Examination of Genetic Components Affecting Human Obesity-Related Quantitative Traits

Obesity increases the risk for several conditions, including type 2 diabetes mellitus, cardiovascular disease, hypertension, osteoarthirits and certain types of cancer. Twin- and family studies have shown that there is a major genetic component in the determination of body mass. In recent years several technological and scientific advance have been made in obesity research. For instance, novel replicated loci have been revealed by a number of genome wide association studies. This thesis aimed to investigate the association of genetic factors and obesity-related quantitative traits. The first study investigated the role of the lactase gene in anthropometric traits. We genetically defined lactose persistence by genotyping 31 720 individuals of European descent. We found that lactase persistence was significantly correlated with weight and body mass index but not with height. In the second study we performed the largest whole genome linkage scan for body mass index to date. The sample consisted of 4401 twin families and 10 535 individuals from six European countries. We found supporting evidence for two loci (3q29 and 7q36). We observed that the heritability estimate increased substantially when additional family members were removed from the analyses, which suggests reduced environmental variance in the twin sample. In the third study we assessed metabonomic, transcriptomic and genomic variation in a Finnish population cohort of 518 individuals. We formed gene expression networks to portray pathways and showed that a set of highly correlated genes of an inflammatory pathway associated with 80 serum metabolites (of 134 quantified measures). Strong association was found, for example, with several lipoprotein subclasses. We inferred causality by using genetic variation as anchors. The expression of the network genes was found to be dependent on the circulatory metabolite concentrations.

Impact of the Human Bacterial Environment on Mycobacteriosis and Allergy

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.

Genetic and environmental influences on human responses to odors

Olfaction, the sense of smell, has many important functions in humans. Human responses to odors show substantial individual variation. Olfactory receptor genes have been identified and other genes may also influence olfaction. However, the proportion of phenotypic variation in odor response due to genetic variation remains largely unknown. Little is also known about which genes modify specific responses to odors. This study aimed to elucidate genetic and environmental influences on human responses to odors. Individuals from Finnish families (n=146) and Australian (n=413), British (n=163), Danish (n=336), and Finnish (n=399) twins rated intensity and pleasantness of a set of 12 (families) or 6 (twins) odors and tried to identify the odors. In addition, the participants rated their own sense of smell and annoyance experienced with different environmental odors. The odor stimuli of a commercial smell test (The Brief Smell Identification Test; banana, chocolate, cinnamon, gasoline, lemon, onion, paint thinner, pineapple, rose, smoke, soap, and turpentine) were presented in the family study. Based on the results of the family study and a literature survey, a new set of odor stimuli (androstenone, chocolate, cinnamon, isovaleric acid, lemon, and turpentine) was designed for the twin studies. In the family sample, heritabilities of the traits were estimated and underlying genomic regions were searched using a genome-wide linkage scan. In the pooled twin sample, variation in the measured traits was decomposed into genetic and environmental components using quantitative genetic modeling. In addition, associations between nongenetic factors (e.g., sex, age, and smoking) and olfactory-related traits were explored. Suggestive evidence for a genetic linkage for pleasantness of cinnamon at a locus on chromosome 4q32.3 emerged from the family sample. High heritability for the pleasantness of cinnamon was found in the family but not the twin study. Heritability of perceived intensity of androstenone odor was determined to be ~30% in the twin sample. A strong genetic correlation between perceived intensity and pleasantness of androstenone, in the absence of any environmental correlation, indicated that only the genetic correlation explained the phenotypic correlation between the traits (r=-0.27) and that the traits were influenced by an overlapping set of genes. Self-rated olfactory function appeared to reflect the odor annoyance experienced rather than actual olfactory acuity or genetic involvement. Results from nongenetic analyses supported the speculated superiority of females' olfactory abilities, the age-related diminishing of olfactory acuity, and the influences of experience-dependent factors on odor responses. This was the first study to estimate heritabilities and perform linkage screens for individual odors. A genetic effect was detected for only a few responses to specific odors, suggesting the predominance of environmental effects in odor perceptions.

