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  • Sand, Andrea (Helsingin yliopisto, 2015)
    In the face of the world's increasing demand for energy, and the need to find sustainable and environmentally friendly ways of producing that energy, fusion power offers an attractive possibility. However, the harsh operating conditions of future fusion devices poses a significant challenge for materials development and engineering. Tungsten (W) and tungsten alloys are current candidate materials for both structural and plasma-facing components, due to favourable properties such as good thermal conductivity, high heat strength and stability, and resistance to erosion. However, fusion reactor components will be subjected to high neutron loads, and little is currently known of the effects of radiation on the mechanical properties of this intrinsically brittle metal. The extreme conditions in a future fusion reactor cannot be reproduced in existing experimental facilities, rendering simulation an invaluable tool in understanding the radiation damage processes. Multiscale methods are necessary to span the length and time scales involved, from the picosecond and nanometer scale of displacement cascades giving rise to the primary damage, to the evolution of the radiation induced microstructure over the seconds of typical in-situ ion irradiation experiments, and further to the years of a reactor component s life time. In order to implement a multiscale simulation method, information must be distilled and transferred from the smaller scale to the larger. Molecular dynamics (MD) simulations are ideal for studying the primary damage, but individual cascades vary greatly, and simulating high energy impacts in MD requires immense computer capacity. It is therefore not possible to simulate directly the whole variety of cascade outcomes. General laws deduced from the MD data can, however, be used to statistically generate varying cascades in the thousands. In this thesis we use MD simulations to study the primary damage in metals, with focus on tungsten. We identify aspects of the simulation methodology which affect the results, and validate our methods by direct comparison to experiments. Detailed analysis of the primary damage from high-energy cascades shows the formation of novel defects, confirming recent experimental observations. We also show that defect cluster sizes follow a general scaling law, which can be used to statistically generate cascade debris as input for microstructural evolution models, circumventing the need to directly simulate thousands of cascades.
  • Zhao, Junlei (2014)
    Metal-induced crystallization (MIC) has been investigated extensively as an alternative crystallization process in the silicon based photovoltaic industry. In this work, we simulate a nanoscale version of this process by using molecular dynamics simulation involving liquid Si nanoclusters inoculated with Ag atoms in Ar thermal bath. The simulations reveal that the energy released during coalescence of the silver silicide region is the main factor to remelt the surface of the Si nanocluster. In an earlier report, Ag nanoparticles is observed to induced crystallization in 12-15 nm- diameter silicon cluster, which upon further cooling results in nano-polycrystalline silicon core and segregated Ag sub-shells. The work focuses on the crucial conditions that influence the MIC process, such as (i) number of Ag atoms per unit volume, (ii) initial temperature of Si cluster, (iii) crystallization temperature and (iv) cooling rate of the Si cluster. The results presented in this study provide insight into the effect of the first three parameters. Also, the results suggest that the coalescence of eutectic phase is the essential step which induces the crystallization.
