Cereulide producing B. cereus and amylosin producing B. subtilis and B. mojavensis : characterization of strains and toxigenicities

B. cereus is one of the most frequent occurring bacteria in foods . It produces several heat-labile enterotoxins and one stable non-protein toxin, cereulide (emetic), which may be pre-formed in food. Cereulide is a heat stable peptide whose structure and mechanism of action were in the past decade elucidated. Until this work, the detection of cereulide was done by biological assays. With my mentors, I developed the first quantitative chemical assay for cereulide. The assay is based on liquid chromatography (HPLC) combined with ion trap mass spectrometry and the calibration is done with valinomycin and purified cereulide. To detect and quantitate valinomycin and cereulide, their [NH4+] adducts, m/z 1128.9 and m/z 1171 respectively, were used. This was a breakthrough in the cereulide research and became a very powerful tool of investigation. This tool made it possible to prove for the first time that the toxin produced by B. cereus in heat-treated food caused human illness. Until this thesis work (Paper II), cereulide producing B. cereus strains were believed to represent a homogenous group of clonal strains. The cereulide producing strains investigated in those studies originated mostly from food poisoning incidents. We used strains of many origins and analyzed them using a polyphasic approach. We found that the cereulide producing B. cereus strains are genetically and biologically more diverse than assumed in earlier studies. The strains diverge in the adenylate kinase (adk) gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three patterns), tyrosin decomposition, haemolysis and lecithine hydrolysis (two phenotypes). Our study was the first demonstration of diversity within the cereulide producing strains of B. cereus. To manage the risk for cereulide production in food, understanding is needed on factors that may upregulate cereulide production in a given food matrix and the environmental factors affecting it. As a contribution towards this direction, we adjusted the growth environment and measured the cereulide production by strains selected for diversity. The temperature range where cereulide is produced was narrower than that for growth for most of the producer strains. Most cereulide was by most strains produced at room temperature (20 - 23ºC). Exceptions to this were two faecal isolates which produced the same amount of cereulide from 23 ºC up until 39ºC. We also found that at 37º C the choice of growth media for cereulide production differed from that at the room temperature. The food composition and temperature may thus be a key for understanding cereulide production in foods as well as in the gut. We investigated the contents of [K+], [Na+] and amino acids of six growth media. Statistical evaluation indicated a significant positive correlation between the ratio [K+]:[Na+] and the production of cereulide, but only when the concentrations of glycine and [Na+] were constant. Of the amino acids only glycine correlated positively with high cereulide production. Glycine is used worldwide as food additive (E 640), flavor modifier, humectant, acidity regulator, and is permitted in the European Union countries, with no regulatory quantitative limitation, in most types of foods. B. subtilis group members are endospore-forming bacteria ubiquitous in the environment, similar to B. cereus in this respect. Bacillus species other than B. cereus have only sporadically been identified as causative agents of food-borne illnesses. We found (Paper IV) that food-borne isolates of B. subtilis and B. mojavensis produced amylosin. It is possible that amylosin was the agent responsible for the food-borne illness, since no other toxic substance was found in the strains. This is the first report on amylosin production by strains isolated from food. We found that the temperature requirement for amylosin production was higher for the B. subtilis strain F 2564/96, a mesophilic producer, than for B. mojavensis strains eela 2293 and B 31, psychrotolerant producers. We also found that an atmosphere with low oxygen did not prevent the production of amylosin. Ready-to-eat foods packaged in micro-aerophilic atmosphere and/or stored at temperatures above 10 °C, may thus pose a risk when toxigenic strains of B. subtilis or B. mojavensis are present.

