51 resultados para sequencing

em Helda - Digital Repository of University of Helsinki


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The growing interest for sequencing with higher throughput in the last decade has led to the development of new sequencing applications. This thesis concentrates on optimizing DNA library preparation for Illumina Genome Analyzer II sequencer. The library preparation steps that were optimized include fragmentation, PCR purification and quantification. DNA fragmentation was performed with focused sonication in different concentrations and durations. Two column based PCR purification method, gel matrix method and magnetic bead based method were compared. Quantitative PCR and gel electrophoresis in a chip were compared for DNA quantification. The magnetic bead purification was found to be the most efficient and flexible purification method. The fragmentation protocol was changed to produce longer fragments to be compatible with longer sequencing reads. Quantitative PCR correlates better with the cluster number and should thus be considered to be the default quantification method for sequencing. As a result of this study more data have been acquired from sequencing with lower costs and troubleshooting has become easier as qualification steps have been added to the protocol. New sequencing instruments and applications will create a demand for further optimizations in future.

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Distraction in the workplace is increasingly more common in the information age. Several tasks and sources of information compete for a worker's limited cognitive capacities in human-computer interaction (HCI). In some situations even very brief interruptions can have detrimental effects on memory. Nevertheless, in other situations where persons are continuously interrupted, virtually no interruption costs emerge. This dissertation attempts to reveal the mental conditions and causalities differentiating the two outcomes. The explanation, building on the theory of long-term working memory (LTWM; Ericsson and Kintsch, 1995), focuses on the active, skillful aspects of human cognition that enable the storage of task information beyond the temporary and unstable storage provided by short-term working memory (STWM). Its key postulate is called a retrieval structure an abstract, hierarchical knowledge representation built into long-term memory that can be utilized to encode, update, and retrieve products of cognitive processes carried out during skilled task performance. If certain criteria of practice and task processing are met, LTWM allows for the storage of large representations for long time periods, yet these representations can be accessed with the accuracy, reliability, and speed typical of STWM. The main thesis of the dissertation is that the ability to endure interruptions depends on the efficiency in which LTWM can be recruited for maintaing information. An observational study and a field experiment provide ecological evidence for this thesis. Mobile users were found to be able to carry out heavy interleaving and sequencing of tasks while interacting, and they exhibited several intricate time-sharing strategies to orchestrate interruptions in a way sensitive to both external and internal demands. Interruptions are inevitable, because they arise as natural consequences of the top-down and bottom-up control of multitasking. In this process the function of LTWM is to keep some representations ready for reactivation and others in a more passive state to prevent interference. The psychological reality of the main thesis received confirmatory evidence in a series of laboratory experiments. They indicate that after encoding into LTWM, task representations are safeguarded from interruptions, regardless of their intensity, complexity, or pacing. However, when LTWM cannot be deployed, the problems posed by interference in long-term memory and the limited capacity of the STWM surface. A major contribution of the dissertation is the analysis of when users must resort to poorer maintenance strategies, like temporal cues and STWM-based rehearsal. First, one experiment showed that task orientations can be associated with radically different patterns of retrieval cue encodings. Thus the nature of the processing of the interface determines which features will be available as retrieval cues and which must be maintained by other means. In another study it was demonstrated that if the speed of encoding into LTWM, a skill-dependent parameter, is slower than the processing speed allowed for by the task, interruption costs emerge. Contrary to the predictions of competing theories, these costs turned out to involve intrusions in addition to omissions. Finally, it was learned that in rapid visually oriented interaction, perceptual-procedural expectations guide task resumption, and neither STWM nor LTWM are utilized due to the fact that access is too slow. These findings imply a change in thinking about the design of interfaces. Several novel principles of design are presented, basing on the idea of supporting the deployment of LTWM in the main task.