Nutrition and bone metabolism : In vivo effects of inorganic phosphate on rats and in vitro effects of bioactive tripeptides on human osteoblasts

Nutrition affects bone health throughout life. To optimize peak bone mass development and maintenance, it is important to pay attention to the dietary factors that enhance and impair bone metabolism. In this study, the in vivo effects of inorganic dietary phosphate and the in vitro effects of bioactive tripeptides, IPP, VPP and LKP were investigated. Dietary phosphate intake is increased through the use of convenience foods and soft drinks rich in phosphate-containing food additives. Our results show that increased dietary phosphate intake hinders mineral deposition in cortical bone and diminishes bone mineral density (BMD) in the aged skeleton in a rodent model (Study I). In the growing skeleton (Study II), increased phosphate intake was observed to reduce bone material and structural properties, leading to diminished bone strength. Studies I and II revealed that a low Ca:P ratio has negative effects on the mature and growing rat skeleton even when calcium intake is sufficient. High dietary protein intake is beneficial for bone health. Protein is essential for bone turnover and matrix formation. In addition, hydrolysis of proteins in the gastrointestinal tract produces short peptides that possess a biological function beyond that of being tissue building blocks. The effects of three bioactive tripeptides, IPP, VPP and LKP, were assessed in short- and long-term in vitro experiments. Short-term treatment (24 h) with tripeptide IPP, VPP or LKP influenced osteoblast gene expression (Study III). IPP in particular, regulates genes associated with cell differentiation, cell growth and cell signal transduction. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization (Study IV). The in vivo effects of IPP on osteoblast differentiation might differ since eating frequency drives food consumption, and protein degradation products, such as bioactive peptides, are available periodically, not continuously as in this study. To sum up, Studies I and II raise concern about the appropriate amount of dietary phosphate to support bone health as excess is harmful. Studies III and IV in turn, support findings of the beneficial effects of dietary protein on bone and provide a mechanistic explanation since cell proliferation and osteoblast function were improved by treatment with bioactive tripeptide IPP.

The effects of human induced disturbances on the sediment geochemistry of Baltic Sea embayments with a focus on eutrophication history

Historical sediment nutrient concentrations and heavy-metal distributions were studied in five embayments in the Gulf of Finland and an adjacent lake. The main objective of the study was to examine the response of these water bodies to temporal changes in human activities. Sediment cores were collected from the sites and dated using 210Pb and 137Cs. The cores were analyzed for total carbon (TC), total nitrogen (TN), total phosphorus (TP), organic phosphorus (OP), inorganic phosphorus (IP), biogenic silica (BSi), loss on ignition (LOI), grain size, Cu, Zn, Al, Fe, Mn, K, Ca, Mg and Na. Principal component analysis (PCA) was used to summarize the trends in the geochemical variables and to compare trends between the different sites. The links between the catchment land use and sediment geochemical data were studied using a multivariate technique of redundancy analysis (RDA). Human activities produce marked geochemical variations in coastal sediments. These variations and signals are often challenging to interpret due to various sedimentological and post-depositional factors affecting the sediment profiles. In general, the sites studied here show significant upcore increases in sedimentation rates, TP and TN concentrations. Also Cu, which is considered to be a good indicator of anthropogenic influence, showed clear increases from 1850 towards the top part of the cores. Based on the RDA-analysis, in the least disturbed embayments with high forest cover, the sediments are dominated by lithogenic indicators Fe, K, Al and Mg. In embayments close to urban settlement, the sediments have high Cu concentrations and a high sediment Fe/Mn ratio. This study suggests that sediment accumulation rates vary significantly from site to site and that the overall sedimentation can be linked to the geomorphology and basin bathymetry, which appear to be the major factors governing sedimentation rates; i.e. a high sediment accumulation rate is not characteristic either to urban or to rural sites. The geochemical trends are strongly site specific and depend on the local geochemical background, basin characteristics and anthropogenic metal and nutrient loading. Of the studied geochemical indicators, OP shows the least monotonic trends in all studied sites. When compared to other available data, OP seems to be the most reliable geochemical indicator describing the trophic development of the study sites, whereas Cu and Zn appear to be good indicators for anthropogenic influence. As sedimentation environments, estuarine and marine sites are more complex than lacustrine basins with multiple sources of sediment input and more energetic conditions in the former. The crucial differences between lacustrine and estuarine/coastal sedimentation environments are mostly related to Fe. P sedimentation is largely governed by Fe redox-reactions in estuarine environments. In freshwaters, presence of Fe is clearly linked to the sedimentation of other lithogenic metals, and therefore P sedimentation and preservation has a more direct linkage to organic matter sedimentation.