Oxidative stability of phytosterols in food models and foods

An important safety aspect to be considered when foods are enriched with phytosterols and phytostanols is the oxidative stability of these lipid compounds, i.e. their resistance to oxidation and thus to the formation of oxidation products. This study concentrated on producing scientific data to support this safety evaluation process. In the absence of an official method for analyzing of phytosterol/stanol oxidation products, we first developed a new gas chromatographic - mass spectrometric (GC-MS) method. We then investigated factors affecting these compounds' oxidative stability in lipid-based food models in order to identify critical conditions under which significant oxidation reactions may occur. Finally, the oxidative stability of phytosterols and stanols in enriched foods during processing and storage was evaluated. Enriched foods covered a range of commercially available phytosterol/stanol ingredients, different heat treatments during food processing, and different multiphase food structures. The GC-MS method was a powerful tool for measuring the oxidative stability. Data obtained in food model studies revealed that the critical factors for the formation and distribution of the main secondary oxidation products were sterol structure, reaction temperature, reaction time, and lipid matrix composition. Under all conditions studied, phytostanols as saturated compounds were more stable than unsaturated phytosterols. In addition, esterification made phytosterols more reactive than free sterols at low temperatures, while at high temperatures the situation was the reverse. Generally, oxidation reactions were more significant at temperatures above 100°C. At lower temperatures, the significance of these reactions increased with increasing reaction time. The effect of lipid matrix composition was dependent on temperature; at temperatures above 140°C, phytosterols were more stable in an unsaturated lipid matrix, whereas below 140°C they were more stable in a saturated lipid matrix. At 140°C, phytosterols oxidized at the same rate in both matrices. Regardless of temperature, phytostanols oxidized more in an unsaturated lipid matrix. Generally, the distribution of oxidation products seemed to be associated with the phase of overall oxidation. 7-ketophytosterols accumulated when oxidation had not yet reached the dynamic state. Once this state was attained, the major products were 5,6-epoxyphytosterols and 7-hydroxyphytosterols. The changes observed in phytostanol oxidation products were not as informative since all stanol oxides quantified represented hydroxyl compounds. The formation of these secondary oxidation products did not account for all of the phytosterol/stanol losses observed during the heating experiments, indicating the presence of dimeric, oligomeric or other oxidation products, especially when free phytosterols and stanols were heated at high temperatures. Commercially available phytosterol/stanol ingredients were stable during such food processes as spray-drying and ultra high temperature (UHT)-type heating and subsequent long-term storage. Pan-frying, however, induced phytosterol oxidation and was classified as a rather deteriorative process. Overall, the findings indicated that although phytosterols and stanols are stable in normal food processing conditions, attention should be paid to their use in frying. Complex interactions between other food constituents also suggested that when new phytosterol-enriched foods are developed their oxidative stability must first be established. The results presented here will assist in choosing safe conditions for phytosterol/stanol enrichment.

Synthesis, Reactivity and Biological Activity of 17β-HSD1 Inhibitors Based on a Thieno[2,3-d]pryrimidin-4(3H)-one Core

Breast cancer is the most common cancer in women in Western countries. In the early stages of development most breast cancers are hormone-dependent, and estrogens, especially estradiol, have a pivotal role in their development and progression. One approach to the treatment of hormone-dependent breast cancers is to block the formation of the active estrogens by inhibiting the action of the steroid metabolising enzymes. 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is a key enzyme in the biosynthesis of estradiol, the most potent female sex hormone. The 17beta-HSD1 enzyme catalyses the final step and converts estrone into the biologically active estradiol. Blocking 17beta-HSD1 activity with a specific enzyme inhibitor could provide a means to reduce circulating and tumour estradiol levels and thus promote tumour regression. In recent years 17beta-HSD1 has been recognised as an important drug target. Some inhibitors of 17beta-HSD1 have been reported, however, there are no inhibitors on the market nor have clinical trials been announced. The majority of known 17beta-HSD1 inhibitors are based on steroidal structures, while relatively little has been reported on non-steroidal inhibitors. As compared with 17beta-HSD1 inhibitors based on steroidal structures, non-steroidal compounds could have advantages of synthetic accessibility, drug-likeness, selectivity and non-estrogenicity. This study describes the synthesis of large group of novel 17beta-HSD1 inhibitors based on a non-steroidal thieno[2,3-d]pyrimidin-4(3H)-one core. An efficient synthesis route was developed for the lead compound and subsequently employed in the synthesis of thieno[2,3-d]pyrimidin-4(3H)-one based molecule library. The biological activities and binding of these inhibitors to 17beta-HSD1 and, finally, the quantitative structure activity relationship (QSAR) model are also reported. In this study, several potent and selective 17beta-HSD1 inhibitors without estrogenic activity were identified. This establishment of a novel class of inhibitors is a progressive achievement in 17beta-HSD1 inhibitor development. Furthermore, the 3D-QSAR model, constructed on the basis of this study, offers a powerful tool for future 17beta-HSD1 inhibitor development. As part of the fundamental science underpinning this research, the chemical reactivity of fused (di)cycloalkeno thieno[2,3-d]pyrimidin-4(3H)-ones with electrophilic reagents, i.e. Vilsmeier reagent and dimethylformamide dimethylacetal, was investigated. These findings resulted in a revision of the reaction mechanism of Vilsmeier haloformylation and further contributed to understanding the chemical reactivity of this compound class. This study revealed that the reactivity is dependent upon a stereoelectronic effect arising from different ring conformations.

