Development of regulatory T cells in the human thymus

The role of the immune system is to protect an organism against pathogens while maintaining tolerance against self. T cells are an essential component of the immune system and they develop in the thymus. The AIRE (autoimmune regulator) gene product plays an important role in T cell development, as it promotes expression of peripheral tissue antigens in the thymus. Developing T cells, thymocytes, which recognize self-antigens with high affinity are deleted. However, this deletion process is not perfect and not all autoreactive T cells are destroyed. When the distinction between self and non-self fails, tolerance breaks and the immune system attacks the host s own tissues. This results in autoimmunity. Regulatory T cells contribute to the maintenance of self-tolerance. They can actively suppress the function of autoreactive cells. Several populations of cells with regulatory properties have been described, but the best characterized population is the natural regulatory T cells (Treg cells), which develop in the thymus and express the transcription factor FOXP3. The thymic development of Treg cells in humans is the subject of this thesis. Thymocytes at different developmental stages were analyzed using flow cytometry. The CD4-CD8- double-negative (DN) thymocytes are the earliest T cell precursors in the T cell lineage. My results show that the Treg cell marker FOXP3 is up-regulated already in a subset of these DN thymocytes. FOXP3+ cells were also found among the more mature CD4+CD8+ double-positive (DP) cells and among the CD4+ and CD8+ single-positive (SP) thymocytes. The different developmental stages of the FOXP3+ thymocytes were isolated and their gene expression examined by quantitative PCR. T cell receptor (TCR) repertoire analysis was used to compare these different thymocyte populations. My data show that in humans commitment to the Treg cell lineage is an early event and suggest that the development of Treg cells follows a linear developmental pathway, FOXP3+ DN precursors evolving through the DP stage to become mature CD4+ Treg cells. Most T cells have only one kind of TCR on their cell surface, but a small fraction of cells expresses two different TCRs. My results show that the expression of two different TCRs is enriched among Treg cells. Furthermore, both receptors were capable of transmitting signals when bound by a ligand. By extrapolating flow cytometric data, it was estimated that the majority of peripheral blood Treg cells are indeed dual-specific. The high frequency of dual-specific cells among human Treg cells suggests that dual-specificity has a role in directing these cells to the Treg cell lineage. It is known that both genetic predisposition and environmental factors influence the development of autoimmunity. It is also known that the dysfunction or absence of Treg cells leads to the development of autoimmune manifestations. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare monogenic autoimmune disease, caused by mutations in the AIRE gene. In the absence of AIRE gene product, deletion of self-specific T cells is presumably disturbed and autoreactive T cells escape to the periphery. I examined whether Treg cells are also affected in APECED. I found that the frequency of FOXP3+ Treg cells and the level of FOXP3 expression were significantly lower in APECED patients than in controls. Additionally, when studied in cell cultures, the suppressive capacity of the patients' Treg cells was impaired. Additionally, repertoire analysis showed that the TCR repertoire of Treg cells was altered. These results suggest that AIRE contributes to the development of Treg cells in humans and the selection of Treg cells is impaired in APECED patients. In conclusion, my thesis elucidates the developmental pathway of Treg cells in humans. The differentiation of Tregs begins early during thymic development and both the cells dual-specificity and AIRE probably affect the final commitment of Treg cells.