The high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.

Microbe-induced Innate Immune Responses in Human Dendritic Cells

Two types of antigen-presenting cells (APCs), macrophages and dendritic cells (DCs), function at the interface of innate and adaptive immunity. Through recognition of conserved microbial patterns, they are able to detect the invading pathogens. This leads to activation of signal transduction pathways that in turn induce gene expression of various molecules required for immune responses and eventually pathogen clearance. Cytokines are among the genes induced upon detection of microbes. They play an important role in regulating host immune responses during microbial infection. Chemotactic cytokines, chemokines, are involved in migratory events of immune cells. Cytokines also promote the differentiation of distinct T cell responses. Because of the multiple roles of cytokines in the immune system, the cytokine network needs to be tightly regulated. In this work, the induction of innate immune responses was studied using human primary macrophages or DCs as cell models. Salmonella enterica serovar Typhimurium served as a model for an intracellular bacterium, whereas Sendai virus was used in virus experiments. The starting point of this study was that DCs of mouse origin had recently been characterized as host cells for Salmonella. However, only little was known about the immune responses initiated in Salmonella-infected human DCs. Thus, cellular responses of macrophages and DCs, in particular the pattern of cytokine production, to Salmonella infection were compared. Salmonella-induced macrophages and DCs were found to produce multiple cytokines including interferon (IFN) -gamma, which is conventionally produced by T and natural killer (NK) cells. Both macrophages and DCs also promoted the intracellular survival of the bacterium. Phenotypic maturation of DCs as characterized by upregulation of costimulatory and human leukocyte antigen (HLA) molecules, and production of CCL19 chemokine, were also detected upon infection with Salmonella. Another focus of this PhD work was to unravel the regulatory events controlling the expression of cytokine genes encoding for CCL19 and type III IFNs, which are central to DC biology. We found that the promoters of CCL19 and type III IFNs contain similar regulatory elements that bind nuclear factor kappaB (NF-kappaB) and interferon regulatory factors (IRFs), which could mediate transcriptional activation of the genes. The regulation of type III IFNs in virus infection resembled that of type I IFNs a cytokine class traditionally regarded as antiviral. The induction of type I and type III IFNs was also observed in response to bacterial infection. Taken together, this work identifies new details about the interaction of Salmonella with its phagocytic host cells of human origin. In addition, studies provide information on the regulatory events controlling the expression of CCL19 and the most recently identified IFN family genes, type III IFN genes.

Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.