  • Samela, Juha (Helsingin yliopisto, 2008)
    This thesis concerns the dynamics of nanoparticle impacts on solid surfaces. These impacts occur, for instance, in space, where micro- and nanometeoroids hit surfaces of planets, moons, and spacecraft. On Earth, materials are bombarded with nanoparticles in cluster ion beam devices, in order to clean or smooth their surfaces, or to analyse their elemental composition. In both cases, the result depends on the combined effects of countless single impacts. However, the dynamics of single impacts must be understood before the overall effects of nanoparticle radiation can be modelled. In addition to applications, nanoparticle impacts are also important to basic research in the nanoscience field, because the impacts provide an excellent case to test the applicability of atomic-level interaction models to very dynamic conditions. In this thesis, the stopping of nanoparticles in matter is explored using classical molecular dynamics computer simulations. The materials investigated are gold, silicon, and silica. Impacts on silicon through a native oxide layer and formation of complex craters are also simulated. Nanoparticles up to a diameter of 20 nm (315000 atoms) were used as projectiles. The molecular dynamics method and interatomic potentials for silicon and gold are examined in this thesis. It is shown that the displacement cascade expansionmechanism and crater crown formation are very sensitive to the choice of atomic interaction model. However, the best of the current interatomic models can be utilized in nanoparticle impact simulation, if caution is exercised. The stopping of monatomic ions in matter is understood very well nowadays. However, interactions become very complex when several atoms impact on a surface simultaneously and within a short distance, as happens in a nanoparticle impact. A high energy density is deposited in a relatively small volume, which induces ejection of material and formation of a crater. Very high yields of excavated material are observed experimentally. In addition, the yields scale nonlinearly with the cluster size and impact energy at small cluster sizes, whereas in macroscopic hypervelocity impacts, the scaling 2 is linear. The aim of this thesis is to explore the atomistic mechanisms behind the nonlinear scaling at small cluster sizes. It is shown here that the nonlinear scaling of ejected material yield disappears at large impactor sizes because the stopping mechanism of nanoparticles gradually changes to the same mechanism as in macroscopic hypervelocity impacts. The high yields at small impactor size are due to the early escape of energetic atoms from the hot region. In addition, the sputtering yield is shown to depend very much on the spatial initial energy and momentum distributions that the nanoparticle induces in the material in the first phase of the impact. At the later phases, the ejection of material occurs by several mechanisms. The most important mechanism at high energies or at large cluster sizes is atomic cluster ejection from the transient liquid crown that surrounds the crater. The cluster impact dynamics detected in the simulations are in agreement with several recent experimental results. In addition, it is shown that relatively weak impacts can induce modifications on the surface of an amorphous target over a larger area than was previously expected. This is a probable explanation for the formation of the complex crater shapes observed on these surfaces with atomic force microscopy. Clusters that consist of hundreds of thousands of atoms induce long-range modifications in crystalline gold.
  • Berzins, Aivars (Helsingin yliopisto, 2010)
    The prevalence, contamination and heat resistance of Listeria monocytogenes were investigated in meat products and meat-processing plants. Moreover, trends of human listeriosis in Latvia were studied over a 10-year period from 1998 to 2007. A high prevalence (40%) of L. monocytogenes was found in cold-smoked meat products compared with other heat-treated ready-to-eat meat products (0.7%) available in retail markets in Latvia. Pulsed-field gel electrophoresis (PFGE) and serotyping were applied to analyse the diversity of L. monocytogenes isolates present in ready-to-eat (RTE) meat products and meat-processing plants. A high genetic diversity was seen among L. monocytogenes isolates from cold-smoked meat products, suggesting the existence of various sources of contamination at different production stages in the meat-processing environment. The manufacture of cold-smoked meat products involves no processing steps to eliminate L. monocytogenes, thus, contamination of the raw meat and contamination during processing can both contribute to L. monocytogenes in the finished product. Logistic multivariable regression model was successfully applied to identify the main factors associated with L. monocytogenes contamination during the manufacturing of cold-smoked pork products. Meat brining by injections was a significant factor (odds ratio 10.66; P<0.05) for contamination of products with L. monocytogenes. Of the cold-smoked meat-processing plant environmental samples, most contaminated sites were associated with brining machine and brining area. Long cold-smoking times (≥ 12 h) also had a significant predictive value (odds ratio 24.38; P<0.014) for a sample testing positive for L. monocytogenes. A genetically diverse population of L. monocytogenes entered the meat-processing plant, where only some of the strains colonized and established a persistent microbial community within the plant over a 5-year period. L. monocytogenes PFGE types belonging to serotypes 1/2a and 4b were isolated from imported, defrosted, raw pork from Germany and Belgium in meat-processing plant B. In total, two L. monocytogenes PFGE types originating from raw meat were found also in finished RTE cold-smoked pork products, whereas one PFGE type was recovered later only from the meat-processing environment. One of the L. monocytogenes PFGE types, belonging to serotype 1/2c, was isolated from RTE cold-smoked meat products and from the feeding teeth of the brining machine, thus showing that improper cleaning, disinfection and poor hygiene design of the brining machine may cause L. monocytogenes contamination over time. Post-package pasteurization of high- and low-fat content cooked sausages at temperatures higher than 55°C was found to be an effective method of post-process thermal treatment to reduce contamination of L. monocytogenes. However, heating to 55°C, 60°C and 62.5°C may not be practical in the meat industry because the process takes too long to reach a 3-log reduction. The formulation of high-fat content RTE cooked sausages may require modification to maintain product quality. During the 10-year study period the overall incidence of listeriosis in Latvia was 0.4 per 100 000 population, with the highest incidences recorded in 2000 and 2002 (1.5 and 0.7 per 100 000 population, respectively). The highest incidence of listeriosis in Latvia was observed in 2000, which significantly exceeded incidence levels in all Baltic and Nordic countries, and was the highest among all EU member states during the same period. A marked clustering of human listeriosis cases was observed from September to December 2000, possibly indicating one large outbreak. The lack of serotyping and molecular typing methods for subtyping of L. monocytogenes isolates in the present surveillance system is one of the main reasons why there have been no officially documented listeriosis outbreaks in Latvia to date. Measures to allow the application of appropriate actions at the food industry level need to be implemented to prevent or significantly reduce the real burden of foodborne listeriosis in Latvia.