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The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

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Congenital lactase deficiency (CLD) (MIM 223000) is a rare autosomal recessive gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. The CLD locus was previously assigned by linkage and linkage disequilibrium analyses on 2q21 in 19 Finnish families. In this study, the molecular background of this disorder is reported. The CLD locus was refined in 32 CLD patients in 24 families by using microsatellite and single nucleotide polymorphism (SNP) haplotypes. Mutation analyses were performed by direct sequencing. We identified 5 distinct mutations in the lactase (LCT) gene, encoding the enzyme that hydrolyzes lactose in the intestinal lumen. These findings facilitate genetic testing of CLD in clinical practice and enable genetic counseling. The present data also provide the basis for detailed characterization of the molecular pathogenesis of this disorder. Adult-type hypolactasia (MIM 223100) (lactase non-persistence, lactose intolerance) is an autosomal recessive gastrointestinal condition that is a result of a decline in the activity of lactase in the intestinal lumen after weaning. Adult-type hypolactasia is considered to be a normal phenomenon among mammals and symptoms are remarkably milder than experienced in CLD. Recently, a variant C/T-13910 was shown to associate with the adult-type hypolactasia trait, locating 13.9 kb upstream of the LCT gene. In this study, the functional significance of the C/T-13910 variant was determined by studying the LCT mRNA levels in intestinal biopsy samples in children and adults with different genotypes. RT-PCR followed by solid-phase minisequencing was applied to determine the relative expression levels of the LCT alleles using an informative SNP located in exon 1. In children, the C-13910 allele was observed to be downregulated after five years of age in parallel with lactase enzyme activity. The expression of the LCT mRNA in the intestinal mucosa in individuals with the T-13910 A-22018 alleles was 11.5 times higher than that found in individuals with the C-13910, G-22018 alleles. These findings suggest that the C/T-13910 associated with adult-type hypolactasia is associated with the transcriptional regulation of the LCT gene. The presence of the T-13910 A-22018 allele also showed significant elevation lactase activity. Galactose, the hydrolysing product of the milk sugar lactose, has been hypothesized to be poisonous to ovarian epithelial cells. Hence, consumption of dairy products and lactase persistence has been proposed to be a risk factor for ovarian carcinoma. To investigate whether lactase persistence is related to the risk of ovarian carcinoma the C/T-13910 genotype was determined in a cohort of 782 women with ovarian carcinoma 1331 individuals serving as controls. Lactase persistence did not associate significantly with the risk for ovarian carcinoma in the Finnish, in the Polish or in the Swedish populations. The findings do not support the hypothesis that lactase persistence increases the risk for ovarian carcinoma.

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Primary pulmonary hypertension (PPH), or according to the recent classification idiopathic pulmonary hypertension (IPAH), is a rare, progressive disease of pulmonary vasculature leading to pulmonary hypertension and right heart failure. Most of the patients are sporadic but in about 6% of cases the disease is familial (FPPH). In 2000 two different groups identified the gene predisposing to PPH. This gene, Bone morphogenetic protein receptor type 2 (BMPR2), encodes a subunit of transforming growth factor β (TGF-β) receptor complex. There is a genetic connection between PPH and hereditary hemorrhagic telangiectasia (HHT), a bleeding disorder characterized by local telangiectasias and sometimes with pulmonary hypertension. In HHT, mutations in ALK1 (activin like kinase type 1) and Endoglin, another members of the TGF-β signaling pathway are found. In this study we identified all of the Finnish PPH patients for the years 1986-1999 using the hospital discharge registries of Finnish university hospitals. During this period we found a total of 59 confirmed PPH patients: 55 sporadic and 4 familial representing 3 different families. In 1999 the prevalence of PPH was 5.8 per million and the annual incidence varied between 0.2-1.3 per million. Among 28 PPH patients studied, heterozygous BMPR2 mutations were found in 12% (3/26) of sporadic patients and in 33% of the PPH families (1/3). All the mutations found were different. Large deletions of BMPR2 were excluded by single-stranded chain polymomorphism analysis. As a candidate gene approach we also studied ALK1, Endoglin, Bone Morphogenetic Receptor Type IA (BMPR1A or ALK3), Mothers Against Decapentaplegic Homolog 4 (SMAD4) and Serotonine Transporter Gene (SLC6A4) using single-strand conformational polymorphism (SSCP) analysis and direct sequencing. Among patients and family members studied, we found two mutations in ALK1 in two unrelated samples. We also identified all the HHT patients treated at the Department of Otorhinolaryngology at Helsinki University Central Hospital between the years of 1990-2005 and 8 of the patients were studied for Endoglin and ALK1 mutations using direct sequencing. A total of seven mutations were found and all the mutations were different. The absence of a founder mutation in the Finnish population in both PPH and HHT was somewhat surprising. This suggests that the mutations of BMPR2, ALK1 and Endoglin are quite young and the older mutations have been lost due to repetitive genetic bottlenecks and/or negative selection. Also, other genes than BMPR2 may be involved in the pathogenesis of PPH. No founder mutations were found in PPH or HHT and thus no simple genetic test is available for diagnostics.