Exposure-related human cancer: Molecular changes in sinonasal cancer and lung cancer, with focus on TP53 mutations

Cancer is a leading cause of death worldwide and the total number of cancer cases continues to increase. Many cancers, for example sinonasal cancer and lung cancer, have clear external risk factors and so are potentially preventable. The occurrence of sinonasal cancer is strongly associated with wood dust exposure and the main risk factor for lung cancer is tobacco smoking. Although the molecular mechanisms involved in lung carcinogenesis have been widely studied, very little is known about the molecular changes leading to sinonasal cancer. In this work, mutations in the tumour suppressor TP53 gene in cases of sinonasal cancer and lung cancer and the associations of these mutations with exposure factors were studied. In addition, another important mechanism in many cancers, inflammation, was explored by analyzing the expression of the inflammation related enzyme, COX-2, in sinonasal cancer. The results demonstrate that TP53 mutations are frequent in sinonasal cancer and lung cancer and in both cancers they are associated with exposure. In sinonasal cancer, the occurrence of TP53 mutation significantly increased in relation to long duration and high level of exposure to wood dust. Smoking was not associated with the overall occurrence of the TP53 mutation in sinonasal cancer, but was associated with multiple TP53 mutations. Furthermore, inflammation appears to play a part in sinonasal carcinogenesis as indicated by our results showing that the expression of COX-2 was associated with adenocarcinoma type of tumours, wood dust exposure and non-smoking. In lung cancer, we detected statistically significant associations between TP53 mutations and duration of smoking, gender and histology. We also found that patients with a tumour carrying a G to T transversion, a mutation commonly found in association with tobacco smoking, had a high level of smoking-related bulky DNA adducts in their non-tumorous lung tissue. Altogether, the information on molecular changes in exposure induced cancers adds to the observations from epidemiological studies and helps to understand the role and impact of different etiological factors, which in turn can be beneficial for risk assessment and prevention.

Nuclear Import Mechanisms of STAT and NF-kB Transcription Factors

The eukaryotic cell nucleoplasm is separated from the cytoplasm by the nuclear envelope. This compartmentation of eukaryotic cells requires that all nuclear proteins must be transported from the cytoplasm into the nucleus. Transport of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). Proteins to be targeted into the nucleus by the classical nuclear import system contain nuclear localization signals (NLSs), which are recognized by importin alpha, the NLS receptor. Importin alpha binds to importin beta, which docks the importin-cargo complex on the cytoplasmic side of the NPC and mediates the movement of the complex into the nucleus. Presently six human importin alpha isoforms have been identified. Transcription factors are among the most important regulators of gene expression in eukaryotic organisms. Transcription factors bind to specific DNA sequences on target genes and modulate the activity of the target gene. Many transcription factors, including signal transducers and activators of transcription (STAT) and nuclear factor kB (NF-kB), reside in the cytoplasm in an inactive form, and upon activation they are rapidly transported into the nucleus. In the nucleus STATs and NF-kB regulate the activity of genes whose products are critical in controlling numerous cellular and organismal processes, such as inflammatory and immune responses, cell growth, differentiation and survival. The aim of this study was to investigate the nuclear import mechanisms of STAT and NF-kB transcription factors. This work shows that STAT1 homodimers and STAT1/STAT2 heterodimers bind specifically and directly to importin alpha5 molecule via unconventional dimer-specific NLSs. Importin alpha molecules have two regions, which have been shown to directly interact with the amino acids in the NLS of the cargo molecule. The Arm repeats 2-4 comprise the N-terminal NLS binding site and Arm repeats 7-8 the C-terminal NLS binding site. In this work it is shown that the binding site for STAT1 homodimers and STAT1/STAT2 heterodimers is composed of Arm repeats 8 and 9 of importin alpha5 molecule. This work demonstrates that all NF-kB proteins are transported into the nucleus by importin alpha molecules. In addition, NLS was identified in RelB protein. The interactions between NF-kB proteins and importin alpha molecules were found to be directly mediated by the NLSs of NF-kB proteins. Moreover, we found that p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. The results from this thesis work identify previously uncharacterized mechanisms in nuclear import of STAT and NF-kB. These findings provide new insights into the molecular mechanisms regulating the signalling cascades of these important transcription factors from the cytoplasm into the nucleus to the target genes.

