Transcriptional regulation by the orphan nuclear receptor ERRgamma

The nuclear receptor (NR) superfamily is comprised of receptors for small lipopfilic ligands such as steroid hormones, thyroid hormone, retinoids, and vitamin D. NRs are ligand-inducible transcription factors capable of both activating and repressing their target gene expression. They control a wide range of biological functions connected to growth, development, and homeostasis. In addition to the ligand-regulated receptors, the family includes a large group of receptors whose physiological ligands are unknown. These receptors are referred to as orphan NRs. Estrogen-related receptor gamma (ERRgamma) belongs to the ERR subfamily of orphan NRs together with the related ERRalpha and ERRbeta. ERRs share amino acid sequence homology with the classical estrogen receptors (ERs) but they are unable to bind natural estrogenic ligands. ERRgamma is expressed in several embryonic and adult tissues but its biological role is still largely unknown. ERRgamma activates reporter gene expression in transfected cells independently of added hormones implying that ERRgamma harbors constitutive activity. However, the intrinsic activity of ERRgamma can be inhibited by synthetic compounds such as the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT). Ligands of NRs can act as agonists that activate transcription, as antagonists that prevent activation of transcription, or as inverse agonists that antagonize the constitutive transcriptional activity of receptor. Most of the synthetic ERRgamma ligands act as inverse agonists but recently, a synthetic ERRgamma agonist GSK4716 was identified. This demonstrates that it is possible to design and identify agonists for ERRgamma. Prior to this thesis work, the structural and functional characteristics of ERRgamma were largely unknown. The aim of this study was to define the functional requirements for ERRgamma-mediated transcriptional regulation and to examine the cross-talk between ERRgamma and other NRs. Due to the fact that natural physiological ligands of ERRgamma are unknown, another aim of this study was to seek new natural compounds that may affect transcriptional activity of ERRgamma. Plant-derived phytoestrogens have previously been shown to act as ligands for ERs and ERRalpha, and therefore the effects of these compounds were also studied on ERRgamma-mediated transcriptional regulation. This work demonstrated that ERRgamma-mediated transcriptional regulation was dependent on DNA-binding, dimerization and activation function-2. Heterodimerization with ERRalpha inhibited the transcriptional activity of ERRgamma. In addition to 4-OHT, another anti-estrogen, 4-hydroxytoremifene (4-OHtor), was identified as an inverse agonist of ERRgamma. Interestingly, ERRgamma activated transcription in the presence of 4-OHT and 4-OHtor on activator protein-1 binding sites. ERRgamma was found to interact with another orphan NR Nurr1 by repressing the ability of Nurr1 to activate transcription of the osteopontin gene. Transcriptional activity of ERRgamma was shown to be stimulated by the phytoestrogen equol. Structural model analysis and mutational experiments indicated that equol was able to bind to the ligand binding domain of ERRgamma. The growth inhibitory effect of ERRgamma on prostate cancer cells was found to be enhanced by equol. In summary, this study demonstrates that despite the absence of an endogenous physiological ligand, the activity of ERRgamma can be modulated in other ways such as dimerization with related receptors or by cross-talk with other transcription factors as well as by binding some synthetic or natural compounds.