Ionic Liquids and Microwaves in Promotion of Organic Synthesis : Friedel-Crafts reaction, deuterolabelling and O-Demethylation

The use of ionic liquids in chemical research has gained considerable interest and activity in recent years. Due to their unique and varied physicochemical properties, in comparison to molecular solvents, the potential applications for ionic liquids are enormous. The use of microwave irradiation, as a powerful dielectric heating technique, in synthetic organic chemistry has been known since 1986. Since then, it has gained significant recognition for its research and application in both academia and industry. The use of either ionic liquids or microwave irradiation in synthetic organic chemistry has been known to afford improved, alternative or complimentary selectivities, in comparison to traditional processes. In this study, the use of ionic liquids as solvents, co-solvents and catalytic media was explored in Friedel-Crafts, deuterolabelling and O-demethylation reactions. Alternative methods for the production of a variety of aromatic ketones using the Friedel-Crafts acylation methodology were investigated using ionic liquid catalyst or ionic liquid acidic additive systems. The disclosed methods, i.e. metal bistriflamides and chloroindate ionic liquids systems, possessed good catalytic activity in the synthesis of typical benzophenones. These catalytic systems were also recyclable. Microwave irradiation was found to be useful in the synthesis of various polyhydroxydeoxybenzoins and arylpropanones as synthetic precursors to naturally occurring or potentially bioactive compounds. Under optimized condition, the reaction occurred in only four minutes using systems such as [bmim][NTf2]/HNTf2 and [bmim][BF4]/BF3·OEt2. Naturally occurring polyphenols, such as isoflavones, can possess various types of biological or pharmacological activity. In particular, some are noted for their beneficial effects on human health. Isotopically labelled analogues of polyphenols are valuable as analytical standards in the quantification of these compounds from biological matrices. A new strategy for deuterolabelling of polyphenols was developed using ionic liquids as co-solvents and 35% DCl/D2O, as a cheap deuterium source, under microwave irradiation. Under these conditions, perdeuterated compounds were achieved in short reaction times, in high isotopic purity and in excellent yields. An O-demethylation reaction was developed, using an ionic liquid reaction medium with BBr3 for the deprotection of a variety methyl protected polyphenolic compounds, such as isoflavons and lignans. This deprotection procedure was found to be very practical as the reaction occurred under mild reaction conditions and in short reaction times. The isolation and purification steps were particularly straightforward and high yielding, in comparison to traditional methods.