Genetic analysis of chromosomal regions 2q33, 7q32 and 19q13 in multiple sclerosis susceptibility

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) affecting 0.1-0.2% of Northern European descent population. MS is considered to be a multifactorial disease, both environment and genetics play a role in its pathogenesis. Despite several decades of intense research, the etiological and pathogenic mechanisms underlying MS remain still largely unknown and no curative treatment exists. The genetic architecture underlying MS is complex with multiple genes involved. The strongest and the best characterized predisposing genetic factors for MS are located, as in other immune-mediated diseases, in the major histocompatibility complex (MHC) on chromosome 6. In humans MHC is called human leukocyte antigen (HLA). Alleles of the HLA locus have been found to associate strongly with MS and remained for many years the only consistently replicable genetic associations. However, recently other genes located outside the MHC region have been proposed as strong candidates for susceptibility to MS in several studies. In this thesis a new genetic locus located on chromosome 7q32, interferon regulatory factor 5 (IRF5), was identified in the susceptibility to MS. In particular, we found that common variation of the gene was associated with the disease in three different populations, Spanish, Swedish and Finnish. We also suggested a possible functional role for one of the risk alleles with impact on the expression of the IRF5 locus. Previous studies have pointed out a possible role played by chromosome 2q33 in the susceptibility to MS and other autoimmune disorders. The work described here also investigated the involvement of this chromosomal region in MS predisposition. After the detection of genetic association with 2q33 (article-1), we extended our analysis through fine-scale single nucleotide polymorphism (SNP) mapping to define further the contribution of this genomic area to disease pathogenesis (article-4). We found a trend (p=0.04) for association to MS with an intronic SNP located in the inducible T-cell co-stimulator (ICOS) gene, an important player in the co-stimulatory pathway of the immune system. Expression analysis of ICOS revealed a novel, previously uncharacterized, alternatively spliced isoform, lacking the extracellular domain that is needed for ligand binding. The stability of the newly-identified transcript variant and its subcellular localization were analyzed. These studies indicated that the novel isoform is stable and shows different subcellular localization as compared to full-length ICOS. The novel isoform might have a regulatory function, but further studies are required to elucidate its function. Chromosome 19q13 has been previously suggested as one of the genomic areas involved in MS predisposition. In several populations, suggestive linkage signals between MS predisposition and 19q13 have been obtained. Here, we analysed the role of allelic variation in 19q13 by family based association analysis in 782 MS families collected from Finland. In this dataset, we were not able to detect any statistically significant associations, although several previously suggested markers were included to the analysis. Replication of the previous findings on the basis of linkage disequilibrium between marker allele and disease/risk allele appears notoriously difficult because of limitations such as allelic heterogeneity. Re-sequencing based approaches may be required for elucidating the role of chromosome 19q13 with MS. This thesis has resulted in the identification of a new MS susceptibility locus (IRF5) previously associated with other inflammatory or autoimmune disorders, such as SLE. IRF5 is one of the mediators of interferons biological function. In addition to providing new insight in the possible pathogenetic pathway of the disease, this finding suggests that there might be common mechanisms between different immune-mediated disorders. Furthermore the work presented here has uncovered a novel isoform of ICOS, which may play a role in regulatory mechanisms of ICOS, an important mediator of lymphocyte activation. Further work is required to uncover its functions and possible involvement of the ICOS locus in MS susceptibility.

Placental abruption : Studies on incidence, risk factors and potential predictive biomarkers