The Pharmacology of Oxycodone : Studies In Vitro, In Vivo and In Humans

The antinociceptive properties of oxycodone and its metabolites were studied in models of thermal and mechanical nociception and in the spinal nerve ligation (SNL) model of neuropathic pain in rats. Oxycodone induced potent antinociception after subcutaneous (s.c.) administration in all models of nociception used in rats compared with morphine, methadone and its enantiomers. In the SNL model of neuropathic pain in rats, oxycodone produced dose dependent antinociception after s.c. administration. The antinociceptive effects of s.c. oxycodone were antagonized by naloxone but not by nor-binaltorphimine (Nor-BNI) a selective κ-opioid receptor antagonist indicating that the antinociceptive properties of oxycodone are predominantly μ-opioid receptor-mediated. The antinociceptive activity of oxymorphone, noroxycodone, and noroxymorphone, oxidative metabolites of oxycodone, were studied to determine their role in the oxycodone-induced antinociception in the rat. Of the metabolites of oxycodone s.c. administration of oxymorphone produced potent thermal and mechanical antinociception. Noroxycodone had a poor antinociceptive effect and noroxymorphone was inactive. Oxycodone produced naloxone-reversible antinociception after intrathecal (i.t) administration with a poor potency compared with morphine and oxymorphone. This seems to be related to the low efficacy and potency of oxycodone to stimulate μ-opioid receptor activation in the spinal cord in μ-opioid receptor agonist-stimulated (GTP)γ[S] autoradiography, compared with morphine and oxymorphone. All metabolites studied were more potent than oxycodone after i.t. administration. I.t. noroxymorphone induced a significantly longer lasting antinociceptive effect compared with the other drugs studied. The role of cytochrome P450 (CYP) 2D6-mediated metabolites on the analgesic activity of oxycodone in humans was studied by blocking the CYP2D6-mediated metabolism of oxycodone with paroxetine. Paroxetine co-administration had no effect on the analgesic effect of oxycodone compared with placebo in chronic pain patients, indicating that oxycodone-induced analgesia and adverse-effects are not dependent of the CYP2D6-mediated metabolism in humans. Although oxycodone has many pharmacologically active metabolites, they seem to have an insignificant role in oxycodone-induced antinociception in humans and rats.

Probiotic activity of Lactobacillus delbrueckii subsp. bulgaricus in the oral cavity

Yogurt consumption has been related to longevity of some populations living on the Balkans. Yogurt starter L. delbrueckii subsp. bulgaricus and Str. thermophilus have been recognized as probiotics with verified beneficial health effects. The oral cavity emerges as a arget for probiotic applications. Probiotics have demonstrated promising results in controlling dental diseases and oral yeast infections. However, L. bulgaricus despite its broad availability in dairy products has not been evaluated for probiotic activity in the mouth. These series of studies investigated in vitro properties of L. bulgaricus to outline its potential as an oral probiotic. Prerequisite probiotic properties in the mouth are resistance to oral defense mechanisms, adherence to saliva-coated surfaces, and inhibition of oral pathogens. L. bulgaricus strains showed a strain-dependent inhibition of oral streptococci and Aggregatibacter actinomycetemcomitans, whereas none of the dairy starter strains could affect growth of Porphyromonas gingivalis and Fusobacterium nucleatum. Adhesion is a factor contributing to colonization of the species at the target site. Radiolabeled L. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated surfaces. The effects of lysozyme on adhesion and adhesion of Streptococcus sanguinis after lactobacilli pretreatment were also assessed. Adhesion of L. bulgaricus remained lower in comparison to L. rhamnosus GG. One L. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment significantly increased Lactobacillus adhesion. Low gelatinolytic activity was observed for all strains and no conversion of proMMP-9 to its active form was induced by L. bulgaricus. Safety assessment ruled out deleterious effects of L. bulgaricus on extracellular matrix structures. Cytokine response of oral epithelial cells was assessed by measuring IL-8 and TNF-α in cell culture supernatants. The effect of P. gingivalis on cytokine secretion after lactobacilli pretreatment was also assessed. A strain- and time-dependent induction of IL-8 was observed with live bacteria inducing the highest levels of cytokine secretion. Levels of TNF-α were low and only one of ten L. bulgaricus strains stimulated TNF-α secretion similar to positive control. The addition of P. gingivalis produced immediate reduction of cytokine levels within the first hours of incubation irrespective of lactobacilli strains co-cultured with epithelial cells. According to these studies strains among the L. delbrueckii subsp. bulgaricus species may have beneficial probiotic properties in the mouth. Their potential in prevention and management of common oral infectious diseases needs to be further studied.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

High-density lipoprotein 17beta-estradiol fatty acyl esters and phytoestrogens : Impact on reverse cholesterol transport and women's cardiovascular health