The Myopathic Protein Myotilin in Developing Mouse and in Muscle Function

Skeletal muscle cells are highly specialised in order to accomplish their function. During development, the fusion of hundreds of immature myoblasts creates large syncytial myofibres with a highly ordered cytoplasm filled with packed myofibrils. The assembly and organisation of contractile myofibrils must be tightly controlled. Indeed, the number of proteins involved in sarcomere building is impressive, and the role of many of them has only recently begun to be elucidated. Myotilin was originally identified as a high affinity a-actinin binding protein in yeast twohybrid screen. It was then found to interact also with filamin C, actin, ZASP and FATZ-1. Human myotilin is mainly expressed in striated muscle and induces efficient actin bundling in vitro and in cells. Moreover, mutations in myotilin cause different forms of muscle disease, now collectively known as myotilinopathies. In this thesis, consisting of three publications, the work on the mouse orthologue is presented. First, the cloning and molecular characterisation of the mouse myotilin gene showed that human and mouse myotilin share high sequence homology and a similar expression pattern and gene regulation. Functional analysis of the mouse promoter revealed the myogenic factor-binding elements that are required for myotilin gene transcription. Secondly, expression of myotilin was studied during mouse embryogenesis. Surprisingly, myotilin was expressed in a wide array of tissues at some stages of development; its expression pattern became more restricted at perinatal stages and in adult life. Immunostaining of human embryos confirmed broader myotilin expression compared to the sarcomeric marker titin. Finally, in the third article, targeted deletion of myotilin gene in mice revealed that it is not essential for muscle development and function. These data altogether indicate that the mouse can be used as a model for human myotilinopathy and that loss of myotilin does not alter significantly muscle structure and function. Therefore, disease-associated mutant myotilin may act as a dominant myopathic factor.

Agroforestry systems for sustainable livelihoods and improved land management in the East Usambara Mountains, Tanzania

Forest destruction for agriculture continues to be a major threat to the rich biological diversity in the East Usambara Mountains in the north-eastern corner of Tanzania. The highest ratio of endemic plant and animal species found on 100 km2 anywhere in the world is depending on the remaining natural forests. Forests are vitally important for the local population in many different ways, and nationally they are an important source of water and hydroelectricity. The soils, of low fertility and mostly acidic Ferrasols, mainly have the nutrients in the topsoil. After clear-cutting, the soils soon become poor when the topsoil is eroded. High-value cardamom is nowadays unsustainably cultivated in the natural forests of the East Usambaras. The general aim was to study the possibilities to develop new profitable and sustainable agroforestry systems for the benefit of the local people that could contribute to relieving the pressure on the remaining natural forests in the East Usambara Mountains. Results from a spice crop agroforestry trial, established in cooperation with a local farmer, showed a clear advantage of intercropping cardamom (Elettaria cardamomum) and black pepper (Piper nigrum) with trees, especially with Grevillea robusta. The nitrogen fixing tree species Gliricidia sepium also improved the nitrogen and organic matter content of the soil over levels found in the natural forest. With improved agroforestry methods for spice production the households generated as much as13 times the net income obtained with traditional forest cultivation practices. There are thus sustainable and profitable ways to cultivate spices as cash crops in well-managed homegardens. However, the farmers need stable markets, access to credit and comprehensive extension services. The soil fertility depletion should be reversed with organic manure application and an enabling policy environment for the smallholder-farming sector. Strong farmers organisations and equal rights to resources and decision-making are needed. Organic spices have an increasing demand, and their export would be profitable for these farmers. What is, however, most needed for a change is a political will of a government that understands the importance of agricultural and forestry development for poverty reduction.

What was behind the bark? : An assessment of decay among urban Tilia, Betula and Acer trees felled as hazardous in the Helsinki City area

Old trees growing in urban environments are often felled due to symptoms of mechanical defects that could be hazardous to people and property. The decisions concerning these removals are justified by risk assessments carried out by tree care professionals. The major motivation for this study was to determine the most common profiles of potential hazard characteristics for the three most common urban tree genera in Helsinki City: Tilia, Betula and Acer, and in this way improve management practices and protection of old amenity trees. For this research, material from approximately 250 urban trees was collected in cooperation with the City of Helsinki Public Works Department during 2001 - 2004. From the total number of trees sampled, approximately 70% were defined as hazardous. The tree species had characteristic features as potential hazard profiles. For Tilia trees, hollowed heartwood with low fungal activity and advanced decay caused by Ganoderma lipsiense were the two most common profiles. In Betula spp., the primary reason for tree removal was usually lowered amenity value in terms of decline of the crown. Internal cracks, most often due to weak fork formation, were common causes of potential failure in Acer spp. Decay caused by Rigidoporus populinus often increased the risk of stem breakage in these Acer trees. Of the decay fungi observed, G. lipsiense was most often the reason for the increased risk of stem collapse. Other fungi that also caused extensive decay were R. populinus, Inonotus obliquus, Kretzschmaria deusta and Phellinus igniarius. The most common decay fungi in terms of incidence were Pholiota spp., but decay caused by these species did not have a high potential for causing stem breakage, because it rarely extended to the cambium. The various evaluations used in the study suggested contradictions in felling decisions based on trees displaying different stages of decay. For protection of old urban trees, it is crucial to develop monitoring methods so that tree care professionals could better analyse the rate of decay progression towards the sapwood and separate those trees with decreasing amounts of sound wood from those with decay that is restricted to the heartwood area.