  • Savolainen-Kopra, Carita (Helsingin yliopisto, 2006)
    The first part of this work investigates the molecular epidemiology of a human enterovirus (HEV), echovirus 30 (E-30). This project is part of a series of studies performed in our research team analyzing the molecular epidemiology of HEV-B viruses. A total of 129 virus strains had been isolated in different parts of Europe. The sequence analysis was performed in three different genomic regions: 420 nucleotides (nt) in the VP4/VP2 capsid protein coding region, the entire VP1 capsid protein coding gene of 876 nt, and 150 nt in the VP1/2A junction region. The analysis revealed a succession of dominant sublineages within a major genotype. The temporally earlier genotypes had been replaced by a genetically homogenous lineage that has been circulating in Europe since the late 1970s. The same genotype was found by other research groups in North America and Australia. Globally, other cocirculating genetic lineages also exist. The prevalence of a dominant genotype makes E-30 different from other previously studied HEVs, such as polioviruses and coxsackieviruses B4 and B5, for which several coexisting genetic lineages have been reported. The second part of this work deals with molecular epidemiology of human rhinoviruses (HRVs). A total of 61 field isolates were studied in the 420-nt stretch in the capsid coding region of VP4/VP2. The isolates were collected from children under two years of age in Tampere, Finland. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades. Seasonal clustering was found. Also, several distinct serotype-like clusters were found to co-circulate during the same epidemic season. Reappearance of a cluster after disappearing for a season was observed. The molecular epidemiology of the analyzed strains turned out to be complex, and we decided to continue our studies of HRV. Only five previously published complete genome sequences of HRV prototype strains were available for analysis. Therefore, all designated HRV prototype strains (n=102) were sequenced in the VP4/VP2 region, and the possibility of genetic typing of HRV was evaluated. Seventy-six of the 102 prototype strains clustered in HRV genetic group A (HRV-A) and 25 in group B (HRV-B). Serotype 87 clustered separately from other HRVs with HEV species D. The field strains of HRV represented as many as 19 different genotypes, as judged with an approximate demarcation of a 20% nt difference in the VP4/VP2 region. The interserotypic differences of HRV were generally similar to those reported between different HEV serotypes (i.e. about 20%), but smaller differences, less than 10%, were also observed. Because some HRV serotypes are genetically so closely related, we suggest that the genetic typing be performed using the criterion "the closest prototype strain". This study is the first systematic genetic characterization of all known HRV prototype strains, providing a further taxonomic proposal for classification of HRV. We proposed to divide the genus Human rhinoviruses into HRV-A and HRV-B. The final part of the work comprises a phylogenetic analysis of a subset (48) of HRV prototype strains and field isolates (12) in the nonstructural part of the genome coding for the RNA-dependent RNA polymerase (3D). The proposed division of the HRV strains in the species HRV-A and HRV-B was also supported by 3D region. HRV-B clustered closer to HEV species B, C, and also to polioviruses than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D coding region than in the VP4/VP2 coding region, in contrast to HEV. Moreover, the diversity of HRV in 3D exceeded that of HEV. One group of HRV-A, designated HRV-A', formed a separate cluster outside other HRV-A in the 3D region. It formed a cluster also in the capsid region, but located within HRV-A. This may reflect a different evolutionary history of distinct genomic regions among HRV-A. Furthermore, the tree topology within HRV-A in the 3D region differed from that in the VP4/VP2, suggesting possible recombination events in the evolution of the strains. No conflicting phylogenies were observed in any of the 12 field isolates. Possible recombination was further studied using the Similarity and Bootscanning analyses of the complete genome sequences of HRV available in public databases. Evidence for recombination among HRV-A was found, as HRV2 and HRV39 showed higher similarity in the nonstructural part of the genome. Whether HRV2 and HRV39 strains - and perhaps also some other HRV-A strains not yet completely sequenced - are recombinants remains to be determined.