Reverse cholesterol transport (RCT) is an important function of high-density lipoproteins (HDL) in the protection of atherosclerosis. RCT is the process by which HDL stimulates cholesterol removal from peripheral cells and transports it to the liver for excretion. Premenopausal women have a reduced risk for atherosclerosis compared to age-matched men and there exists a positive correlation for serum 17β-estradiol (E2) and HDL levels in premenopausal women supporting the role of E2 in atherosclerosis prevention. In premenopausal women, E2 associates with HDL as E2 fatty acyl esters. Discovery of the cellular targets, metabolism, and assessment of the macrophage cholesterol efflux potential of these HDL-associated E2 fatty acyl esters were the major objectives of this thesis (study I, III, and IV). Soy phytoestrogens, which are related to E2 in both structure and function, have been proposed to be protective against atherosclerosis but the evidence to support these claims is conflicting. Therefore, another objective of this thesis was to assess the ability of serum from postmenopausal women, treated with isoflavone supplements (compared to placebo), to promote macrophage cholesterol efflux (study II). The scope of this thesis was to cover the roles that HDL-associated E2 fatty acyl esters have in the cellular aspects of RCT and to determine if soy isoflavones can also influence RCT mechanisms. SR-BI was a pivotal cellular receptor, responsible for hepatic and macrophage uptake and macrophage cholesterol efflux potential of HDL-associated E2 fatty acyl esters. Functional SR-BI was also critical for proper LCAT esterification activity which could impact HDL-associated E2 fatty acyl ester assembly and its function. In hepatic cells, LDL receptors also contributed to HDL-associated E2 fatty acyl esters uptake and in macrophage cells, estrogen receptors (ERs) were necessary for both HDL-associated E2 ester-specific uptake and cholesterol efflux potential. HDL-containing E2 fatty acyl esters (E2-FAE) stimulated enhanced cholesterol efflux compared to male HDL (which are deficient in E2) demonstrating the importance of the E2 ester in this process. To support this, premenopausal female HDL, which naturally contains E2, showed greater macrophage cholesterol efflux compared to males. Additionally, hepatic and macrophage cells hydrolyzed the HDL-associated E2 fatty acyl ester into unesterified E2. This could have important biological ramifications because E2, not the esterified form, has potent cellular effects which may influence RCT mechanisms. Lastly, soy isoflavone supplementation in postmenopausal women did not modulate ABCA1-specific macrophage cholesterol efflux but did increase production of plasma pre-β HDL levels, a subclass of HDL. Therefore, the impact of isoflavones on RCT and cardiovascular health needs to be further investigated. Taken as a whole, HDL-associated E2 fatty acyl esters from premenopausal women and soy phytoestrogen treatment in postmenopausal women may be important factors that increase the efficiency of RCT through cellular lipoprotein-related processes and may have direct implications on the cardiovascular health of women.

