdrawal signs: stress, re-exposure to the drug, and environmental stimuli (cues) that have been previously associated with drug use. The neurotransmitters dopamine and glutamate have been implicated in the modulation of drug-seeking behavior. The aim of this project was to examine the role of glutamatergic neurotransmission in relapse triggered by conditioned drug-associated stimuli. The focus was on clarifying whether relapse to drug seeking can be attenuated by blockade of glutamate receptors. In addition, as the nucleus accumbens has been proposed to participate in the modulation of drug-seeking behavior, the effects of glutamate receptor blockade in this brain structure on cue-induced relapse were investigated. The studies employed animals models in which rats were trained to press a lever in a test cage to obtain alcohol or intravenous cocaine. Drug availability was paired with distinct olfactory, auditory, or visual stimuli. This phase was followed by extinction training, during which lever presses did not result in the presentation of the drug or the drug-associated stimuli. Extinction training led to a gradual decrease in the number of lever presses during test sessions. Relapse was triggered by presenting the rats with the drug-associated stimuli in the absence of alcohol or cocaine. The drug-associated stimuli were alone capable of inducing resumption of lever pressing and maintaining this behavior during repeated testing. The number of lever presses during a session represented the intensity of drug-seeking and relapse behavior. The results suggest that glutamatergic neurotransmission is involved in the modulation of drug-seeking behavior. Both alcohol and cocaine relapse were attenuated by systemic pretreatment with glutamate receptor antagonists. However, differences were found in the ability of ionotropic AMPA/kainate and NMDA receptor antagonists to regulate drug-seeking behavior. The AMPA/kainate antagonists CNQX and NBQX, and L-701,324, an antagonist with affinity for the glycine site of the NMDA receptor, attenuated cue-induced drug seeking, whereas the competitive NMDA antagonist CGP39551 and the NMDA channel blocker MK-801 were without effect. MPEP, an antagonist at metabotropic mGlu5 glutamate receptors, also decreased drug seeking, but its administration was found to lead to conditioned suppression of behavior during subsequent treatment sessions, suggesting that MPEP may have undesirable side effects. The mGluR2/3 agonist LY379268 and the mGluR8 agonist (S)-3,4-DCPG decreased both cue-induced relapse to alcohol drinking and alcohol consumption. Control experiments showed however that administration of the agonists was accompanied by motor suppression limiting their usefulness. Administration of the AMPA/kainate antagonist CNQX, the NMDA antagonist D-AP5, and the mGluR5 antagonist MPEP into the nucleus accumbens resulted also in a decrease in drug-seeking behavior, suggesting that the nucleus accumbens is at least one of the anatomical sites regulating drug seeking and mediating the effects of glutamate receptor antagonists on this behavior.