Potato virus A as a heterologous protein expression tool in plants

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä, joka pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena kaikkia proteiineja tuotetaan kasvisoluissa samansuuruinen määrä. Lisäksi, viruksen proteiinikuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman että viruksen tartutuskyky merkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa. Tämän työn tarkoituksena oli muuntaa PVA:n genomia siten, että virus soveltuisi yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden välistä kohtaa ja ihmisestä peräisi olevaa geeniä, joka tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi). Sen aktiivisuuden rajoittaminen auttaa Parkinsonintaudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. PVA:n P1-proteiinia koodaavalta alueelta oli paikannettu kohta, johon ehkä voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Samaan PVA-genomiin siirrettiin kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta: GFP-geeni P1:n sisälle, merivuokon lusiferaasigeeni P1/HCpro-kohtaan ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiini-kohtaan. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen n. 15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia esiintyivät lehdissä aktiivisina.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

The Impact of Emissions Trading on the Price of Electricity in Nord Pool : Market Power and Price Determination in the Nordic Electricity Market

The objective of this thesis is to find out how dominant firms in a liberalised electricity market will react when they face an increase in the level of costs due to emissions trading, and how this will effect the price of electricity. The Nordic electricity market is chosen as the setting in which to examine the question, since recent studies on the subject suggest that interaction between electricity markets and emissions trading is very much dependent on conditions specific to each market area. There is reason to believe that imperfect competition prevails in the Nordic market, thus the issue is approached through the theory of oligopolistic competition. The generation capacity available at the market, marginal cost of electricity production and seasonal levels of demand form the data based on which the dominant firms are modelled using the Cournot model of competition. The calculations are made for two levels of demand, high and low, and with several values of demand elasticity. The producers are first modelled under no carbon costs and then by adding the cost of carbon dioxide at 20€/t to those technologies subject to carbon regulation. In all cases the situation under perfect competition is determined as a comparison point for the results of the Cournot game. The results imply that the potential for market power does exist on the Nordic market, but the possibility for exercising market power depends on the demand level. In season of high demand the dominant firms may raise the price significantly above competitive levels, and the situation is aggravated when the cost of carbon dioixide is accounted for. Under low demand leves there is no difference between perfect and imperfect competition. The results are highly dependent on the price elasticity of demand.

Middleware for Mobile Sensing Applications in Urban Environments

Sensor networks represent an attractive tool to observe the physical world. Networks of tiny sensors can be used to detect a fire in a forest, to monitor the level of pollution in a river, or to check on the structural integrity of a bridge. Application-specific deployments of static-sensor networks have been widely investigated. Commonly, these networks involve a centralized data-collection point and no sharing of data outside the organization that owns it. Although this approach can accommodate many application scenarios, it significantly deviates from the pervasive computing vision of ubiquitous sensing where user applications seamlessly access anytime, anywhere data produced by sensors embedded in the surroundings. With the ubiquity and ever-increasing capabilities of mobile devices, urban environments can help give substance to the ubiquitous sensing vision through Urbanets, spontaneously created urban networks. Urbanets consist of mobile multi-sensor devices, such as smart phones and vehicular systems, public sensor networks deployed by municipalities, and individual sensors incorporated in buildings, roads, or daily artifacts. My thesis is that "multi-sensor mobile devices can be successfully programmed to become the underpinning elements of an open, infrastructure-less, distributed sensing platform that can bring sensor data out of their traditional close-loop networks into everyday urban applications". Urbanets can support a variety of services ranging from emergency and surveillance to tourist guidance and entertainment. For instance, cars can be used to provide traffic information services to alert drivers to upcoming traffic jams, and phones to provide shopping recommender services to inform users of special offers at the mall. Urbanets cannot be programmed using traditional distributed computing models, which assume underlying networks with functionally homogeneous nodes, stable configurations, and known delays. Conversely, Urbanets have functionally heterogeneous nodes, volatile configurations, and unknown delays. Instead, solutions developed for sensor networks and mobile ad hoc networks can be leveraged to provide novel architectures that address Urbanet-specific requirements, while providing useful abstractions that hide the network complexity from the programmer. This dissertation presents two middleware architectures that can support mobile sensing applications in Urbanets. Contory offers a declarative programming model that views Urbanets as a distributed sensor database and exposes an SQL-like interface to developers. Context-aware Migratory Services provides a client-server paradigm, where services are capable of migrating to different nodes in the network in order to maintain a continuous and semantically correct interaction with clients. Compared to previous approaches to supporting mobile sensing urban applications, our architectures are entirely distributed and do not assume constant availability of Internet connectivity. In addition, they allow on-demand collection of sensor data with the accuracy and at the frequency required by every application. These architectures have been implemented in Java and tested on smart phones. They have proved successful in supporting several prototype applications and experimental results obtained in ad hoc networks of phones have demonstrated their feasibility with reasonable performance in terms of latency, memory, and energy consumption.