Ode to a Skylark: Agricultural intensification and farmland birds in the Baltic region

Intensification of agricultural land-use has been was shown to be the key reason behind declines in wildlife species associated with farmland. Accession to the European Union is regarded as a potential threat to the farmland biota of its new member states. In my thesis I looked at scenarios of agricultural development across the Baltic states of Estonia, Latvia and Lithuania, and the ways they are seen to affect farmed environments as a habitat of farmland bird species. I looked at the effects of major farmed habitats across the region, and assessed the role of spatial organisation of farmed habitats. I also evaluated the direction and magnitude of changes in bird communities following progression of farmland land-use from a relatively less intensive to the most intensive type within each country. Different aspects of the structural complexity of farmland were critical for supporting farmland birds. There was a clear indication that the more intensively farmed areas across the region provided habitat for fewer bird species and individuals, and intensification of field management was reflected in a tangible decrease in farmland bird abundance. The second part of the thesis, based on interviews in Estonia and Finland, is devoted to farmers interest in and knowledge of farmland wildlife, their understanding of the concept of biodiversity, and awareness of causes behind species declines. I examined the relationship between farmers interest and their willingness to undertake practices favouring farmland wildlife. Many farmers viewed biodiversity from a narrow perspective. In Finland farmers expressed higher concern about the decline in common farmland species than in Estonia. In both countries farmers rated intensification of agriculture as the major driving force behind declines. The expressed interest in wildlife positively correlated with willingness to undertake wildlife-friendly measures. Only farmers with agri-environment contracts targeted specifically at biodiversity were more knowledgeable about practical on-farm activities favouring wildlife, and were more willing to employ them that the rest. The results suggest that, by contributing to simplification of the farmland structure, homogenisation of crops, and increase in intensity of field use, EU agricultural policies will have a detrimental effect on farmland bird populations in Eastern Europe. Farmers are on the whole positive to the idea of supporting wildlife on their farms, and are concerned about declines, but they require payments to offset their income loss and extra work. I propose ways of further improving and better targeting of the agri-environment schemes in the region. I argue that with a foreseen tripling of cereal yields across the region, the EU Council s target of halting biodiversity decline in the EU by 2010 may not be realistic unless considerable improvements are made in conservation safeguards within the EU agricultural policy for the region.

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Poverty and the Environment : Investment Poverty and the Role of Assets in Generating Welfare for Farmer Households in the Province of Herrera, Panama

