Lipid-Containing Icosahedral dsDNA Bacteriophages: Entry, Exit and Structure

In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.

Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.

Structural studies on members of the picornavirus superfamily

The pathogenic members of the picornavirus superfamily have adverse effects on humans, their crops and their livestock. As structure is related to function, detailed structural studies on these viruses are important not only for fundamental understanding of the viral life cycle, but also for the rational design of vaccines and inhibitors for disease control. These viruses have positive sense, single-stranded RNA genomes enclosed in a protein capsid. X-ray crystallography and cryo-electron microscopy studies have revealed that the isometric members of this group have icosahedrally-symmetric capsids made up of 60 copies of each of the structural proteins. The members that infect animal cells often employ one or more cellular receptors to facilitate cell entry which in some cases is known to initiate the uncoating sequence of the genome. The nature of the interactions between individual viruses and alternative cellular receptors has rarely been probed. The capsid assembly of the members of the picornavirus superfamily is considered to be cooperative and the interactions of RNA and capsid proteins are thought to play an important role in orchestrating virus assembly. The major aims of this thesis were to solve the structures of blackcurrant reversion virus (BRV), human parechovirus 1 (HPEV1) and coxsackievirus A7 (CAV7), as well as the structure of HPEV1 complexed with two of its cellular receptors using cryo-electron microscopy, three-dimensional image reconstruction and homology modeling. Each of the selected viruses represents a taxonomic group where little or no structural data was previously available. The results enabled the detailed comparison of the new structures to those of known picornaviruses, the identification of surface-exposed epitopes potentially important for host interaction, the mapping of RNA-capsid protein interactions and the elucidation of the basis for the specificity of two different receptor molecules for the same capsid. This work will form the basis for further studies on the influence of RNA on parechovirus assembly as a potential target for drug design.

Increasing Knowledge Focus - a Means for Entrepreneurs to Remain on a Growth Path: Essays on the Role and Nature of Knowledge Management in Finnish SMEs (summary section only)

This study explores the role and nature of knowledge management (KM) in small and medium-sized companies (SMEs). Even though the role of knowledge as a competitive advantage is commonly recognized in the SME sector, almost no attention has been paid to the managing and developing of knowledge in SMEs. This thesis consists of three different sub-studies that were reported in four individual essays. The results of the questionnaire study indicate that nearly all companies that responded to the questionnaire (N = 108) found intangible assets, i.e. knowledge resources to be their main source of competitive advantage. However, only less than a third of the companies actively deal with knowledge management. The results also indicate a significant correlation between activity in knowledge management and sustainable organic growth of the company. The interview study (N = 10) explored the context and motives of the SMEs for managing their intangible assets, and the concrete practices of knowledge management. It turned out that KM facilitated change management, clarification of the vision and new strategy formulation. All the interviewed companies were aiming at improved innovation process, new ways of doing business and attaining an increased “knowledge focus” in their business. Nearly all also aspired to grow significantly. Thus, KM provides a strategy for these SMEs to guarantee their survival and sustainability in the turbulent markets. The action research was a process to assess and develop intangible resources in three companies. The experienced benefits were the clarification of future focus and strategy, creation of a common language to discuss strategic issues within the company, as well as improved balance of different categories of intangible assets. After the process all the case companies had developed in the chosen key areas. Thus, by systematic knowledge management the implementation of new strategic orientation (knowledge focusing) was facilitated. The findings can be summarized in two main points. First, knowledge management seems to serve the purpose of change, renewal and new strategic orientation in the SMEs. It also seems to be closely related to organic growth and innovation. All of these factors can be considered dimensions of entrepreneurship. Second, the conscious development of intangible assets can increase the balance of different categories of intangible assets and the overall knowledge focusing of business. In the case companies, this in turn facilitated the path to the improved overall performance.