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Colorectal cancer (CRC) is the third most common cancer in Finland. Of all CRC tumors, 15% display microsatellite-instability (MSI) caused by defective cellular mismatch repair. Cells displaying MSI accumulate a high number of mutations genome-wide, especially in short repeat areas, microsatellites. When targeting genes essential for cell growth or death, MSI can promote tumorigenesis. In non-coding areas, microsatellite mutations are generally considered as passenger events. Since the discovery of MSI and its linkage to cancer, more that 200 genes have been investigated for a role in MSI tumorigenesis. Although various criteria have been suggested for MSI target gene identification, the challenge has been to distinguish driver mutations from passenger mutations. This study aimed to clarify these key issues in the research field of MSI cancer. Prior to this, background mutation rate in MSI cancer has not been studied in a large-scale. We investigated the background mutation rate in MSI CRC by analyzing the spectrum of microsatellite mutations in non-coding areas. First, semenogelin I was studied for a possible role in MSI carcinogenesis. The intronic T9 repeat of semenogelin I was frequently mutated but no evidence for selection during tumorigenesis was obtained. Second, a sequencing approach was utilized to evaluate the general background mutation rate in MSI CRC. Both intronic and intergenic repeats harbored extremely high mutation rates of ≤ 87% and intergenic repeats were more unstable than the intronic repeats. As mutation rates of presumably neutral microsatellites can be high in MSI CRC in the absence of apparent selection pressure, high mutation frequency alone is not sufficient evidence for identification of driver MSI target genes. Next, an unbiased approach was designed to identify the mutatome of MSI CRC. By combining expression array data and a database search we identified novel genes possibly related to MSI CRC carcinogenesis. One of the genes was studied further. In the functional analysis this gene was observed to cause an abnormal cancer-prone cellular phenotype, possibly through altered responses to DNA damage. In our recent study, smooth muscle myosin heavy chain 11 (MYH11) was identified as a novel MSI CRC gene. Additionally, MYH11 has a well established role in acute myeloid leukemia (AML) through an oncogenic fusion protein CBFB-MYH11. We investigated further the role of MYH11 in AML by sequencing. Three novel missense variants of MYH11 were identified. None of the variants were present in the population-based control material. One of the identified variants, V71A, lies in the N-terminal SH3-like domain of MYH11 of unknown function. The other two variants, K1059E and R1792Q are located in the coil-coiled myosin rod essential for the regulation and filament formation of MYH11. The variant K1059E lies in the close proximity of the K1044N that has been functionally assessed in our earlier work of CRC and has been reported to cause total loss of MYH11 protein regulation. As the functional significance of the three novel variants examined in this work remains unknown, future studies should clarify the further role of MYH11 in AML leukaemogenesis and in other malignancies.