Köyhiä maanviljelijöitä on usein syytetty kehitysmaiden ympäristöongelmista. On väitetty, että eloonjäämistaistelu pakottaa heidät käyttämään maata ja muita luonnonvaroja lyhytnäköisesti. Harva asiaa koskeva tutkimus on kuitenkaan tukenut tätä väitettä; perheiden köyhyyden astetta ja heidän aiheuttamaansa ympäristövaikutusta ei ole kyetty kytkemään toisiinsa. Selkeyttääkseen köyhyys-ympäristö –keskustelua, Thomas Reardon ja Steven Vosti kehittivät investointiköyhyyden käsitteen. Se tunnistaa sen kenties suuren joukon maanviljelijäperheitä, jotka eivät ole köyhiä perinteisten köyhyysmittareiden mukaan, mutta joiden hyvinvointi ei ole riittävästi köyhyysrajojen yläpuolella salliakseen perheen investoida kestävämpään maankäyttöön. Reardon ja Vosti korostivat myös omaisuuden vaikutusta perheiden hyvinvointiin, ja uskoivat sen vaikuttavan tuotanto- ja investointipäätöksiin. Tässä tutkimuksessa pyritään vastaamaan kahteen kysymykseen: Miten investointiköyhyyttä voidaan ymmärtää ja mitata? Ja, mikä on viljelijäperheiden omaisuuden hyvinvointia lisäävä vaikutus? Tätä tutkimusta varten haastateltiin 402 maanviljelijäperhettä Väli-Amerikassa, Panaman tasavallan Herreran läänissä. Näiden perheiden hyvinvointia mitattiin heidän kulutuksensa mukaan, ja paikalliset köyhyysrajat laskettiin paikallisen ruoan hinnan mukaan. Herrerassa ihminen tarvitsee keskimäärin 494 dollaria vuodessa saadakseen riittävän ravinnon, tai 876 dollaria vuodessa voidakseen ravinnon lisäksi kattaa muitakin välttämättömiä menoja. Ruoka- eli äärimmäisen köyhyyden rajan alle jäi 15,4% tutkituista perheistä, ja 33,6% oli jokseenkin köyhiä, eli saavutti kyllä riittävän ravitsemuksen, muttei kyennyt kustantamaan muita perustarpeitaan. Molempien köyhyysrajojen yläpuolelle ylsi siis 51% tutkituista perheistä. Näiden köyhyysryhmien välillä on merkittäviä eroavaisuuksia ei vain perheiden varallisuuden, tulojen ja investointistrategioiden välillä, mutta myös perheiden rakenteessa, elinympäristössä ja mahdollisuuksissa saada palveluja. Investointiköyhyyden mittaaminen osoittautui haastavaksi. Herrerassa viljelijät eivät tee investointeja puhtaasti ympäristönsuojeluun, eikä maankäytön kestävyyttä muutenkaan pystytty yhdistämään perheiden hyvinvoinnin tasoon. Siksi investointiköyhyyttä etsittiin sellaisena hyvinvoinnin tasona, jonka alapuolella elävien perheiden parissa tuottavat maanparannusinvestoinnit eivät enää ole suorassa suhteessa hyvinvointiin. Tällaisia investointeja ovat mm. istutetut aidat, lannoitus ja paranneltujen laiduntyyppien viljely. Havaittiin, että jos perheen hyvinvointi putoaa alle 1000 dollarin/henkilö/vuosi, tällaiset tuottavat maanparannusinvestoinnit muuttuvat erittäin harvinaisiksi. Investointiköyhyyden raja on siis noin kaksi kertaa riittävän ravitsemuksen hinta, ja sen ylitti 42,3% tutkituista perheistä. Heille on tyypillistä, että molemmat puolisot käyvät työssä, ovat korkeasti koulutettuja ja yhteisössään aktiivisia, maatila tuottaa paremmin, tilalla kasvatetaan vaativampia kasveja, ja että he ovat kerryttäneet enemmän omaisuutta kuin investointi-köyhyyden rajan alla elävät perheet. Tässä tutkimuksessa kyseenalaistettiin yleinen oletus, että omaisuudesta olisi poikkeuksetta hyötyä viljelijäperheelle. Niinpä omaisuuden vaikutusta perheiden hyvinvointiin tutkittiin selvittämällä, mitä reittejä pitkin perheiden omistama maa, karja, koulutus ja työikäiset perheenjäsenet voisivat lisätä perheen hyvinvointia. Näiden hyvinvointi-mekanismien ajateltiin myös riippuvan monista väliin tulevista tekijöistä. Esimerkiksi koulutus voisi lisätä hyvinvointia, jos sen avulla saataisiin paremmin palkattuja töitä tai perustettaisiin yritys; mutta näihin mekanismeihin saattaa vaikuttaa vaikkapa etäisyys kaupungeista tai se, omistaako perhe ajoneuvon. Köyhimpien perheiden parissa nimenomaan koulutus olikin ainoa tutkittu omaisuuden muoto, joka edisti perheen hyvinvointia, kun taas maasta, karjasta tai työvoimasta ei ollut apua köyhyydestä nousemiseen. Varakkaampien perheiden parissa sen sijaan korkeampaa hyvinvointia tuottivat koulutuksen lisäksi myös maa ja työvoima, joskin monesta väliin tulevasta muuttujasta, kuten tuotantopanoksista riippuen. Ei siis ole automaatiota, jolla omaisuus parantaisi perheiden hyvinvointia. Vaikka rikkailla onkin yleensä enemmän karjaa kuin köyhemmillä, ei tässä aineistossa löydetty yhtään mekanismia, jota kautta karjan määrä tuottaisi korkeampaa hyvinvointia viljelijäperheille. Omaisuuden keräämisen ja hyödyntämisen strategiat myös muuttuvat hyvinvoinnin kasvaessa ja niihin vaikuttavat monet ulkoiset tekijät. Ympäristön ja köyhyyden suhde on siis edelleen epäselvä. Köyhyyden voittaminen vaatii pitkällä tähtäimellä sitä, että viljelijäperheet nousisivat investointiköyhyyden rajan yläpuolelle. Näin heillä olisi varaa alkaa kartuttaa omaisuutta ja investoida kestävämpään maankäyttöön. Tällä hetkellä kuitenkin isolle osalle herreralaisia perheitä tuo raja on kaukana tavoittamattomissa. Miten päästä yli tuhannen dollarin kulutukseen perheenjäsentä kohden, mikäli elintaso ei yllä edes riittävään ravitsemukseen? Ja sittenkin, vaikka hyvinvointi kohenisi, ei ympäristön kannalta parannuksia ole välttämättä odotettavissa, mikäli karjalaumat kasvavat ja eroosioalttiit laitumet leviävät.