Trade Liberalization, Heterogeneous Firms and Industrial Dynamics

This master thesis studies how trade liberalization affects the firm-level productivity and industrial evolution. To do so, I built a dynamic model that considers firm-level productivity as endogenous to investigate the influence of trade on firm’s productivity and the market structure. In the framework, heterogeneous firms in the same industry operate differently in equilibrium. Specifically, firms are ex ante identical but heterogeneity arises as an equilibrium outcome. Under the setting of monopolistic competition, this type of model yields an industry that is represented not by a steady-state outcome, but by an evolution that rely on the decisions made by individual firms. I prove that trade liberalization has a general positive impact on technological adoption rates and hence increases the firm-level productivity. Besides, this endogenous technology adoption model also captures the stylized facts: exporting firms are larger and more productive than their non-exporting counterparts in the same sector. I assume that the number of firms is endogenous, since, according to the empirical literature, the industrial evolution shows considerably different patterns across countries; some industries experience large scale of firms’ exit in the period of contracting market shares, while some industries display relative stable number of firms or gradually increase quantities. The special word “shakeout” is used to describe the dramatic decrease in the number of firms. In order to explain the causes of shakeout, I construct a model where forward-looking firms decide to enter and exit the market on the basis of their state of technology. In equilibrium, firms choose different dates to adopt innovation which generate a gradual diffusion process. It is exactly this gradual diffusion process that generates the rapid, large-scale exit phenomenon. Specifically, it demonstrates that there is a positive feedback between firm’s exit and adoption, the reduction in the number of firms increases the incentives for remaining firms to adopt innovation. Therefore, in the setting of complete information, this model not only generates a shakeout but also captures the stability of an industry. However, the solely national view of industrial evolution neglects the importance of international trade in determining the shape of market structure. In particular, I show that the higher trade barriers lead to more fragile markets, encouraging the over-entry in the initial stage of industry life cycle and raising the probability of a shakeout. Therefore, more liberalized trade generates more stable market structure from both national and international viewpoints. The main references are Ederington and McCalman(2008,2009).

Applications of Treatment Effects Models and Semiparametric Estimation

This thesis is composed of an introductory chapter and four applications each of them constituting an own chapter. The common element underlying each of the chapters is the econometric methodology. The applications rely mostly on the leading econometric techniques related to estimation of causal effects. The first chapter introduces the econometric techniques that are employed in the remaining chapters. Chapter 2 studies the effects of shocking news on student performance. It exploits the fact that the school shooting in Kauhajoki in 2008 coincided with the matriculation examination period of that fall. It shows that the performance of men declined due to the news of the school shooting. For women the similar pattern remains unobserved. Chapter 3 studies the effects of minimum wage on employment by employing the original Card and Krueger (1994; CK) and Neumark and Wascher (2000; NW) data together with the changes-in-changes (CIC) estimator. As the main result it shows that the employment effect of an increase in the minimum wage is positive for small fast-food restaurants and negative for big fast-food restaurants. Therefore, it shows that the controversial positive employment effect reported by CK is overturned for big fast-food restaurants and that the NW data are shown, in contrast to their original results, to provide support for the positive employment effect. Chapter 4 employs the state-specific U.S. data (collected by Cohen and Einav [2003; CE]) on traffic fatalities to re-evaluate the effects of seat belt laws on the traffic fatalities by using the CIC estimator. It confirms the CE results that on the average an implementation of a mandatory seat belt law results in an increase in the seat belt usage rate and a decrease in the total fatality rate. In contrast to CE, it also finds evidence on compensating-behavior theory, which is observed especially in the states by the border of the U.S. Chapter 5 studies the life cycle consumption in Finland, with the special interest laid on the baby boomers and the older households. It shows that the baby boomers smooth their consumption over the life cycle more than other generations. It also shows that the old households smoothed their life cycle consumption more as a result of the recession in the 1990s, compared to young households.