Q-evankeliumi ja psykohistoria : Uuden testamentin eksegetiikan väitöskirja

In the course of my research for my thesis The Q Gospel and Psychohistory, I moved on from the accounts of the Cynics ideals to psychohistorical explanations. Studying the texts dealing with the Cynics and the Q Gospel, I was amazed by the fact that these texts actually portrayed people living in greater poverty than they had to. I paid particular attention to the fact that the Q Gospel was born in traumatising, warlike circumstances. Psychiatric traumatology helped me understand the Q Gospel and other ancient documents using historical approaches in a way that would comply with modern behavioural science. Even though I found some answers to the questions I had posed in my research, the main result of my research work is the justification of the question: Is it important to ask whether there is a connection between the ethos expressed by means of the religious language of the Q Gospel and the predominantly war-related life experiences typical to Palestine at the time. As has been convincingly revealed by a number of studies, traumatic events contribute to the development of psychotic experiences. I approached the problematic nature, significance and complexity of the ideal of poverty and this warlike environment by clarifying the history of psychohistorical literary research and the interpretative contexts associated with Sigmund Freud, Jacques Lacan and Melanie Klein. It is justifiable to question abnormal mentality, but there is no reliable return from the abnormal mentality described in any particular text to the only affecting factor. The popular research tendency based on the Oedipus complex is just as controversial as the Oedipus complex itself. The sociological frameworks concerning moral panics and political paranoia of an outer and inner danger fit quite well with the construction of the Q Gospel. Jerrold M. Post, M.D., Professor of Psychiatry, Political Psychology and Interna-tional Affairs at George Washington University, and founder and director of the Center for the Analysis of Personality and Political Behavior for the Central Intelligence Agency, has focused on the role played by charisma in the attracting of followers and detailed the psychological styles of a "charismatic" leader. He wrote the books Political Paranoia and Leaders and Their Followers in a Dangerous World: the Psychology of Political Behavior among others. His psychoanalytic vocabulary was useful for my understanding of the minds and motivations involved in the Q Gospel s formation. The Q sect began to live in a predestined future, with the reality and safety of this world having collapsed in both their experience and their fantasies. The deep and clear-cut divisions into good and evil that are expressed in the Q Gospel reveal the powerful nature of destructive impulses, envy and overwhelming anxiety. Responsible people who influenced the Q Gospel's origination tried to mount an ascetic defense against anxiety, denying their own needs, focusing their efforts on another objective (God s Kingdom) and a regressive, submissive earlier phase of development (a child s carelessness). This spiritual process was primarily an ecclesiastic or group-dynamical tactic to give support to the power of group leaders.

EU Multi-level Governance in the Making - The Community Initiative LEADER+ in Finland and Germany

In order to fully understand the process of European integration it is of paramount importance to consider developments at the sub-national and local level. EU integration scholars shifted their attention to the local level only at the beginning of the 1990s with the concept of multi-level governance (MLG). While MLG is the first concept to scrutinise the position of local levels of public administration and other actors within the EU polity, I perceive it as too optimistic in the degree of influence it ascribes to local levels. Thus, learning from and combining MLG with other concepts, such as structural constructivism, helps to reveal some of the hidden aspects of EU integration and paint a more realistic picture of multi-level interaction. This thesis also answers the call for more case studies in order to conceptualise MLG further. After a critical study of theories and concepts of European integration, above all, MLG, I will analyse sub-national and local government in Finland and Germany. I show how the sub-national level and local governments are embedded in the EU s multi-level structure of governance and how, through EU integration, those levels have been empowered but also how their scope of action has partially decreased. After theoretical and institutional contextualisation, I present the results of my empirical study of the EU s Community Initiative LEADER+. LEADER stands for Liaison Entre Actions de Développement de l'Économie Rurale , and aims at improving the economic conditions in Europe s rural areas. I was interested in how different actors construct and shape EU financed rural development, especially in how local actors organised in so-called local action groups (LAGs) cooperate with other administrative units within the LEADER+ administrative chain. I also examined intra-institutional relations within those groups, in order to find out who are the most influential and powerful actors within them. Empirical data on the Finnish and German LAGs was first gathered through a survey, which was then supplemented and completed by interviewing LAG members, LAG-managers, several civil servants from Finnish and German decision-making and managing authorities and a civil servant from the EU Commission. My main argument is that in both Germany and Finland, the Community Initiative LEADER+ offered a space for multi-level interaction and local-level involvement, a space that on the one hand consists of highly motivated people actively contributing to the improvement of the quality of life and economy in Europe s countryside but which is dependent and also restricted by national administrative practices, implementation approaches and cultures on the other. In Finland, the principle of tri-partition (kolmikantaperiaatte) in organising the executive committees of LAGs is very noticeable. In comparison to Germany, for instance, the representation of public administration in those committees is much more limited due to this principle. Furthermore, the mobilisation of local residents and the bringing together of actors from the local area with different social and institutional backgrounds to become an active part of LEADER+ was more successful in Finland than in Germany. Tri-partition as applied in Finland should serve as a model for similar policies in other EU member states. EU integration changed the formal and informal inter-institutional relations linking the different levels of government. The third sector including non-governmental institutions and interest groups gained access to policy-making processes and increasingly interact with government institutions at all levels of public administration. These developments do not necessarily result in the empowering of the local level.