Power Aware Real Time Scheduling in Constrained Devices

Real-time scheduling algorithms, such as Rate Monotonic and Earliest Deadline First, guarantee that calculations are performed within a pre-defined time. As many real-time systems operate on limited battery power, these algorithms have been enhanced with power-aware properties. In this thesis, 13 power-aware real-time scheduling algorithms for processor, device and system-level use are explored.

Power and Authority. Birgitta of Sweden and her Revelations

In the High Middle Ages female saints were customarily noble virgins. Thus, as a wife and a mother of eight children, the Swedish noble lady Birgitta (1302/3 1373) was an atypical candidate for sanctity. However, in 1391 she was canonized only 18 years after her death and became a role model for many late medieval women, who were mothers and widows. The dissertation Power and Authority Birgitta of Sweden and Her Revelations investigates how Birgitta went about establishing her power and authority during the first ten years of her career as a living saint, in 1340 1349. It is written from the perspectives of gender, authority, and power. The sources consist of approximately seven hundred revelations, hagiographical texts and other medieval documents. This work concentrates on the interaction between Birgitta and her audience. During her lifetime Birgitta was already regarded as a holy woman, as a living saint. A living saint could be given no formal papal or other recognition, for one could never be certain about his or her future activities. Thus, the living saint needed an audience for whom to perform signs of sanctity. In this study particular attention is paid to situations within which the power relations between the living saint and her audience can be traced and are open to critical analysis. Situations of conflict that arose in Birgitta s life are especially fruitful for this purpose. During the Middle Ages, institutional power and authority were exclusively in the hands of secular male leaders and churchmen. In this work it is argued, however, that Birgitta used different kinds of power than men. It is evident that she exercized influence on lay people as well as on secular and clerical authorities. The second, third, and fourth chapter of this study examine the beginning of Birgitta s career as a visionary, what factors and influences lay behind it, and what kind of roles they played in establishing her religious authority. The fifth, sixth, and seventh chapter concentrate on Birgitta s exercising of power in specific situations during her time in Sweden until she left on a pilgrimage to Rome in 1349. The central question is how she exercised power with different people. As a result, this book will offer a narrative of Birgitta s social interactions in Sweden seen from the perspectives of power and authority. Along with the concept of power, authority is a key issue. By definition, one who has power also has authority but a person who does not have official power can, nevertheless, have authority. Authority in action is defined here as meaning that a person was listened to. Birgitta acted both in situations of open conflict and where no conflict was evident. Her strategies included, for example, inducement, encouragement and flattery. In order to make people do as she felt was right she also threatened them openly with divine wrath. Sometimes she even used both positive persuasion and threats. Birgitta s power seems very similar to that of priests and ascetics. Common to all of them was that their power demanded interaction with other people and audiences. Because Birgitta did not have power and authority ex officio she had to persuade people to believe in her powers. She did this because she was convinced of her mission and sought to make people change their lives. In so doing, she moved from the domestic field to the public fields of religion and politics.