Branding: The Past, Present, and Future: A Study of the Evolution and Future of Branding

Branding, as any other concept, has evolved over time: from the days when sheep of one herd started to be branded to distinguish them from another herd to the current era when everything, from water and flowers to clothes and food, is branded. Throughout these times, there have been numerous theories to describe and understand the underlying nuances. This paper finds the relationships in previous literature and reveals how these theories see branding from various perspectives and how they can be integrated to form a coherent view. It is also discussed how branding and society affect each other. Based on the knowledge of how branding theories have been developed as dependent variables of each other and the society, we are able to form a better understanding of the past, the present, and the future of branding.

Birth of the European individual : Outline of a theory of legal practice

The modern subject is what we can call a self-subjecting individual. This is someone in whose inner reality has been implanted a more permanent governability, a governability that works inside the agent. Michel Foucault s genealogy of the modern subject is the history of its constitution by power practices. By a flight of imagination, suppose that this history is not an evolving social structure or cultural phenomenon, but one of those insects (moth) whose life cycle consists of three stages or moments: crawling larva, encapsulated pupa, and flying adult. Foucault s history of power-practices presents the same kind of miracle of total metamorphosis. The main forces in the general field of power can be apprehended through a generalisation of three rationalities functioning side-by-side in the plurality of different practices of power: domination, normalisation and the law. Domination is a force functioning by the rationality of reason of state: the state s essence is power, power is firm domination over people, and people are the state s resource by which the state s strength is measured. Normalisation is a force that takes hold on people from the inside of society: it imposes society s own reality its empirical verity as a norm on people through silently working jurisdictional operations that exclude pathological individuals too far from the average of the population as a whole. The law is a counterforce to both domination and normalisation. Accounting for elements of legal practice as omnihistorical is not possible without a view of the general field of power. Without this view, and only in terms of the operations and tactical manoeuvres of the practice of law, nothing of the kind can be seen: the only thing that practice manifests is constant change itself. However, the backdrop of law s tacit dimension that is, the power-relations between law, domination and normalisation allows one to see more. In the general field of power, the function of law is exactly to maintain the constant possibility of change. Whereas domination and normalisation would stabilise society, the law makes it move. The European individual has a reality as a problem. What is a problem? A problem is something that allows entry into the field of thought, said Foucault. To be a problem, it is necessary for certain number of factors to have made it uncertain, to have made it lose familiarity, or to have provoked a certain number of difficulties around it . Entering the field of thought through problematisations of the European individual human forms, power and knowledge one is able to glimpse the historical backgrounds of our present being. These were produced, and then again buried, in intersections between practices of power and games of truth. In the problem of the European individual one has suitable circumstances that bring to light forces that have passed through the individual through centuries.

The Transcriptional Regulation of Retrotransposon BARE

The purpose of this research project was to understand the steps of the retrotransposon BARE (BArley REtrotransposon) life cycle, from regulation of transcription to Virus-Like Particle (VLP) formation and ultimate integration back into the genome. Our study concentrates mainly on BARE1 transcriptional regulation because transcription is the crucial first step in the retrotransposon life cycle. The BARE element is a Class I LTR (Long Terminal Repeat) retrotransposon belonging to the Copia superfamily and was originally isolated in our research group. The LTR retrotransposons are transcribed from promoters in the LTRs and encode proteins for packaging of their transcripts, the reverse transcription of the transcripts into cDNA, and integration of the cDNA back into the genome. BARE1 is translated as a single polyprotein and cleaved into the capsid protein (GAG), integrase (IN), and reverse transcriptase-RNaseH (RT-RH) by the integral aspartic proteinase (AP). The BARE retrotransposon family comprises more than 104 copies in the barley (Hordeum vulgare) genome. The element is bound by long terminal repeats (LTRs, 1829 bp) containing promoters required for replication, signals for RNA processing, and motifs necessary for the integration of the cDNA. Members of the BARE1 subfamily are transcribed, translated, and form virus-like particles. Several basic questions concerning transcription are explored in the thesis: BARE1 transcription control, promoter choice in different barley tissues, start and termination sites for BARE transcripts, and BARE1 transcript polyadenylation (I). Polyadenylation is an important step during mRNA maturation, and determines its stability and translatability among other characteristics. Our work has found a novel way used by BARE1 to make extra GAG protein, which is critical for VLP formation. The discovery that BARE1 uses one RNA population for protein synthesis and another RNA population for making cDNA has established the most important step of the BARE1 life cycle (III). The relationship between BARE1 and BARE2 has been investigated. Besides BARE, we have examined the retrotransposon Cassandra (II), which uses a very different transcriptional mechanism and a fully parasitic life cycle. In general, this work is focused on BARE1 promoter activity, transcriptional regulation including differential promoter usage and RNA pools, extra GAG protein production and VLP formation. The results of this study give new insights into transcription regulation of LTR retrotransposons.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