Essays on Banking Crises

This thesis aims at finding the role of deposit insurance scheme and central bank (CB) in keeping the banking system safe. The thesis also studies the factors associated with long-lasting banking crises. The first essay analyzes the effect of using explicit deposit insurance scheme (EDIS), instead of using implicit deposit insurance scheme (IDIS), on banking crises. The panel data for the period of 1980-2003 includes all countries for which the data on EDIS or IDIS exist. 70% of the countries in the sample are less developed countries (LDCs). About 55% of the countries adopting EDIS also come from LDCs. The major finding is that the using of EDIS increases the crisis probability at a strong significance level. This probability is greater if the EDIS is inefficiently designed allowing higher scope of moral hazard problem. Specifically, the probability is greater if the EDIS provides higher coverage to deposits and if it is less powerful from the legal point of view. This study also finds that the less developed a country is to handle EDIS, the higher the chance of banking crisis. Once the underdevelopment of an economy handling the EDIS is controlled, the EDIS separately is no longer a significant factor of banking crises. The second essay aims at determining whether a country s powerful CB can lessen the instability of the banking sector by minimizing the likelihood of a banking crisis. The data used include indicators of the CB s autonomy for a set of countries over the period of 1980-89. The study finds that in aggregate a more powerful CB lessens the probability of banking crisis. When the CB s authority is disentangled with respect to its responsibilities, the study finds that the longer tenure of CB s chief executive officer and the greater power of CB in assigning interest rate on government loans are necessary for reducing the probability of banking crisis. The study also finds that the probability of crisis reduces more if an autonomous CB can perform its duties in a country with stronger law and order tradition. The costs of long-lasting banking crises are high because both the depositors and the investors lose confidence in the banking system. For a rapid recovery of a crisis, the government very often undertakes one or more crisis resolution policy (CRP) measures. The third essay examines the CRP and other explanatory variables correlated with the durations of banking crises. The major finding is that the CRP measure allowing the regulation forbearance to keep the insolvent banks operative and the public debt relief program are respectively strongly and weakly significant to increase the durations of crises. Some other explanatory variables, which were found by previous studies to be related with the probability of crises to occur, are also correlated with the durations of crises.

Dynamics of inflation expectations in the euro area

The present study examines empirically the inflation dynamics of the euro area. The focus of the analysis is on the role of expectations in the inflation process. In six articles we relax rationality assumption and proxy expectations directly using OECD forecasts or Consensus Economics survey data. In the first four articles we estimate alternative Phillips curve specifications and find evidence that inflation cannot instantaneously adjust to changes in expectations. A possible departure of expectations from rationality seems not to be powerful enough to totally explain the persistence of euro area inflation in the New Keynesian framework. When expectations are measured directly, the purely forward-looking New Keynesian Phillips curve is outperformed by the hybrid Phillips curve with an additional lagged inflation term and the New Classical Phillips curve with a lagged expectations term. The results suggest that the euro area inflation process has become more forward-looking in the recent years of low and stable inflation. Moreover, in low inflation countries, the inflation dynamics have been more forward-looking already since the late 1970s. We find evidence of substantial heterogeneity of inflation dynamics across the euro area countries. Real time data analysis suggests that in the euro area real time information matters most in the expectations term in the Phillips curve and that the balance of expectations formation is more forward- than backward-looking. Vector autoregressive (VAR) models of actual inflation, inflation expectations and the output gap are estimated in the last two articles.The VAR analysis indicates that inflation expectations, which are relatively persistent, have a significant effect on output. However,expectations seem to react to changes in both output and actual inflation, especially in the medium term. Overall, this study suggests that expectations play a central role in inflation dynamics, which should be taken into account in conducting monetary policy.