Growth differentiation factor 9 signalling in the ovary

In the ovary, two new members of the large TGF-beta superfamily of growth factors were discovered in the 1990s. The oocyte was shown to express two closely related growth factors that were named growth differentiation factor 9 (GDF-9) and growth differentiation factor 9B (GDF-9B). Both of these proteins are required for normal ovarian follicle development although their individual significance varies between species. GDF-9 and GDF-9B mRNAs are expressed in the human oocytes from the primary follicle stage onwards. This thesis project was aimed to define the signalling mechanisms utilized by the oocyte secreted GDF-9. We used primary cultures of human granulosa luteal cells (hGL) as our cell model, and recombinant adenovirus-mediated gene transfer in manipulating the TGF-b family signalling cascade molecules in these cells. Overexpression of the constitutively active forms of the seven type I receptors, the activin receptor-like kinases 1-7 (ALK1-7), using recombinant adenoviruses caused a specific activation of either the Smad1 or Smad2 pathway proteins depending on the ALK used. Activation of both Smad1 and Smad2 proteins also stimulated the expression of dimeric inhibin B protein in hGL cells. Treatment with recombinant GDF-9 protein induced the specific activation of the Smad2 pathway and stimulated the expression of inhibin betaB subunit mRNA as well as inhibin B protein secretion in our cell model. Recombinant GDF-9 also activated the Smad3-responsive CAGA-luciferase reported construct, and the GDF-9 response in hGL cells was markedly potentiated upon the overexpression of Alk5 by adenoviral gene transduction. Alk5 overexpression also enhanced the GDF-9 induced inhibin B secretion by these cells. Similarly, in a mouse teratocarcinoma cell line P19, GDF-9 could activate the Smad2/3 pathway, and overexpression of ALK5 in COS7 cells rendered them responsive to GDF-9. Furthermore, transfection of rat granulosa cells with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 effects. In conclusion, this thesis shows that both Smad1 and Smad2 pathways are involved in controlling the regulation of inhibin B secretion. Therefore, in addition to endocrine control of inhibin production by the pituitary gonadotropins, also local paracrine factors within in the ovary, like the oocyte-derived growth factors, may contribute to controlling inhibin secretion. This thesis shows as well that like other TGF-beta family ligands, also GDF-9 signalling is mediated by the canonical type I and type II receptors with serine/threonine kinase activity, and the intracellular transcription factors, the Smads. Although GDF-9 binds to the BMP type II receptor, its downstream actions are specifically mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins.

In vitro development of islets from human adult pancreatic tissues

Transplantation of isolated islets from cadaver pancreas is a promising possibility for the optimal treatment of type 1 diabetes. The lack of islets is a major problem. Here we have investigated the possibility of generating islets in tissue culture of human pancreatic cells. We first reproduced a previously reported method of in vitro generation of endocrine cells from human adult pancreatic tissue. By tracing the bromodeoxyuridine-labeled cells in differentiated islet buds, we found that the pancreatic progenitor cells represented a subpopulation of cytokeratin 19 (CK19)-positive ductal cells. Serum-free medium and Matrigel overlay were essential for the endocrine differentiation. We then examined the involvement of preexisting islet cells in islet neogenesis. About 6-10% of endocrine cells dedifferentiated and acquired a transitional phenotype by coexpressing CK19. Significant cell proliferation was only observed in CK19-positive cells, but not in chromogranin A-positive endocrine cells. The in vitro-derived human islets were morphologically and functionally immature when compared with normal islets. Their insulin mRNA levels were only 4-5% of that found in fresh human islets, and glucose-stimulated insulin release was 3 times lower than that of control islets. Moreover, some immature endocrine cells coexpressed insulin and glucagon. After transplantation in nude mice, the in vitro-generated islets became mature with one type of hormone per endocrine cell. In addition, we also found that also in both fresh islet transplants many cells coexpressed endocrine markers and ductal marker CK19 as a sign of ductal to endocrine cell transition. Finally, we studied the effects of clinically used immunosuppressive drugs on precursor cell proliferation and differentiation. Mycophenolate mofetil (MMF) severely hampered duct-cell proliferation, and significantly reduced the total DNA content indicating its antiproliferative effect on the precursors. Tacrolimus mainly affected differentiated beta cells by decreasing the insulin content per DNA as well as the proportion of insulin-positive cells. Sirolimus and daclizumab did not show any individual or synergistic side effects suggesting that these drugs are amenable for use in clinical islet transplantation. In summary, we confirm the capacity of endocrine differentiation from progenitors present in the adult human pancreas. The plasticity of differentiated cell types of human pancreas may be a potential mechanism of human pancreas regeneration. Ductal cell differentiation into endocrine cells in transplanted islets may be an important factor in sustaining the long-term function of islet transplants. The immunosuppressive protocol is likely to be an important determinant of long-term clinical islet graft function. Moreover, these results provide new information on the mechanisms of pancreatic islet regeneration and provide the basis for the development of new strategies for the treatment of insulin deficient diabetes mellitus.