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Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

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Positional cloning has enabled hypothesis-free, genome-wide scans for genetic factors contributing to disorders or traits. Traditionally linkage analysis has been used to identify regions of interest, followed by meticulous fine mapping and candidate gene screening using association methods and finally sequencing of regions of interest. More recently, genome-wide association analysis has enabled a more direct approach to identify specific genetic variants explaining a part of the variance of the phenotype of interest. Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization. Here, we have utilized modern molecular genetic methods combined with the advantages provided by the special population structure in Finland to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within a population isolate. Stratification based on clinical phenotype resulted in encouraging results, as previously identified linkage to 3p14-p24 was replicated in an independent family set of families with Asperger syndrome, but no other ASDs. Fine-mapping of the previously identified linkage peak for ASDs at 3q25-q27 revealed association between autism and a subunit of the 5-hydroxytryptamine receptor 3C (HTR3C). We also used dense, genome-wide single nucleotide polymorphism (SNP) data to characterize the population structure of Finns. We observed significant population substructure which correlates with the known history of multiple consecutive bottle-necks experienced by the Finnish population. We used this information to ascertain a genetically homogenous subset of autism families to identify possible rare, enriched risk variants using genome-wide SNP data. No rare enriched genetic risk factors were identified in this dataset, although a subset of families could be genealogically linked to form two extended pedigrees. The lack of founder mutations in this isolated population suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. The results of this study are consistent with others in the field. The underlying genetic architecture for this group of disorders appears highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and only explain a subset of the genetic risk in the general population in spite of their high penetrance within individual families. The results of this study, together with other results obtained in this field, indicate that family specific linkage, homozygosity mapping and resequencing efforts are needed to identify these rare genetic risk factors.

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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