Living on voles - plastic life of the Ural owl

Individuals face variable environmental conditions during their life. This may be due to migration, dispersion, environmental changes or, for example, annual variation in weather conditions. Genetic adaptation to a novel environment happens through natural selection. Phenotypic plasticity allows, however, a quick individual response to a new environment. Phenotypic plasticity may also be beneficial for individual if the environment is highly variable. For example, eggs are costly to produce. If the food conditions vary significantly between breeding seasons it is useful to be able to adjust the clutch and egg size according to the food abundance. In this thesis I use Ural owl vole system to study phenotypic plasticity and natural selection using a number of reproduction related traits. The Ural owl (Strix uralensis) is a long-lived and sedentary species. The reproduction and survival of the Ural owl, in fact their whole life, is tied to the dramatically fluctuating vole densities. Ural owls do not cause vole cycles but they have to adjust their behaviour to the rather predictable population fluctuations of these small mammals. Earlier work with this system has shown that Ural owl laying date and clutch size are plastic in relation to vole abundance. Further, individual laying date clutch size reaction norms have been shown to vary in the amount of plasticity. My work extends the knowledge of natural selection and phenotypic plasticity in traits related to reproduction. I show that egg size, timing of the onset of incubation and nest defense aggressiveness are plastic traits with fitness consequences for the Ural owl. Although egg size is in general thought to be a fixed characteristic of an individual, this highly heritable trait in the Ural owl is also remarkably plastic in relation to the changes in vole numbers, Ural owls are laying the largest eggs when their prey is most abundant. Timing of the onset of incubation is an individual-specific property and plastic in relation to clutch size. Timing of incubation is an important underlying cause for asynchronous hatching in birds. Asynchronous hatching is beneficial to offspring survival in Ural owl. Hence, timing of the onset of incubation may also be under natural selection. Ural owl females also adjust their nest defense aggressiveness according to the vole dynamics, being most aggressive in years when they produce the largest broods. Individual females show different levels of nest defense aggressiveness. Aggressiveness is positively correlated with the phenotypic plasticity of aggressiveness. As elevated nest defense aggressiveness is selected for, it may promote the plasticity of aggressive nest defense behaviour. All the studied traits are repeatable or heritable on individual level, and their expression is either directly or indirectly sensitive to changes in vole numbers. My work considers a number of important fitness-related traits showing phenotypic plasticity in all of them. Further, in two chapters I show that there is individual variation in the amount of plasticity exhibited. These findings on plasticity in reproduction related traits suggest that variable environments indeed promote plasticity.

Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

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.

Farmland birds and habitat heterogeneity in intensively cultivated boreal agricultural landscapes

The biodiversity of farmland ecosystems has decreased remarkably during the latter half of the 20th century, and this development is due to intensive farming with its various environmental effects. In the countries of the EU the Common Agricultural Policy (CAP) is the main determinant affecting farmland biodiversity, since the agricultural policy defines guidelines of agricultural practices. In addition to policies promoting intensive farming, CAP also includes national agri-environment schemes (AES), in which a part of subsidies paid to farmers is directed to acts that are presumed to promote environmental protection and biodiversity. In order to shape AES into relevant and powerful tools for biodiversity protection, detailed studies on the effects of agriculture on species and species assemblages are needed. In my thesis I investigated the importance of habitat heterogeneity and effects of different habitat and landscape characteristics on farmland bird abundance and diversity in typical cereal cultivation-dominated southern Finnish agricultural environments. The extensive data used were collected by territory mapping. My two main study species were the drastically declined ortolan bunting (Emberiza hortulana) and the phenomenally increased tree sparrow (Passer montanus); in addition I studied assemblages of 20 species breeding in open arable and edge/bush habitats. In light of my results I discuss whether the Finnish AES take into account the habitat needs of farmland birds, and I provide suggestions for improvement of the future AES. My results show that heterogeneity of both uncultivated and cultivated habitats increases abundance and species richness among farmland birds, but in this respect the amount and diversity of uncultivated habitats are essential. Ditches in particular are a keystone structure for farmland birds in boreal landscapes. Ditches lined by trees or bushes increased ortolan bunting abundance. Loss of that kind of ditches (and clearance of forest and bush patches), reduced breeding ortolan buntings, mainly by decreasing availability of song-posts that are important for the breeding groups of the species. Heterogeneity of uncultivated habitats, most importantly open ditches and the habitat patch richness, increased densities and species richnesses of species assemblages of open arable and edge/bush habitats. Human impact (winter-feeding, nest-boxes) affected favourably the tree sparrow s rapid range expansion in southern Finland, but any habitat types had no significant effects. At the moment the Finnish agri-environmental policy does not conserve farmland ditches as a habitat type. Instead, sub-surface drainage is financially promoted. This is a fatal mistake as far as farmland biodiversity is concerned. In addition to the maintenance of ditches, at least the following aspects should be included more than is done previously in the measures of the future AES: 1) promotion of diverse crop rotation (especially by promoting animal husbandry), 2) maintenance of tree and bush vegetation in islets and along ditches, 3) promotion of organic farming.