Livelihood, land use and environment interactions in the highlands of East Africa

This study aims at improving understanding of the interactions of livelihoods and the environment focusing on both socio-economic and biodiversity implications of land use change in the context of population pressure, global and local markets, climate change, cultural and regional historical factors in the highlands of East Africa. The study is based on three components (1) two extensive livelihood surveys, one on Mt. Kilimanjaro in Tanzania and the other in the Taita Hills of Kenya, (2) a land use change study of the southern slopes of Mt. Kilimanjaro focusing on land use trends between 1960s and 1980s and 1980s and 2000 and (3) a bird diversity study focusing on the potential impacts of the future land use change on birds in the main land use types on the slopes and the adjacent plains of Mt. Kilimanjaro. In addition, information on the highlands in Embu and the adjacent lowlands in Mbeere of Kenya are added to the discussion. Some general patterns of livelihood, land use and environment interactions can be found in the three sites. However, the linkages are very complex. Various external factors at different times in history have influenced most of the major turning points. Farmers continually make small adaptations to their farming practices, but the locally conceived alternatives are too few. Farmers lack specific information and knowledge on the most suitable crops, market opportunities and the quality requirements for growing the crops for markets. Population growth emerges as the most forceful driver of land use and environmental change. The higher altitudes have become extremely crowded with population densities in some areas higher than typical urban population densities. Natural vegetation has almost totally been replaced by farmland. Decreasing farm size due to population pressure is currently threatening the viability of whole farming systems. In addition, capital-poor intensification has lead to soil fertility depletion. Agricultural expansion to the agriculturally marginal lowlands has created a new and distinct group of farmers struggling constantly with climate variability causing frequent crop failures. Extensification to the fragile drylands is the major cause of fragmentation and loss of wildlife habitat. The linkages between livelihoods, land use and the environment generally point to degradation of the environment leading to reduced environmental services and ecosystem functions. There is no indication that the system is self-regulating in this respect. Positive interventions will be needed to maintain ecosystem integrity.