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My work describes two sectors of the human bacterial environment: 1. The sources of exposure to infectious non-tuberculous mycobacteria. 2. Bacteria in dust, reflecting the airborne bacterial exposure in environments protecting from or predisposing to allergic disorders. Non-tuberculous mycobacteria (NTM) transmit to humans and animals from the environment. Infection by NTM in Finland has increased during the past decade beyond that by Mycobacterium tuberculosis. Among the farm animals, porcine mycobacteriosis is the predominant NTM disease in Finland. Symptoms of mycobacteriosis are found in 0.34 % of slaughtered pigs. Soil and drinking water are suspected as sources for humans and bedding materials for pigs. To achieve quantitative data on the sources of human and porcine NTM exposure, methods for quantitation of environmental NTM are needed. We developed a quantitative real-time PCR method, utilizing primers targeted at the 16S rRNA gene of the genus of Mycobacterium. With this method, I found in Finnish sphagnum peat, sandy soils and mud high contents of mycobacterial DNA, 106 to 107 genome equivalents per gram. A similar result was obtained by a method based on the Mycobacterium-specific hybridization of 16S rRNA. Since rRNA is found mainly in live cells, this result shows that the DNA detected by qPCR mainly represented live mycobacteria. Next, I investigated the occurrence of environmental mycobacteria in the bedding materials obtained from 5 pig farms with high prevalence (>4 %) of mycobacteriosis. When I used for quantification the same qPCR methods as for the soils, I found that piggery samples contained non-mycobacterial DNA that was amplified in spite of several mismatches with the primers. I therefore improved the qPCR assay by designing Mycobacterium-specific detection probes. Using the probe qPCR assay, I found 105 to 107 genome equivalents of mycobacterial DNA in unused bedding materials and up to 1000 fold more in the bedding collected after use in the piggery. This result shows that there was a source of mycobacteria in the bedding materials purchased by the piggery and that mycobacteria increased in the bedding materials during use in the piggery. Allergic diseases have reached epidemic proportions in urbanized countries. At the same time, childhood in rural environment or simple living conditions appears to protect against allergic disorders. Exposure to immunoreactive microbial components in rural environments seems to prevent allergies. I searched for differences in the bacterial communities of two indoor dusts, an urban house dust shown to possess immunoreactivity of the TH2-type and a farm barn dust with TH1-activity. The immunoreactivities of the dusts were revealed by my collaborators, in vitro in human dendritic cells and in vivo in mouse. The dusts accumulated >10 years in the respiratory zone (>1.5 m above floor), thus reflecting the long-term content of airborne bacteria at the two sites. I investigated these dusts by cloning and sequencing of bacterial 16S rRNA genes from dust contained DNA. From the TH2-active urban house dust, I isolated 139 16S rRNA gene clones. The most prevalent genera among the clones were Corynebacterium (5 species, 34 clones), Streptococcus (8 species, 33 clones), Staphylococcus (5 species, 9 clones) and Finegoldia (1 species, 9 clones). Almost all of these species are known as colonizers of the human skin and oral cavity. Species of Corynebacterium and Streptococcus have been reported to contain anti-inflammatory lipoarabinomannans and immunmoreactive beta-glucans respectively. Streptococcus mitis, found in the urban house dust is known as an inducer of TH2 polarized immunity, characteristic of allergic disorders. I isolated 152 DNA clones from the TH1-active farm barn dust and found species quite different from those found from the urban house dust. Among others, I found DNA clones representing Bacillus licheniformis, Acinetobacter lwoffii and Lactobacillus each of which was recently reported to possess anti-allergy immunoreactivity. Moreover, the farm barn dust contained dramatically higher bacterial diversity than the urban house dust. Exposure to this dust thus stimulated the human dendritic cells by multiple microbial components. Such stimulation was reported to promote TH1 immunity. The biodiversity in dust may thus be connected to its immunoreactivity. Furthermore, the bacterial biomass in the farm barn dust consisted of live intact bacteria mainly. In the urban house dust only ~1 % of the biomass appeared as intact bacteria, as judged by microscoping. Fragmented microbes may possess bioactivity different from that of intact cells. This was recently shown for moulds. If this is also valid for bacteria, the different immunoreactivities of the two dusts may be explained by the intactness of dustborne bacteria. Based on these results, we offer three factors potentially contributing to the polarized immunoreactivities of the two dusts: (i) the species-composition, (ii) the biodiversity and (iii) the intactness of the dustborne bacterial biomass. The risk of childhood atopic diseases is 4-fold lower in the Russian compared with the Finnish Karelia. This difference across the country border is not explainable by different geo-climatic factors or genetic susceptibilities of the two populations. Instead, the explanation must be lifestyle-related. It has already been reported that the microbiological quality of drinking water differs on the two sides of the borders. In collaboration with allergists, I investigated dusts collected from homes in the Russian Karelia and in the Finnish Karelia. I found that bacterial 16S rRNA genes cloned from the Russian Karelian dusts (10 homes, 234 clones) predominantly represented Gram-positive taxa (the phyla Actinobacteria and Firmicutes, 67%). The Russian Karelian dusts contained nine-fold more of muramic acid (60 to 70 ng mg-1) than the Finnish Karelian dusts (3 to 11 ng mg-1). Among the DNA clones isolated from the Finnish side (n=231), Gram-negative taxa (40%) outnumbered the Gram-positives (34%). Out of the 465 DNA clones isolated from the Karelian dusts, 242 were assigned to cultured validly described bacterial species. In Russian Karelia, animal-associated species e.g. Staphylococcus and Macrococcus were numerous (27 clones, 14 unique species). This finding may connect to the difference in the prevalence of allergy, as childhood contacts with pets and farm animals have been connected with low allergy risk. Plant-associated bacteria and plant-borne 16S rRNA genes (chloroplast) were frequent among the DNA clones isolated from the Finnish Karelia, indicating components originating from plants. In conclusion, my work revealed three major differences between the bacterial communtites in the Russian and in the Finnish Karelian homes: (i) the high prevalence of Gram-positive bacteria on the Russian side and of Gram-negative bacteria on the Finnish side and (ii) the rich presence of animal-associated bacteria on the Russian side whereas (iii) plant-associated bacteria prevailed on the Finnish side. One or several of these factors may connect to the differences in the prevalence of allergy.