Short- and long-term consequences of food resources on Ural owl Strix uralensis reproduction

Life-history theory states that although natural selection would favour a maximisation of both reproductive output and life-span, such a combination can not be achieved in any living organism. According to life-history theory the reason for the fact that not all traits can be maximised simultaneously is that different traits compete with each other for resources. These relationships between traits that constrain the simultaneous evolution of two or more traits are called trade-offs. Therefore, during different life-stages an individual needs to optimise its allocation of resources to life-history components such as growth, reproduction and survival. Resource limitation acts on these traits and therefore investment in one trait, e.g. reproduction, reduces the resources available for investment in another trait, e.g. residual reproduction or survival. In this thesis I study how food resources during different stages of the breeding event affect reproductive decisions in the Ural owl (Strix uralensis) and the consequences of these decisions on parents and offspring. The Ural owl is a suitable study species for such studies in natural populations since they are long-lived, site-tenacious, and feed on voles. The vole populations in Fennoscandia fluctuate in three- to four-year cycles, which create a variable food environment for the Ural owls to cope with. The thesis gives new insight in reproductive costs and their consequences in natural animal populations with emphasis on underlying physiological mechanisms. I found that supplementary fed Ural owl parents invest supplemented food resources during breeding in own self-maintenance instead of allocating those resources to offspring growth. This investment in own maintenance instead of improving current reproduction had carry-over effects to the following year in terms of increased reproductive output. Therefore, I found evidence that reduced reproductive costs improves future reproductive performance. Furthermore, I found evidence for the underlying mechanism behind this carry-over effect of supplementary food on fecundity. The supplementary-fed parents reduced their feeding investment in the offspring compared to controls, which enabled the fed female parents to invest the surplus resources in parasite resistance. Fed female parents had lower blood parasite loads than control females and this effect lasted until the following year when also reproductive output was increased. Hence, increased investment in parasite resistance when resources are plentiful has the potential to mediate positive carry-over effects on future reproduction. I further found that this carry-over effect was only present when potentials for future reproduction were good. The thesis also provides new knowledge on resource limitation on maternal effects. I found that increased resources prior to egg laying improve the condition and health of Ural owl females and enable them to allocate more resources to reproduction than control females. These additional resources are not allocated to increase the number of offspring, but instead to improve the quality of each offspring. Fed Ural owl females increased the size of their eggs and allocated more health improving immunological components into the eggs. Furthermore, the increased egg size had long-lasting effects on offspring growth, as offspring from larger eggs were heavier at fledging. Limiting resources can have different short- and long-term consequences on reproductive decisions that affect both offspring number and quality. In long-lived organisms, such as the Ural owl, it appears to be beneficial in terms of fitness to invest in long breeding life-span instead of additional investment in current reproduction. In Ural owls, females can influence the phenotypic quality of the offspring by transferring additional resources to the eggs that can have long-lasting effects on growth.