  • Maunula, Leena (Helsingin yliopisto, 2001)
  • Fredriksson-Ahomaa, Maria (Helsingin yliopisto, 2001)
  • Suomalainen, Marjo (Helsingin yliopisto, 2014)
    Omptins are a family of conserved, integral outer membrane proteases and widely distributed within Gram-negative bacterial species. The family offers a good example of the evolution and the adaptation of a protein to novel functions and to differing pathogenic bacterial life-styles. This work investigates three different omptins: Pla of Yersinia pestis, PgtE of Salmonella enterica and OmpT of Escherichia coli. The omptin proteases differ in substrate specificity and need lipopolysaccharide (LPS) for activity. My thesis work addressed two main questions in omptin function: what is the molecular basis of the dissimilar substrate selectivity in the structurally very similar omptins; and what are the structural features in LPS that affect omptin activity. I studied the LPS dependency of omptins by expressing the proteins in bacterial cells that differ in LPS structure and by reconstituting purified, detergent-solubilized omptin protein with characterized, purified LPS molecules. Y. pestis alters its LPS structure in response to change of temperature from 20°C to 37°C, which reflects the transfer from a flea to a mammalian host. I found that the activity of Pla in cells from 20°C was very low, whereas cells from 37°C expressed high activity. I reconstituted detergent-purified His6-Pla protein with various model LPS structures and with LPSs of Y. pestis grown at different temperatures. Adding Y. pestis LPS from 37°C to the nonfunctional Pla protein induced high proteolytic activity, whereas 20°C-LPS gave very low activity, indicating that the activity of Pla is controlled by LPS. Similarly, I found that the activity of PgtE was high with rough LPS and low with smooth LPS; the difference mimics the LPS of intracellular (rough) and extracellular (smooth) S. enterica. Thus, in both bacterial species the omptin activity is controlled by the LPS type that the bacteria express during infection in mammals. I further studied the fine structure of Y. pestis LPS that affects Pla activity. This was done by reconstituting Pla activity with various structurally characterized Y. pestis and E. coli LPSs. I found that lower levels of lipid A acylation and phosphate substitution by aminoarabinose, are important for Pla activity, these features are characteristic for Y. pestis LPS from 37°C. A common and conserved feature in omptin structure is the presence of LPS-binding motif in protein barrel. Disrupting of the lipid A-binding motifs in PgtE and Pla abolished their proteolytic activity, emphasizing the importance of the LPS binding site for omptin activity. Omptins have a highly spatically conserved active center and catalytic domains but express functional heterogeneity. The omptin transmembrane barrel contains five surface-exposed loops that show slightly higher sequence variation than the transmembrane protein regions. To study the effect of loop structures in omptin proteolytic specificity, I changed OmpT of E. coli to a Pla-like enzyme by a stepwise substitution of the loop areas. The proteins were characterized by their ability to activate the human protease precursor plasminogen(Plg) to the active serine protease plasmin and to inactivate the main plasmin inhibitor, α2-antiplasmin(α2AP); both functions are important for bacterial virulence. Pla cleaves very efficiently both substrates, whereas OmpT is only poorly active with them. I showed that OmpT could be converted into a Pla-like enzyme by cumulative substitutions at the loop areas, especially the loops L3-L5 were important. The successful conversion of OmpT towards Pla indicates that the loop structures are critical for omptin activity by allowing correct recognition of the polypeptide substrate. More detailed substitution analysis was taken to identify the catalytic residues in Pla. My thesis demonstrates that the omptin proteolytic activity depends on two things: their specific interaction with LPS and the structure of their surface-exposed loops. The thesis offers an example of omptins extensive evolvability and of how they adapt to the lifestyle of their host bacterium.