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Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

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The present study focuses on the translational strategies of Cocksfoot mottle virus (CfMV, genus Sobemovirus), which infects monocotyledonous plants. CfMV RNA lacks the 5'cap and the 3'poly(A) tail that ensure efficient translation of cellular messenger RNAs (mRNAs). Instead, CfMV RNA is covalently linked to a viral protein VPg (viral protein, genome-linked). This indicates that the viral untranslated regions (UTRs) must functionally compensate for the lack of the cap and poly(A) tail. We examined the efficacy of translation initiation in CfMV by comparing it to well-studied viral translational enhancers. Although insertion of the CfMV 5'UTR (CfMVe) into plant expression vectors improved gene expression in barley more than the other translational enhancers examined, studies at the RNA level showed that CfMVe alone or in combination with the CfMV 3'UTR did not provide the RNAs translational advantage. Mutation analysis revealed that translation initiation from CfMVe involved scanning. Interestingly, CfMVe also promoted translation initiation from an intercistronic position of dicistronic mRNAs in vitro. Furthermore, internal initiation occurred with similar efficacy in translation lysates that had reduced concentrations of eukaryotic initiation factor (eIF) 4E, suggesting that initiation was independent of the eIF4E. In contrast, reduced translation in the eIF4G-depleted lysates indicated that translation from internally positioned CfMVe was eIF4G-dependent. After successful translation initiation, leaky scanning brings the ribosomes to the second open reading frame (ORF). The CfMV polyprotein is produced from this and the following overlapping ORF via programmed -1 ribosomal frameshift (-1 PRF). Two signals in the mRNA at the beginning of the overlap program approximately every fifth ribosome to slip one nucleotide backwards and continue translation in the new -1 frame. This leads to the production of C-terminally extended polyprotein, which encodes the viral RNA-dependent RNA polymerase (RdRp). The -1 PRF event in CfMV was very efficient, even though it was programmed by a simple stem-loop structure instead of a pseudoknot, which is usually required for high -1 PRF frequencies. Interestingly, regions surrounding the -1 PRF signals improved the -1 PRF frequencies. Viral protein P27 inhibited the -1 PRF event in vivo, putatively by binding to the -1 PRF site. This suggested that P27 could regulate the occurrence of -1 PRF. Initiation of viral replication requires that viral proteins are released from the polyprotein. This is catalyzed by viral serine protease, which is also encoded from the polyprotein. N-terminal amino acid sequencing of CfMV VPg revealed that the junction of the protease and VPg was cleaved between glutamate (E) and asparagine (N) residues. This suggested that the processing sites used in CfMV differ from the glutamate and serine (S) or threonine (T) sites utilized in other sobemoviruses. However, further analysis revealed that the E/S and E/T sites may be used to cleave out some of the CfMV proteins.