Struggles over resources and representations in territorial conflicts in the Northern Autonomous Atlantic Region (RAAN), Nicaragua

Territoriality is a central issue in indigenous peoples struggles. The territorial struggles involve struggles over the control of natural resources and over political participation and representation, but also over the perception of territorial rights and the symbolic representation of the territory. These struggles are carried through both in material and symbolic ways through recurring to different discourses and representations that provide legitimation for the territorial claims of the group. The study is located in the Northern Autonomous Atlantic Region of Nicaragua. The study concerns the territorial strategies, conceptions and practices of the indigenous people and other actors. Territorial conflicts exist between the autonomous region and the central government of Nicaragua, between mestizo settlers and indigenous people, between different indigenous groups, and between these and development agents such as conservation projects. The study focuses on how territorial discourses and representations are used to legitimate territorial control. Environmental, historical and cartographical discourses are the most important discourses recurred to. The influence of discourses and representations on the territorial practices and policies of the different actors, the links between the local struggles and global processes, and the broader structural factors impacting on the territorial struggles are also analysed. Among the structural factors are the problems related to land tenure and management and the use of natural resources, the advance of the agricultural frontier, the institutional weaknesses of the central and regional governments and the legislative processes. The territorial discourses are both recurred to in a strategic way and also grounded in local ideals and practices. The discourses have produced real effects for example in legislation, land tenure systems, political representation and environmental practices. Although the use of discourses and representations are an important power tool in territorial struggles, territorial control cannot be effectively accomplished merely through representing territorial claims in a legitimate way or through reforming legislation, as the conflicts are also largely a result of structural factors affecting the region. The fieldwork was carried out during a total of twelve months between 2000 and 2002. The research methods used were semi-structured interviews, participant observation and participatory research methods. A broad range of literary sources were also used to collect data. The study is located within the field of critical political geography with a discursive political ecology approach. It can be called a critical realist approach to the discursive analysis of indigenous territoriality.

Cryptographic Software Export Controls in the EU

Certain software products employing digital techniques for encryption of data are subject to export controls in the EU Member States pursuant to Community law and relevant laws in the Member States. These controls are agreed globally in the framework of the so-called Wassenaar Arrangement. Wassenaar is an informal non-proliferation regime aimed at promoting international stability and responsibility in transfers of strategic (dual-use) products and technology. This thesis covers provisions of Wassenaar, Community export control laws and export control laws of Finland, Sweden, Germany, France and United Kingdom. This thesis consists of five chapters. The first chapter discusses the ratio of export control laws and the impact they have on global trade. The ratio is originally defence-related - in general to prevent potential adversaries of participating States from having the same tools, and in particular in the case of cryptographic software to enable signals intelligence efforts. Increasingly as the use of cryptography in a civilian context has mushroomed, export restrictions can have negative effects on civilian trade. Information security solutions may also be took weak because of export restrictions on cryptography. The second chapter covers the OECD's Cryptography Policy, which had a significant effect on its member nations' national cryptography policies and legislation. The OECD is a significant organization,because it acts as a meeting forum for most important industrialized nations. The third chapter covers the Wassenaar Arrangement. The Arrangement is covered from the viewpoint of international law and politics. The Wassenaar control list provisions affecting cryptographic software transfers are also covered in detail. Control lists in the EU and in Member States are usually directly copied from Wassenaar control lists. Controls agreed in its framework set only a minimum level for participating States. However, Wassenaar countries can adopt stricter controls. The fourth chapter covers Community export control law. Export controls are viewed in Community law as falling within the domain of Common Commercial Policy pursuant to Article 133 of the EC Treaty. Therefore the Community has exclusive competence in export matters, save where a national measure is authorized by the Community or falls under foreign or security policy derogations established in Community law. The Member States still have a considerable amount of power in the domain of Common Foreign and Security Policy. They are able to maintain national export controls because export control laws are not fully harmonized. This can also have possible detrimental effects on the functioning of internal market and common export policies. In 1995 the EU adopted Dual-Use Regulation 3381/94/EC, which sets common rules for exports in Member States. Provisions of this regulation receive detailed coverage in this chapter. The fifth chapter covers national legislation and export authorization practices in five different Member States - in Finland, Sweden, Germany, France and in United Kingdom. Export control laws of those Member States are covered when the national laws differ from the uniform approach of the Community's acquis communautaire. Keywords: export control, encryption, software, dual-use, license, foreign trade, e-commerce, Internet