  • Österman, Janina (Helsingin yliopisto, 2015)
    Nitrogen is an indispensable element for plants and animals to be able to synthesise essential biological compounds such as amino acids and nucleotides. Although there is plenty of nitrogen in the form of nitrogen gas (N2) in the Earth s atmosphere, it is not readily available to plants but needs to be converted (fixed) into ammonia before it can be utilised. Nitrogen-fixing bacteria living freely in the soil or in symbiotic association with legume plants, fix N2 into ammonia used by the plants. This is known as biological nitrogen fixation (BNF). In contrast to industrial nitrogen fixation, an energy-demanding process using high temperature and pressure to produce chemical fertilizers, BNF makes use of solar energy alone to complete the same reaction. However, the requirements on compatibility of plants and nitrogen-fixing micro-organism, the rate of conversion and the ability of the micro-organisms to survive in stressful environments are limiting factors of this system. The current demand for more sustainable food production makes BNF an attractive alternative. However, optimization of existing BNF systems as well as development of new highly productive ones is necessary, to be able to replace the use of chemical fertilisers. In order to develop new alternatives, we need to gain more knowledge on the requirements set by both plants and micro-organisms for successful and efficient nitrogen fixation to occur. In this thesis, the nitrogen-fixing legume host Galega (goat s rue) and its symbiotic microbial partner Neorhizobium galegae were used as a model system to investigate the features defining good symbiotic nitrogen fixation. Studies of genetic diversity within the host plant showed that there are genetic traits making a distinction between the two species G. orientalis and G. officinalis, both at a whole-genome level and at the level of specific symbiosis-related genes. Genome sequencing of ten strains of N. galegae provided a useful dataset for studying i) the genomic features separating N. galegae from related nitrogen-fixing bacteria (rhizobia) and ii) the genetically encoded characteristics that divide strains of N. galegae into two separate symbiovars (symbiotic variants that show different phenotypes on the two different Galega host plant species). These studies provided new information on genes possibly involved in determining host specificity and efficiency of nitrogen fixation. In addition, previously unrecognised genetic contents provided insight into the ecology of N. galegae. Most importantly, genome sequencing enabled identification of the noeT gene, responsible for acetylation of the N. galegae Nod factor (signal molecule required for symbiosis). Although the noeT gene did not turn out to be the crucial determinant enabling nodulation of Galega spp. as previously anticipated, these results are important for future studies on mechanisms behind the selectiveness (host specificity) observed in nitogen-fixing symbioses between Galega and N. galegae.