The mast cell as a regulator of atherosclerotic plaque stability

Atherosclerosis is an inflammatory disease progressing over years via the accumulation of cholesterol in arterial intima with subsequent formation of atherosclerotic plaques. The stability of a plaque is determined by the size of its cholesterol-rich necrotic lipid core and the thickness of the fibrous cap covering it. The strength and thickness of the cap are maintained by smooth muscle cells and the extracellular matrix produced by them. A plaque with a large lipid core and a thin cap is vulnerable to rupture that may lead to acute atherothrombotic events, such as myocardial infarction and stroke. In addition, endothelial erosion, possibly induced by apoptosis of endothelial cells, may lead to such clinical events. One of the major causes of plaque destabilization is inflammation induced by accumulated and modified lipoproteins, and exacerbated by local aberrant shear stress conditions. Macrophages, T-lymphocytes and mast cells infiltrate particularly into the plaque’s shoulder regions prone to atherothrombotic events, and they are present at the actual sites of plaque rupture and erosion. Two major mechanisms of plaque destabilization induced by inflammation are extracellular matrix remodeling and apoptosis. Mast cells are bone marrow-derived inflammatory cells that as progenitors upon chemotactic stimuli infiltrate the target tissues, such as the arterial wall, differentiate in the target tissues and mediate their effects via the release of various mediators, typically in a process called degranulation. The released preformed mast cell granules contain proteases such as tryptase, chymase and cathepsin G bound to heparin and chondroitin sulfate proteoglycans. In addition, various soluble mediators such as histamine and TNF-alpha are released. Mast cells also synthesize many mediators such as cytokines and lipid mediators upon activation. Mast cells are capable of increasing the level of LDL cholesterol in the arterial intima by increasing accumulation and retention of LDL and by decreasing removal of cholesterol by HDL in vitro. In addition, by secreting proinflammatory mediators and proteases, mast cells may induce plaque destabilization by inducing apoptosis of smooth muscle and endothelial cells. Also in vivo data from apoE-/- and ldlr-/- mice suggest a role for mast cells in the progression of atherosclerosis. Furthermore, mast cell-deficient mice have become powerful tools to study the effects of mast cells in vivo. In this study, evidence suggesting a role for mast cells in the regulation of plaque stability is presented. In a mouse model genetically susceptible to atherosclerosis, mast cell deficiency (ldlr-/-/KitW-sh/W-sh mice) was associated with a less atherogenic lipid profile, a decreased level of lipid accumulation in the aortic arterial wall and a decreased level of vascular inflammation as compared to mast-cell competent littermates. In vitro, mast cell chymase-induced smooth muscle cell apoptosis was mediated by inhibition of NF-kappaB activity, followed by downregulation of bcl-2, release of cytochrome c, and activation of caspase-8, -9 and -3. Mast cell-induced endothelial cell apoptosis was mediated by chymase and TNF-alpha, and involved chymase-mediated degradation of fibronectin and vitronectin, and inactivation of FAK- and Akt-mediated survival signaling. Subsequently, mast cells induced inhibition of NF-kappaB activity and activation of caspase-8 and -9. In addition, possible mast cell protease-mediated mechanisms of endothelial erosion may include degradation of fibronectin and VE-cadherin. Thus, the present results suggest a role for mast cells in destabilization of atherosclerotic plaques.

Functional genes and gene array analysis as tools for monitoring hydrocarbon biodegradation

Bioremediation, which is the exploitation of the intrinsic ability of environmental microbes to degrade and remove harmful compounds from nature, is considered to be an environmentally sustainable and cost-effective means for environmental clean-up. However, a comprehensive understanding of the biodegradation potential of microbial communities and their response to decontamination measures is required for the effective management of bioremediation processes. In this thesis, the potential to use hydrocarbon-degradative genes as indicators of aerobic hydrocarbon biodegradation was investigated. Small-scale functional gene macro- and microarrays targeting aliphatic, monoaromatic and low molecular weight polyaromatic hydrocarbon biodegradation were developed in order to simultaneously monitor the biodegradation of mixtures of hydrocarbons. The validity of the array analysis in monitoring hydrocarbon biodegradation was evaluated in microcosm studies and field-scale bioremediation processes by comparing the hybridization signal intensities to hydrocarbon mineralization, real-time polymerase chain reaction (PCR), dot blot hybridization and both chemical and microbiological monitoring data. The results obtained by real-time PCR, dot blot hybridization and gene array analysis were in good agreement with hydrocarbon biodegradation in laboratory-scale microcosms. Mineralization of several hydrocarbons could be monitored simultaneously using gene array analysis. In the field-scale bioremediation processes, the detection and enumeration of hydrocarbon-degradative genes provided important additional information for process optimization and design. In creosote-contaminated groundwater, gene array analysis demonstrated that the aerobic biodegradation potential that was present at the site, but restrained under the oxygen-limited conditions, could be successfully stimulated with aeration and nutrient infiltration. During ex situ bioremediation of diesel oil- and lubrication oil-contaminated soil, the functional gene array analysis revealed inefficient hydrocarbon biodegradation, caused by poor aeration during composting. The functional gene array specifically detected upper and lower biodegradation pathways required for complete mineralization of hydrocarbons. Bacteria representing 1 % of the microbial community could be detected without prior PCR amplification. Molecular biological monitoring methods based on functional genes provide powerful tools for the development of more efficient remediation processes. The parallel detection of several functional genes using functional gene array analysis is an especially promising tool for monitoring the biodegradation of mixtures of hydrocarbons.