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The chemical composition of breast milk has been studied in detail in the past decades. Hundreds of new antibacterial and antiviral components have been found. Several molecules have been found to promote the proper function of neonatal intestine. However, microbiological studies of breast milk have been, until recently, focused mainly on detecting harmful and pathogenic bacteria and viruses. Natural microbial diversity of human milk has not been widely studied before the work reported in this thesis. This is mainly because breast milk has traditionally been thought to be sterile - even if a certain amount of commensal bacteria have usually been detected in milk samples. The first part of this licentiate thesis contains a short literature review about the anatomy and physiology of breast feeding, human milk chemical and microbiological composition, mastitis, intestinal flora and bacteriocins. The second part reports on the experiments of the licentiate work, concentrating on the microbial diversity in the milk of healthy breast-feeding mothers, and the ability of these bacteria to produce antibacterial substances against pathogenic bacteria. The results indicate that human milk is a source of commensal bacteria for infant intestine. 509 random isolates from 40 breast milk samples were isolated and identified by 16S rRNA sequencing. Median bacterial count was about 600 colony forming units per milliliter. Over half of the isolates were staphylococci, and almost one third streptococci. The most common species were skin bacteria Staphylococcus epidermidis and oral bacteria Streptococcus salivarius and Streptococcus mitis. Lactic acid bacteria, identified as members of Lactobacillus-, Lactococcus- and Leuconostoc -genera, were found in five milk samples. Enterococci were found in three samples. A novel finding in this study is the capability of these commensal bacteria to inhibit the growth of pathogens. In 90 precent of the milk samples commensal bacteria inhibiting the growth of Staphylococcus aureus were found. In 40 precent of samples the colonies could block the growth completely. One fifth of the isolated Staph. epidermidis strains, half of Str. salivarius strains, and all lactic acid bacteria and enterococci could inhibit or block the growth of Staph. aureus. In further study also Listeria innocua- and Micrococcus luteus active isolates were found in 33 and 11 precent of milk samples (out of 140). Furthermore, two Lactococcus lactis isolates from the breast milk were shown to produce bacteriocin nisin, which is an antimicrobial molecule used as a food preservative. The importance of these human milk commensal bacteria in the development of newborn intestinal flora and immune system, as well as in preventing maternal breast infections, should be further explored.

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Kontrolloidut radikaalipolymerointimenetelmät, kuten RAFT-polymerointi, ovat moderni tapa valmistaa polymeerejä säädellysti. RAFT-polymeroinnilla polymeerien ketjunpituutta, moolimassajakaumaa, mikrorakennetta (taktisuus, järjestys), koostumusta ja funktionaalisuutta kyetään hallitsemaan. Siten menetelmällä voidaan valmistaa uudenlaisia polymeeriarkkitektuureja, kuten blokki- ja tähtipolymeerejä, sekä hybridimateriaaleja ja biokonjugaatteja. Polymeeristen rakennuspalikoiden itsejärjestyminen, missä huolellisesti syntetisoidut polymeerit järjestyvät halutulla tavalla nanoskaalassa, on suosittu tutkimuskohde materiaalitieteessä. On huomattava, että blokkipolymeerien itsejärjestyminen on vielä suhteellisen nuori tutkimusaihe. Tämän hetkiset polymeeriset nanomateriaalit ovat suhteellisen yksinkertaisia luonnon luomuksiin verrattuina, tarjoten jatkuvasti uusia mahdollisuuksia seuraavan sukupolven polymeereille. Tässä työssä RAFT-polymeroinnilla syntetisoitiin amfifiilisiä di- ja triblokkikopolymeerejä sekä tutkittiin niiden järjestymistä nanorakenteiksi. Kaikissa blokkikopolymeereissä käytettiin lämpöherkkää poly(N-isopropyyliakryyliamidia). Siten polymeerit ja tutkitut materiaalit reagoivat lämpötilanmuutokseen ympäristössä eli ovat ns. ympäristöherkkiä. Työssä tutkittiin taktisuuden kontrollointia N-isopropyyliakryyliamidin RAFT-polymeroinnissa. Polymeerin taktisuutta sekä ketjunpituutta ja blokkijärjestystä säätämällä voitiin hallita polymeerin itsejärjestymistä vesiliuoksessa. Amfifiiliset polymeerit järjestyivät laimeissa vesiliuoksissa erilaisiksi misellirakenteiksi, muodostaen ns. mikrosäiliöitä. Tällaisilla polymeereillä odotetaan olevan sovelluksia esim. lääkeainevapautuksessa. Amfifiilejä käytetään myös esimerkiksi apuaineina pinnoitteissa ja kosmetiikassa. Kiinteässä tilassa tutkitut triblokkikopolymeerit muodostivat teoreettisesti ennustettuja morfologioita. Lämpöherkän materiaalin hydrogeelit toimivat suodatinmembraanina nanokokoluokassa. RAFT-polymeroinnilla syntetisoituja polymeereja voidaan sellaisenaan käyttää kultananopartikkeleiden päällystämiseen. Kultananopartikkelit ovat erittäin kiinostavia mm. niiden stabiilisuuden ja ainutlaatuisten pintaominaisuuksien vuoksi. Kun amfifiilisiä polymeerejä kiinnitettiin kultapartikkelin pinnalle, sen liuos- ja optisia ominaisuuksia voitiin säädellä pH:n ja lämpötilan avulla. Tällaisilla kultananopartikkeleilla on sovelluksia mm. diagnostiikassa, sensoreina ja solukuvauksessa.