  • Österman, Janina (Helsingin yliopisto, 2015)
    Nitrogen is an indispensable element for plants and animals to be able to synthesise essential biological compounds such as amino acids and nucleotides. Although there is plenty of nitrogen in the form of nitrogen gas (N2) in the Earth s atmosphere, it is not readily available to plants but needs to be converted (fixed) into ammonia before it can be utilised. Nitrogen-fixing bacteria living freely in the soil or in symbiotic association with legume plants, fix N2 into ammonia used by the plants. This is known as biological nitrogen fixation (BNF). In contrast to industrial nitrogen fixation, an energy-demanding process using high temperature and pressure to produce chemical fertilizers, BNF makes use of solar energy alone to complete the same reaction. However, the requirements on compatibility of plants and nitrogen-fixing micro-organism, the rate of conversion and the ability of the micro-organisms to survive in stressful environments are limiting factors of this system. The current demand for more sustainable food production makes BNF an attractive alternative. However, optimization of existing BNF systems as well as development of new highly productive ones is necessary, to be able to replace the use of chemical fertilisers. In order to develop new alternatives, we need to gain more knowledge on the requirements set by both plants and micro-organisms for successful and efficient nitrogen fixation to occur. In this thesis, the nitrogen-fixing legume host Galega (goat s rue) and its symbiotic microbial partner Neorhizobium galegae were used as a model system to investigate the features defining good symbiotic nitrogen fixation. Studies of genetic diversity within the host plant showed that there are genetic traits making a distinction between the two species G. orientalis and G. officinalis, both at a whole-genome level and at the level of specific symbiosis-related genes. Genome sequencing of ten strains of N. galegae provided a useful dataset for studying i) the genomic features separating N. galegae from related nitrogen-fixing bacteria (rhizobia) and ii) the genetically encoded characteristics that divide strains of N. galegae into two separate symbiovars (symbiotic variants that show different phenotypes on the two different Galega host plant species). These studies provided new information on genes possibly involved in determining host specificity and efficiency of nitrogen fixation. In addition, previously unrecognised genetic contents provided insight into the ecology of N. galegae. Most importantly, genome sequencing enabled identification of the noeT gene, responsible for acetylation of the N. galegae Nod factor (signal molecule required for symbiosis). Although the noeT gene did not turn out to be the crucial determinant enabling nodulation of Galega spp. as previously anticipated, these results are important for future studies on mechanisms behind the selectiveness (host specificity) observed in nitogen-fixing symbioses between Galega and N. galegae.
  • Niittymäki, Iina (2011)
    Both inherited genetic variations and somatically acquired mutations drive cancer development. The aim of this thesis was to gain insight into the molecular mechanisms underlying colorectal cancer (CRC) predisposition and tumor progression. Whereas one-third of CRC may develop in the context of hereditary predisposition, the known highly penetrant syndromes only explain a small fraction of all cases. Genome-wide association studies have shown that ten common single nucleotide polymorphisms (SNPs) modestly predispose to CRC. Our population-based sample series of around thousand CRC cases and healthy controls was genotyped for these SNPs. Tumors of heterozygous patients were analyzed for allelic imbalance, in an attempt to reveal the role of these SNPs in somatic tumor progression. The risk allele of rs6983267 at 8q24 was favored in the tumors significantly more often than the neutral allele, indicating that this germline variant is somatically selected for. No imbalance targeting the risk allele was observed in the remaining loci, suggesting that most of the low-penetrance CRC SNPs mainly play a role in the early stages of the neoplastic process. The ten SNPs were further analyzed in 788 CRC cases, 97 of which had a family history of CRC, to evaluate their combined contribution. A significant association appeared between the overall number of risk alleles and familial CRC and these ten SNPs seem to explain around 9% of the familial clustering of CRC. Finding more CRC susceptibility alleles may facilitate individualized risk prediction and cancer prevention in the future. Microsatellite instability (MSI), resulting from defective mismatch repair function, is a hallmark of Lynch syndrome and observed in a subset of all CRCs. Our aim was to identify microsatellite frameshift mutations that inactivate tumor suppressor genes in MSI CRCs. By sequencing microsatellite repeats of underexpressed genes we found six novel MSI target genes that were frequently mutated in 100 MSI CRCs: 51% in GLYR1, 47% in ABCC5, 43% in WDTC1, 33% in ROCK1, 30% in OR51E2, and 28% in TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in homozygously mutated tumors, providing further support for the loss of function hypothesis. Another mutation screening effort sought to identify MSI target genes with putative oncogenic functions. Microsatellites were similarly sequenced in genes that were overexpressed and, upon mutation, predicted to avoid nonsense-mediated mRNA decay. The mitotic checkpoint kinase TTK harbored protein-elongating mutations in 59% of MSI CRCs and the mutant protein was detected in heterozygous MSI CRC cells. No checkpoint dysregulation or defective protein localization was observable however, and the biological relevance of this mutation may hence be related to other mechanisms. In conclusion, these two large-scale and unbiased efforts identified frequently mutated genes that are likely to contribute to the development of this cancer type and may be utilized in developing diagnostic and therapeutic applications.