Placental abruption, one of the most significant causes of perinatal mortality and maternal morbidity, occurs in 0.5-1% of pregnancies. Its etiology is unknown, but defective trophoblastic invasion of the spiral arteries and consequent poor vascularization may play a role. The aim of this study was to define the prepregnancy risk factors of placental abruption, to define the risk factors during the index pregnancy, and to describe the clinical presentation of placental abruption. We also wanted to find a biochemical marker for predicting placental abruption early in pregnancy. Among women delivering at the University Hospital of Helsinki in 1997-2001 (n=46,742), 198 women with placental abruption and 396 control women were identified. The overall incidence of placental abruption was 0.42%. The prepregnancy risk factors were smoking (OR 1.7; 95% CI 1.1, 2.7), uterine malformation (OR 8.1; 1.7, 40), previous cesarean section (OR 1.7; 1.1, 2.8), and history of placental abruption (OR 4.5; 1.1, 18). The risk factors during the index pregnancy were maternal (adjusted OR 1.8; 95% CI 1.1, 2.9) and paternal smoking (2.2; 1.3, 3.6), use of alcohol (2.2; 1.1, 4.4), placenta previa (5.7; 1.4, 23.1), preeclampsia (2.7; 1.3, 5.6) and chorioamnionitis (3.3; 1.0, 10.0). Vaginal bleeding (70%), abdominal pain (51%), bloody amniotic fluid (50%) and fetal heart rate abnormalities (69%) were the most common clinical manifestations of placental abruption. Retroplacental blood clot was seen by ultrasound in 15% of the cases. Neither bleeding nor pain was present in 19% of the cases. Overall, 59% went into preterm labor (OR 12.9; 95% CI 8.3, 19.8), and 91% were delivered by cesarean section (34.7; 20.0, 60.1). Of the newborns, 25% were growth restricted. The perinatal mortality rate was 9.2% (OR 10.1; 95% CI 3.4, 30.1). We then tested selected biochemical markers for prediction of placental abruption. The median of the maternal serum alpha-fetoprotein (MSAFP) multiples of median (MoM) (1.21) was significantly higher in the abruption group (n=57) than in the control group (n=108) (1.07) (p=0.004) at 15-16 gestational weeks. In multivariate analysis, elevated MSAFP remained as an independent risk factor for placental abruption, adjusting for parity ≥ 3, smoking, previous placental abruption, preeclampsia, bleeding in II or III trimester, and placenta previa. MSAFP ≥ 1.5 MoM had a sensitivity of 29% and a false positive rate of 10%. The levels of the maternal serum free beta human chorionic gonadotrophin MoM did not differ between the cases and the controls. None of the angiogenic factors (soluble endoglin, soluble fms-like tyrosine kinase 1, or placental growth factor) showed any difference between the cases (n=42) and the controls (n=50) in the second trimester. The levels of C-reactive protein (CRP) showed no difference between the cases (n=181) and the controls (n=261) (median 2.35 mg/l [interquartile range {IQR} 1.09-5.93] versus 2.28 mg/l [IQR 0.92-5.01], not significant) when tested in the first trimester (mean 10.4 gestational weeks). Chlamydia pneumoniae specific immunoglobulin G (IgG) and immunoglobulin A (IgA) as well as C. trachomatis specific IgG, IgA and chlamydial heat-shock protein 60 antibody rates were similar between the groups. In conclusion, although univariate analysis identified many prepregnancy risk factors for placental abruption, only smoking, uterine malformation, previous cesarean section and history of placental abruption remained significant by multivariate analysis. During the index pregnancy maternal alcohol consumption and smoking and smoking by the partner turned out to be the major independent risk factors for placental abruption. Smoking by both partners multiplied the risk. The liberal use of ultrasound examination contributed little to the management of women with placental abruption. Although second-trimester MSAFP levels were higher in women with subsequent placental abruption, clinical usefulness of this test is limited due to low sensitivity and high false positive rate. Similarly, angiogenic factors in early second trimester, or CRP levels, or chlamydial antibodies in the first trimester failed to predict placental abruption.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Hormone therapy in elderly women; a comparative study with alendronate of the effects on bone, cardiovascular risk factors, periodontal conditions and quality of life