Naudanlihan jalostusketjun hiilijalanjälki Suomessa

Kirjallisuustutkimuksen tavoitteena oli perehtyä kasvihuoneilmiön taustoihin ja kartoittaa aiempia tutkimuksia naudan- ja muiden lihatuotteiden kasvihuonekaasupäästöistä. Lisäksi kirjallisuustutkimuksessa perehdyttiin aiemmissa tutkimuksissa elintarvikkeiden hiilijalanjäljen laskemisessa sovellettuun elinkaarianalyysiin ISO 14040-standardin mukaisesti. Kokeellisen osion tavoitteena oli määrittää naudanlihan hiilijalanjälki Suomessa maatilan portilta kuluttajan ruokapöytään. Tavoitteena oli myös ymmärtää jalostusketjun päästöjen merkitys verrattuna koko naudanlihan tuotantoketjuun ja määrittää jalostusketjun vaiheiden merkitys ketjussa. Työn toiminnallisena yksikkönä toimi kilo naudanlihaa. Työ toteutettiin perehtymällä yksityiskohtaisesti yhteen naudanlihan jalostusketjuun Suomessa. Päästöt laskettiin todellisten yhteistyöyritykseltä saatujen prosessitietojen perusteella. Tiedot kerättiin tiedonkeruulomakkeella vierailemalla yhteistyöyrityksen kahdessa tuotantolaitoksessa ja täydentämällä tietoja haastatteluilla. Naudanlihan jalostusketjun päästöt olivat 1240 g CO2-ekv/lihakilo. Eniten päästöjä tuottivat jalostusvaihe (310 g CO2-ekv/lihakilo), teurastus (280 g CO2-ekv/lihakilo) ja lihatuotteiden kuljetus kuluttajalle (210 g CO2-ekv/lihakilo). Koko naudanlihan tuotantoketjusta jalostusketjun päästöt muodostivat alle 4 %, sillä syntymästä maatilan portille syntyviksi päästöiksi laskettiin kirjallisuuden perusteella yli 30 000 g CO2-ekv/lihakilo. Jatkossa naudanlihan hiilijalanjälkeä voitaisiin pääasiassa pienentää kehittämällä prosessia maatilan portille asti. Tämän työn tulokset olivat hyvin samansuuruiset verrattuna aiempaan tutkimukseen broilerin jalostusketjun päästöistä Suomessa (Katajajuuri ym. 2008). Tämä vastasi ennakko-odotuksia, sillä jalostusketjujen vaiheissa ei ollut merkittäviä eroja. Aiempia tutkimuksia naudanlihan jalostusketjun päästöistä ei ollut saatavilla.