  • Roth, Stina (Helsingin yliopisto, 2000)
  • Koski, Taru (Helsingin yliopisto, 2010)
    Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a hereditary tumour predisposition syndrome. Its phenotype includes benign cutaneous and uterine leiomyomas (CLM, ULM) with high penetrance and rarer renal cell cancer (RCC), most commonly of papillary type 2 subtype. Over 130 HLRCC families have been identified world-wide but the RCC phenotype seems to concentrate in families from Finland and North America for unknown reasons. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. FH encodes the enzyme fumarase from mitochondrial citric acid cycle. Fumarase enzyme activity or type or site of the FH mutation are unassociated with disease phenotype. The strongest evidence for tumourigenesis mechanism in HLRCC supports a hypoxia inducible factor driven process called pseudohypoxia resulting from accumulation of the fumarase substrate fumarate. In this study, to assess the importance of gene- or exon-level deletions or amplifications of FH in patients with HLRCC-associated phenotypes, multiplex ligation-dependent probe amplification (MLPA) method was used. One novel FH mutation, deletion of exon 1, was found in a Swedish male patient with an evident HLRCC phenotype with CLM, RCC, and a family history of ULM and RCC. Six other patients with CLM and 12 patients with only RCC or uterine leiomyosarcoma (ULMS) remained FH mutation-negative. These results suggest that copy number aberrations of FH or its exons are an infrequent cause of HLRCC and that only co-occurrence of benign tumour types justifies FH-mutation screening in RCC or ULMS patients. Determination of the genomic profile of 11 HLRCC-associated RCCs from Finnish patients was performed by array comparative genomic hybridization. The most common copy number aberrations were gains of 2, 7, and 17 and losses of 13q12.3-q21.1, 14, 18, and X. When compared to aberrations of sporadic papillary RCCs, HLRCC-associated RCCs harboured a distinct DNA copy number profile and lacked many of the changes characterizing the sporadic RCCs. The findings suggest a divergent molecular pathway for tumourigenesis of papillary RCCs in HLRCC. In order to find a genetic modifier of RCC risk in HLRCC, genome-wide linkage and identical by descent (IBD) analysis studies were performed in Finnish HLRCC families with microsatellite marker mapping and SNP-array platforms. The linkage analysis identified only one locus of interest, the FH gene locus in 1q43, but no mutations were found in the genes of the region. IBD analysis yielded no convincing haplotypes shared by RCC patients. Although these results do not exclude the existence of a genetic modifier for RCC risk in HLRCC, they emphasize the role of FH mutations in the malignant tumourigenesis of HLRCC. To study the benign tumours in HLRCC, genome-wide DNA copy number and gene expression profiles of sporadic and HLRCC ULMs were defined with modern SNP- and gene-expression array platforms. The gene expression array suggests novel genes involved in FH-deficient ULM tumourigenesis and novel genes with putative roles in propagation of sporadic ULM. Both the gene expression and copy number profiles of HLRCC ULMs differed from those of sporadic ULMs indicating distinct molecular basis of the FH-deficient HLRCC tumours.
  • Sironen, Tarja (Helsingin yliopisto, 2007)
    Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome. Finland has the highest documented incidence of NE with around 1000 cases diagnosed annually. PUUV is also found in other Scandinavian countries, Central Europe and the European part of Russia. PUUV belongs to the genus Hantavirus in the family Bunyaviridae. Hantaviruses are rodent-borne viruses each carried by a specific host that is persistently and asymptomatically infected by the virus. PUUV is carried by the bank voles (Myodes glareolus, previously known as Clethrionomys glareolus). Hantaviruses have co-evolved with their carrier rodents for millions of years and these host animals are the evolutionary scene of hantaviruses. In this study, PUUV sequences were recovered from bank voles captured in Denmark and Russian Karelia to study the evolution of PUUV in Scandinavia. Phylogenetic analysis of these strains showed a geographical clustering of genetic variants following the presumable migration pattern of bank voles during the recolonization of Scandinavia after the last ice age approximately 10 000 years ago. The currently known PUUV genome sequences were subjected to in-depth phylogenetic analyses and the results showed that genetic drift seems to be the major mechanism of PUUV evolution. In general, PUUV seems to evolve quite slowly following a molecular clock. We also found evidence for recombination in the evolution of some genetic lineages of PUUV. Viral microevolution was studied in controlled virus transmission in colonized bank voles and changes in quasispecies dynamics were recorded as the virus was transmitted from one animal to another. We witnessed PUUV evolution in vivo, as one synonymous mutation became repeatedly fixed in the viral genome during the experiment. The detailed knowledge on the PUUV diversity was used to establish new sensitive and specific detection methods for this virus. Direct viral invasion of the hypophysis was demonstrated for the first time in a lethal case of NE. PUUV detection was done by immunohistochemistry, in situ hybridization and RT-nested-PCR of the autopsy tissue samples.