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The purpose of this study is to describe the development of application of mass spectrometry for the structural analyses of non-coding ribonucleic acids during past decade. Mass spectrometric methods are compared of traditional gel electrophoretic methods, the characteristics of performance of mass spectrometric, analyses are studied and the future trends of mass spectrometry of ribonucleic acids are discussed. Non-coding ribonucleic acids are short polymeric biomolecules which are not translated to proteins, but which may affect the gene expression in all organisms. Regulatory ribonucleic acids act through transient interactions with key molecules in signal transduction pathways. Interactions are mediated through specific secondary and tertiary structures. Posttranscriptional modifications in the structures of molecules may introduce new properties to the organism, such as adaptation to environmental changes or development of resistance to antibiotics. In the scope of this study, the structural studies include i) determination of the sequence of nucleobases in the polymer chain, ii) characterisation and localisation of posttranscriptional modifications in nucleobases and in the backbone structure, iii) identification of ribonucleic acid-binding molecules and iv) probing of higher order structures in the ribonucleic acid molecule. Bacteria, archaea, viruses and HeLa cancer cells have been used as target organisms. Synthesised ribonucleic acids consisting of structural regions of interest have been frequently used. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) have been used for ionisation of ribonucleic analytes. Ammonium acetate and 2-propanol are common solvents for ESI. Trihydroxyacetophenone is the optimal MALDI matrix for ionisation of ribonucleic acids and peptides. Ammonium salts are used in ESI buffers and MALDI matrices as additives to remove cation adducts. Reverse phase high performance liquid chromatography has been used for desalting and fractionation of analytes either off-line of on-line, coupled with ESI source. Triethylamine and triethylammonium bicarbonate are used as ion pair reagents almost exclusively. Fourier transform ion cyclotron resonance analyser using ESI coupled with liquid chromatography is the platform of choice for all forms of structural analyses. Time-of-flight (TOF) analyser using MALDI may offer sensitive, easy-to-use and economical solution for simple sequencing of longer oligonucleotides and analyses of analyte mixtures without prior fractionation. Special analysis software is used for computer-aided interpretation of mass spectra. With mass spectrometry, sequences of 20-30 nucleotides of length may be determined unambiguously. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Sequencing in conjunction with other structural studies enables accurate localisation and characterisation of posttranscriptional modifications and identification of nucleobases and amino acids at the sites of interaction. High throughput screening methods for RNA-binding ligands have been developed. Probing of the higher order structures has provided supportive data for computer-generated three dimensional models of viral pseudoknots. In conclusion. mass spectrometric methods are well suited for structural analyses of small species of ribonucleic acids, such as short non-coding ribonucleic acids in the molecular size region of 20-30 nucleotides. Structural information not attainable with other methods of analyses, such as nuclear magnetic resonance and X-ray crystallography, may be obtained with the use of mass spectrometry. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Ligand screening may be used in the search of possible new therapeutic agents. Demanding assay design and challenging interpretation of data requires multidisclipinary knowledge. The implement of mass spectrometry to structural studies of ribonucleic acids is probably most efficiently conducted in specialist groups consisting of researchers from various fields of science.