Rapsista tuotetun biodieselin ja vehnästä tuotetun bioetanolin energiavirrat ja ympäristövaikutukset Euroopan unionissa

The aim of this thesis was to study the crops currently used for biofuel production from the following aspects: 1. what should be the average yield/ ha to reach an energy balance at least 0 or positive 2. what are the shares of the primary and secondary energy flows in agriculture, transport, processing and usage, and 3. overall effects of biofuel crop cultivation, transport, processing and usage. This thesis concentrated on oilseed rape biodiesel and wheat bioethanol in the European Union, comparing them with competing biofuels, such as corn and sugarcane-based ethanol, and the second generation biofuels. The study was executed by comparing Life Cycle Assessment-studies from the EU-region and by analyzing them thoroughly from the differences viewpoint. The variables were the following: energy ratio, hectare yield (l/ha), impact on greenhouse gas emissions (particularly CO2), energy consumption in crop growing and processing one hectare of a particular crop to biofuel, distribution of energy in processing and effects of the secondary energy flows, like e.g. wheat straw. Processing was found to be the most energy consuming part in the production of biofuels. So if the raw materials will remain the same, the development will happen in processing. First generation biodiesel requires esterification, which consumes approximately one third of the process energy. Around 75% of the energy consumed in manufacturing the first generation wheat-based ethanol is spent in steam and electricity generation. No breakthroughs are in sight in the agricultural sector to achieve significantly higher energy ratios. It was found out that even in ideal conditions the energy ratio of first generation wheat-based ethanol will remain slightly under 2. For oilseed rape-based biodiesel the energy ratios are better, and energy consumption per hectare is lower compared to wheat-based ethanol. But both of these are lower compared to e.g. sugarcane-based ethanol. Also the hectare yield of wheat-based ethanol is significantly lower. Biofuels are in a key position when considering the future of the world’s transport sector. Uncertainties concerning biofuels are, however, several, like the schedule of large scale introduction to consumer markets, technologies used, raw materials and their availability and - maybe the biggest - the real production capacity in relation to the fuel consumption. First generation biofuels have not been the expected answer to environmental problems. Comparisons made show that sugarcane-based ethanol is the most prominent first generation biofuel at the moment, both from energy and environment point of view. Also palmoil-based biodiesel looks promising, although it involves environmental concerns as well. From this point of view the biofuels in this study - wheat-based ethanol and oilseed rape-based biodiesel - are not very competitive options. On the other hand, crops currently used for fuel production in different countries are selected based on several factors, not only based on thier relative general superiority. It is challenging to make long-term forecasts for the biofuel sector, but it can be said that satisfying the world's current and near future traffic fuel consumption with biofuels can only be regarded impossible. This does not mean that biofuels shoud be rejected and their positive aspects ignored, but maybe this reality helps us to put them in perspective. To achieve true environmental benefits through the usage of biofuels there must first be a significant drop both in traffic volumes and overall fuel consumption. Second generation biofuels are coming, but serious questions about their availability and production capacities remain open. Therefore nothing can be taken for granted in this issue, expect the need for development.

Endemic malaria: an 'indoor' disease in northern Europe

Background: Endemic northern malaria reached 68°N latitude in Europe during the 19th century, where the summer mean temperature only irregularly exceeded 16°C, the lower limit needed for sporogony of Plasmodium vivax. Because of the available historical material and little use of quinine, Finland was suitable for an analysis of endemic malaria and temperature. Methods: Annual malaria death frequencies during 1800–1870 extracted from parish records were analysed against long-term temperature records in Finland, Russia and Sweden. Supporting data from 1750–1799 were used in the interpretation of the results. The life cycle and behaviour of the anopheline mosquitoes were interpreted according to the literature. Results: Malaria frequencies correlated strongly with the mean temperature of June and July of the preceding summer, corresponding to larval development of the vector. Hatching of imagoes peaks in the middle of August, when the temperature most years is too low for the sporogony of Plasmodium. After mating some of the females hibernate in human dwellings. If the female gets gametocytes from infective humans, the development of Plasmodium can only continue indoors, in heated buildings. Conclusion: Northern malaria existed in a cold climate by means of summer dormancy of hypnozoites in humans and indoor transmission of sporozoites throughout the winter by semiactive hibernating mosquitoes. Variable climatic conditions did not affect this relationship. The epidemics, however, were regulated by the population size of the mosquitoes which, in turn, ultimately was controlled by the temperatures of the preceding summer.