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.

Causes and consequences of inbreeding in the ant Formica exsecta

Human actions cause destruction and fragmentation of natural habitats, predisposing populations to loss of genetic diversity and inbreeding, which may further decrease their fitness and survival. Understanding these processes is a main concern in conservation genetics. Yet data from natural populations is scarce, particularly on invertebrates, owing to difficulties in measuring both fitness and inbreeding in the wild. Ants are social insects, and a prime example of an ecologically important group for which the effects of inbreeding remain largely unstudied. Social insects serve key roles in all terrestrial ecosystems, and the division of labor between the females in the colonies queens reproduce, workers tend to the developing brood probably is central to their ecological success. Sociality also has important implications for the effects of inbreeding. Despite their relative abundance, the effective population sizes of social insects tend to be small, owing to the low numbers of reproductive individuals relative to the numbers of sterile workers. This may subject social insects to loss of genetic diversity and subsequent inbreeding depression. Moreover, both the workers and queens can be inbred, with different and possibly multiplicative consequences. The aim of this study was to investigate causes and consequences of inbreeding in a natural population of ants. I used a combination of long-term field and genetic data from colonies of the narrow-headed ant Formica exsecta to examine dispersal, mating behavior and the occurrence of inbreeding, and its consequences on individual and colony traits. Mating in this species takes place in nuptial flights that have been assumed to be population-wide and panmictic. My results, however, show that dispersal is local, with queens establishing new colonies as close as 60 meters from their natal colony. Even though actual sib-mating was rare, individuals from different but related colonies pair, which causes the population to be inbred. Furthermore, multiple mates of queens were related to each other, which also indicates localized mating flights. Hence, known mechanisms of inbreeding avoidance, dispersal and multiple mating, were not effective in this population, as neither reduced inbreeding level of the future colony. Inbreeding had negative consequences both at the individual and colony level. A queen that has mated with a related male produces inbred workers, which impairs the colony s reproductive success. The inbred colonies were less productive and, specifically, produced fewer new queens, possibly owing to effects of inbreeding on the caste determination of female larvae. A striking finding was that males raised in colonies with inbred workers were smaller, which reflects an effect of the social environment as males, being haploid, cannot be inbred themselves. The queens produced in the inbred colonies, in contrast, were not smaller, but their immune response was up-regulated. Inbreeding had no effect on queen dispersal, but inbred queens had a lower probability of successfully founding a new colony. Ultimately, queens that survived through the colony founding phase had a shorter lifespan. This supports the idea that inbreeding imposes a genetic stress, leading to inbreeding depression on both the queen and the colony level. My results show that inbreeding can have profound consequences on insects in the wild, and that in social species the effects of inbreeding may be multiplicative and mediated through the diversity of the social environment, as well as the genetic makeup of the individuals themselves. This emphasizes the need to take into account all levels of organization when assessing the effects of genetic diversity in social animals.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

Neurotrophins and neuronal plasticity in the action of antidepressants and morphine