  • Mustajoki, Sami (Helsingin yliopisto, 1999)
  • Seitsonen, Sanna (Helsingin yliopisto, 2008)
    Age-related macular degeneration (AMD; OMIM # 603075) is an eye disease of the elderly, signs of which appear after the age of 50. In the Western world it is a leading cause of permanent visual loss with a prevalence of 8.5% in persons under 54 years of age and of 37% in persons over 75 years of age. Early forms of AMD may be asymptomatic, but in the late forms usually a central scotoma in the visual field follows severely complicating daily tasks. Smoking, age, and genetic predisposition are known risk factors for AMD. Until recently no true susceptibility genes had been identified though the composition of drusen deposits, the hallmarks of AMD, has suggested that the complement system might play a role in the pathogenesis of AMD. When four groups reported in March 2005, that, on chromosome 1q32, a Y402H variant in the complement factor H (CFH) gene confers risk for AMD in independent Caucasian samples, a new period in the field of genetic research of AMD started. CFH is a key regulator of the complement system. Thus, it is logical to speculate, that it plays a role in the pathogenesis of AMD. We performed a case-control association study to analyse whether the CFH Y402H variant contain a risk for AMD in the Finnish population. Although the population of Finland represents a genetic isolate, the CFH Y402H polymorphism was associated with AMD also in our patient sample with similar risk allele frequencies as in the other Caucasian populations. We further evaluated the effects of this variant, but no association between lesion subtype (predominantly classic, minimally classic or occult lesion) or lesion size of neovascular AMD and the CFH Y402H variant was detected. Neither did the variant have an effect on the photodynamic therapy (PDT) outcome. The patients that respond to PDT carried the risk genotype as frequently as those who did not respond, and no difference was found in the number of PDT sessions needed in patients with or without the risk genotypes of CFH Y402H. Functional analyses, however, showed that the binding of C-reactive protein (CRP) to CFH was significantly reduced in patients with the risk genotype of Y402H. In the past two years, the LOC387715/ high-temperature requirement factor A1 (HTRA1) locus on 10q26 has also been repeatedly associated with AMD in several populations. The recent discovery of the LOC387715 protein on the mitochondrial outer membrane suggests that the LOC387715 gene, not HTRA1, is the true predisposing gene in this region, although its biological function is still unknown. In our Finnish patient material, patients with AMD carried the A69S risk genotype of LOC387715 more frequently than the controls. Also, for the first time, an interaction between the CFH Y402H and the LOC387715 A69S variants was found. The most recently detected susceptibilty gene of AMD, the complement component 3 (C3) gene, encodes the central component of the complement system, C3. In our Finnish sample, an additive gene effect for the C3 locus was detected, though weaker than the effects for the two main loci, CFH and LOC387715. Instead, the hemicentin-1 or the elongation of very long chain fatty acids-like 4 genes that have also been suggested as candidate genes for AMD did not carry a risk for AMD in the Finnish population. This was the first series of molecular genetic study of AMD in Finland. We showed that two common risk variants, CFH Y402H and LOC387715 A69S, represent a high risk of AMD also in the isolated Finnish population, and furthermore, that they had a statistical interaction. It was demonstrated that the CFH Y402H risk genotype affects the binding of CFH to CRP thus suggesting that complement indeed plays an important role in the pathogenesis of AMD.