Thirty percent of 70-year-old women have osteoporosis; after age of 80 its prevalence is up to 70%. Postmenopausal women with osteoporosis seem to be at an increased risk for cardiovascular events, and deterioration of oral health, as shown by attachment loss of teeth, which is proportional to the severity of osteoporosis. Osteoporosis can be treated with many different medication, e.g. estrogen and alendronate. We randomized 90 elderly osteoporotic women (65-80 years of age) to receive hormone therapy (HT)(2mg E2+NETA), 10mg alendronate, and their combination for two years and compared their effects on bone mineral density (BMD) and turnover, two surrogate markers of the risk of cardiovascular diseases, C-reactive protein (CRP) and E-selectin, as well as oral health. The effect of HT on health-related quality of life (HRQoL) was studied in the population-based cohort of 1663 postmenopausal women (mean age 68 yr) (585 estrogen users and 1078 non-users). BMD was measured with dual-energy X-ray absorptiometry (DXA) at 0, 12 and 24 months. Urinary N-telopeptide (NTX) of type I collagen, a marker of bone resorption, and serum aminoterminal propeptide of human type I procollagen (PINP), a marker of bone formation, were measured every six months of treatment. Serum CRP and E-selectin, were measured at 0, 6, and 12 months. Dental, and periodontal conditions, and gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 levels were studied to evaluate the oral health status and for the mouth symptoms a structured questionnaire was used. The HRQoL was measured with 15D questionnaire. Lumbar spine BMD increased similarly in all treatment groups (6.8-8.4% and 9.1-11.2%). Only HT increased femoral neck BMD at both 12 (4.9%) and 24 months (5.8%), at the latter time point the HT group differed significantly from the other groups. HT reduced bone marker levels of NTX and PINP significantly less than other two groups.Oral HT significantly increased serum CRP level by 76.5% at 6 and by 47.1% (NS) at 12 months, and decreased serum E-selectin level by 24.3% and 30.0%. Alendronate had no effect on these surrogate markers. Alendronate caused a decrease in the resting salivary flow rate and tended to increase GCF MMP-8 levels. Otherwise, there was no effect on the parameters of oral health. HT improved the HRQoL of elderly women significantly on the dimensions of usual activities, vitality and sexual activity, but the overall improvement in HRQoL was neither statistically significant nor clinically important. In conclusion, bisphosphonates might be the first option to start the treatment of postmenopausal osteoporosis in the old age.

Functional imaging of peripheral vision and dorsal stream function in the human cerebral cortex

Visual information processing in brain proceeds in both serial and parallel fashion throughout various functionally distinct hierarchically organised cortical areas. Feedforward signals from retina and hierarchically lower cortical levels are the major activators of visual neurons, but top-down and feedback signals from higher level cortical areas have a modulating effect on neural processing. My work concentrates on visual encoding in hierarchically low level cortical visual areas in human brain and examines neural processing especially in cortical representation of visual field periphery. I use magnetoencephalography and functional magnetic resonance imaging to measure neuromagnetic and hemodynamic responses during visual stimulation and oculomotor and cognitive tasks from healthy volunteers. My thesis comprises six publications. Visual cortex forms a great challenge for modeling of neuromagnetic sources. My work shows that a priori information of source locations are needed for modeling of neuromagnetic sources in visual cortex. In addition, my work examines other potential confounding factors in vision studies such as light scatter inside the eye which may result in erroneous responses in cortex outside the representation of stimulated region, and eye movements and attention. I mapped cortical representations of peripheral visual field and identified a putative human homologue of functional area V6 of the macaque in the posterior bank of parieto-occipital sulcus. My work shows that human V6 activates during eye-movements and that it responds to visual motion at short latencies. These findings suggest that human V6, like its monkey homologue, is related to fast processing of visual stimuli and visually guided movements. I demonstrate that peripheral vision is functionally related to eye-movements and connected to rapid stream of functional areas that process visual motion. In addition, my work shows two different forms of top-down modulation of neural processing in the hierachically lowest cortical levels; one that is related to dorsal stream activation and may reflect motor processing or resetting signals that prepare visual cortex for change in the environment and another local signal enhancement at the attended region that reflects local feed-back signal and may perceptionally increase the stimulus saliency.