Neuronal plasticity is a well characterized phenomenon in the developing and adult brain. It refers to capasity of a single neuron to modify morphology, synaptic connections and activity. Neuronal connections and capacity for plastic events are compromised in several pathological disorders, such as major depression. In addition, neuronal atrophy has been reported in depressive patients. Neurotrophins are a group of secretory proteins functionally classified as neuronal survival factors. Neurotrophins, especially brain derived neurotrophic factor (BDNF), have also been associated with promoting neuronal plasticity in dysfunctional neuronal networks. Chronic antidepressant treatment increases plastic events including neurogenesis and arborization and branching of neurites in distinct brain areas, such as the hippocampus. One suggested mode of action is where the antidepressants elevate the synaptic levels of BDNF thus further activating several signaling cascades via trkB-receptor. In our studies we have tried to clarify the mechanisms of action for antidepressants and to resolve the role of BDNF in this process. We found that chronic antidepressant treatment increases amount of markers of neuronal plasticity in both hippocampus and in the medial prefrontal cortex, both of which are closely linked to the etiology of major depression. Secondary actions of antidepressants include rapid activation of the trkB receptor followed by a phosphorylation of transcription factor CREB. In addition, activation of CREB by phosphorylation appears responsible for the regulation of the expression of the BDNF gene. Using transgenic mice we found that BDNF-induced trkB-mediated signaling proved crucial for the behavioral effects of antidepressants in the forced swimming test and for the survival of newly-born neurons in the adult hippocampus. Antidepressants not only increased neurogenesis in the adult hippocampus but also elevated the turnover of hippocampal neurons. During these studies we also discovered that another trkB ligand, NT-4, is involved in morphine-mediated anti-nociception and tolerance. These results present a novel role for trkB-mediated signaling in plastic events present in the opioid system. This thesis evaluates neuronal plasticity and trkB as a target for future antidepressant treatments.

Marking one-summer old whitefish with fluorescent pigment spraying method and results of whitefish stockings in the Gulf of Bothnia

Anadromous whitefish is one of the most important fish species in the Finnish coastal fisheries in the Gulf fo Bothnia. To compensate the lost reproduction due to river damming and to support the fisheries, several million one-summer old whitefish are released yearly into the Gulf of Bothnia. Since there are naturally reproducing whitefish in the Gulf as well, and the wild and stocked fish can not be separated in the catch, stocking impact can only be estimated by marking the stocked fish. Due to the small size and large number of released whitefish, the scattered fishery and large area where the whitefish migrate, most of the traditionally used fish marking methods were either unsuitable (e.g. Carlin-tags) or proved to be too expensive (e.g. coded wire tags). Fluorescent pigment spraying method offers a fast and cost-effective method to mass-mark young fish. However, the results are not always satisfactory due to low long-time retention of the marks in some species. The method has to be tested and proper marking conditions and methods determined for each species. This thesis is based on work that was accomplished while developing the fluorescent pigment spraying method for marking one-summer old whitefish fingerlings, and it draws together the results of mass-marking whitefish fingerlings that were released in the Gulf of Bothnia. Fluorescent pigment spraying method is suitable for one-summer old whitefish larger than 8 cm total length. The water temperature during the marking should not exceed 10o C. Suitable spraying pressure is 6 bars measured in the compressor outlet, and the distance of the spraying gun nozzle should be ca 20 cm from the fish. Under such conditions, the marking results in long-term retention of the mark with low or no mortality. The stress level of the fish (measured as muscle water content) rises during the marking procedure, but if the fish are allowed to recover after marking, the overall stress level remains within the limits observed in normal fish handling during the capture-loading-transport-stocking procedure. The marked whitefish fingerlings are released into the sea at larger size and later in the season than the wild whitefish. However, the stocked individuals migrate to the southern feeding grounds in a similar pattern to the wild ones. The catch produced by whitefish stocking in the Gulf of Bothnia varied between released fingerling groups, but was within the limits reported elsewhere in Finland. The releases in the southern Bothnian Bay resulted in a larger catch than those made in the northern Bothnian Bay. The size of the released fingerlings seemed to have some effect on survival of the fish during the first winter in the sea. However, when the different marking groups were compared, the mean fingerling size was not related to stocking success.