'A responsible nuclear weapons power' : Nuclear Weapons and Indian Foreign Policy

This thesis examines the interrelationship and dynamics between the Indian United Progressive Alliance government’s foreign policy and its nuclear weapons policy. The purpose of the study is to situate nuclear policy within a foreign policy framework, and the fundamental research problem is thus how does the Indian nuclear policy reflect and respond to the Indian foreign policy? The study examines the intentions in the Indian foreign and nuclear policies, and asks whether these intentions are commensurable or incommensurable. Moreover, the thesis asks whether the UPA government differs from its predecessors, most notably the Bharatiya Janata Party-led National Democratic Alliance government in its foreign and nuclear policies. Answers to these questions are based on the interpretation of political texts and speeches as suggested by Quentin Skinner’s notion of meaning3, what does a writer or speaker mean by what he or she says in a given text, and by J.L. Austin’s speech act theory. This linguistic perspective and the approach of intertextualizing, place the political acts within their contingent intellectual and political contexts. The notion of strategic culture is therefore introduced to provide context for these juxtapositions. The thesis firstly analyses the societal, historical and intellectual context of India’s foreign and nuclear policy. Following from this analysis the thesis then examines the foreign and nuclear policies of Prime Minister Manmo-han Singh’s UPA government. This analysis focuses on the texts, speeches and statements of Indian authorities between 2004 and 2008. This study forwards the following claims: firstly, the UPA Government conducts a foreign policy that is mainly and explicitly inclusive, open and enhancing, and it conducts a nuclear policy that is mainly and implicitly excluding, closed and protective. Secondly, despite the fact that the notion of military security is widely appreciated and does not, as such, necessarily collide with foreign policy, the UPA Government conducts a nuclear policy that is incommensurable with its foreign policy. Thirdly, the UPA Gov-ernment foreign and nuclear policies are, nevertheless, commensurable re-garding their internal intentions. Finally, the UPA Government is conduct-ing a nuclear policy that is gradually leading India towards having a triad of nuclear weapons with various platforms and device designs and a function-ing and robust command and control system encompassing political and military planning, decision-making and execution. Regarding the question of the possible differences between the UPA and NDA governments this thesis claims that, despite their different ideological roots and orientations in domestic affairs, the Indian National Congress Party conducts, perhaps surprisingly, quite a similar foreign and nuclear policy to the Bharatiya Janata Party.

Ministers as strategic political leaders? : Strategic political steering after NPM reforms in Finland

This study explores strategic political steering after the New Public Management (NPM) reforms, with emphasis on the new role assigned to Government ministers in Finland. In the NPM model, politicians concentrate on broad, principal issues, while agencies have discretion within the limits set by politicians. In Finland, strategic steering was introduced with Management by Results (MBR), but the actual tools for strategic political steering have been the Government Programme, the Government Strategy Portfolio (GSP) and Frame Budgeting. This study addresses these tools as means of strategic steering conducted by the Cabinet and individual ministers within their respective ministries. The time frame of the study includes the two Lipponen Cabinets between 1995 and 2003. Interviews with fourteen ministers as well as with fourteen top officials were conducted. In addition, administrative reform documents and documents related to strategic steering tools were analysed. The empirical conclusions of the study can be summarised as follows: There were few signs of strategic political steering in the Lipponen Cabinets. Although the Government Programmes of both Cabinets introduced strategic thinking, the strategic guidelines set forth at the beginning of the Programme were not linked to the GSP or to Frame Budgeting. The GSP could be characterised as the collected strategic agendas of each ministry, while there was neither the will nor the courage among Cabinet members to prioritise the projects and to make selections. The Cabinet used Frame Budgeting mainly in the sense of spending limits, not in making strategic allocation decisions. As for the GSP at the departmental level, projects were suggested by top officials, and ministers only approved the suggested list. Frame Budgeting at the departmental level proved to be the most interesting strategic steering tool from ministers viewpoint: they actively participated in defining which issues would need extra financing. Because the chances for extra financing were minimal, ministers had an effect only on a marginal share of the budget. At the departmental level, the study shows that strategic plans were considered the domain of officials. As for strategies concerning specific substances, there was variation in the interest shown by the ministers. A few ministers emphasised the importance of strategic work and led strategy processes. In most cases, however, officials led the process while ministers offered comments on the drafts of strategy documents. The results of this study together with experiences reported in other countries and local politics show that political decision-makers have difficulty operating at the strategic level. The conclusion is that politicians do not have sufficient incentive to perform the strategic role implied by the NPM type of reforms. Overall, the empirical results of the study indicate the power of politics over management reforms.

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.

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.