Novel Multiple Sclerosis Predisposing Genetic Variants Outside the HLA Region

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Both environmental factors and several predisposing genes are required to generate MS. Despite intensive research these risk factors are still largely unknown, the pathogenesis of MS demyelination is poorly understood, and no curative treatment exists. Both prevalence and familial occurrence of MS are exceptionally high in a Finnish population subisolate, Southern Ostrobothnia, presumably due to enrichment of predisposing genetic variants within this region. Previous linkage scan on MS pedigrees from Southern Ostrobothnia detected three main MS loci on chromosomes 5p, 6p (HLA) and 17q. Linkage studies in other populations have also provided independent evidence for the location of MS susceptibility genes in these regions. Further, these loci are syntenic to the experimental autoimmune encephalomyelitis (EAE) susceptibility loci of rodents. In this thesis work an effort was made to localize MS predisposing alleles of the linked loci outside the HLA region by studying familial MS cases from the Southern Ostrobothnia isolate. Analysis of the 5p locus revealed one region, flanking the complement component 7 (C7) gene. The identified relatively rare haplotype seems to have a fairly large effect on genetic susceptibility of MS (frequency MS 12%, controls 4%; p=0.000003, OR=2.73). Evidence for association with alleles of the region and MS was seen also in more heterogeneous populations. Convincingly, plasma C7 protein levels and complement activity correlated with the risk haplotype identified. The finding stimulated us to study other complement cascade genes in MS. No evidence for association could be observed with the complement component coding genes outside 5p. A scan of the 17q locus provided evidence for association with variants of the protein kinase C alpha (PRKCA) gene (p=0.0001). Modest evidence for association with PRKCA was observed also in Canadian MS families. Finally we used a candidate gene based approach to identify potential MS loci. Mutations of DAP12 and TREM2 cause a recessively inherited CNS white matter disease PLOSL. Interestingly, DAP12 and TREM2 are located in MS regions on 6p and 19q, and we tested them as potential candidate genes in the Finnish MS sample. No evidence for association with MS was observed. This thesis provides an example of how extended families from special populations can be utilized in fine-mapping of the linked loci. A first relatively rare MS variant was identified utilizing the strength of a Finnish population subisolate. This variant seems to have an effect on activity of the complement system, which has previously been suggested to have an important role in the pathogenesis of MS.

Latent TGF-β binding proteins -3 and -4 : transcriptional control and extracellular matrix targeting

Extracellular matrix (ECM) is a complex network of various proteins and proteoglycans which provides tissues with structural strength and resilience. By harvesting signaling molecules like growth factors ECM has the capacity to control cellular functions including proliferation, differentiation and cell survival. Latent transforming growth factor β (TGF-β) binding proteins (LTBPs) associate fibrillar structures of the ECM and mediate the efficient secretion and ECM deposition of latent TGF-β. The current work was conducted to determine the regulatory regions of LTBP-3 and -4 genes to gain insight into their tissue-specific expression which also has impact on TGF-β biology. Furthermore, the current research aimed at defining the ECM targeting of the N-terminal variants of LTBP-4 (LTBP-4S and -4L), which is required to understand their functions in tissues and to gain insight into conditions in which TGF-β is activated. To characterize the regulatory regions of LTBP-3 and -4 genes in silico and functional promoter analysis techniques were employed. It was found that the expression of LTBP-4S and -4L are under control of two independent promoters. This finding was in accordance with the observed expression patterns of LTBP-4S and -4L in human tissues. All promoter regions characterized in this study were TATAless, GC-rich and highly conserved between human and mouse species. Putative binding sites for Sp1 and GATA family of transcription factors were recognized in all of these regulatory regions. It is possible that these transcription factors control the basal expression of LTBP-3 and -4 genes. Smad binding element was found within the LTBP-3 and -4S promoter regions, but it was not present in LTBP-4L promoter. Although this element important for TGF-β signaling was present in LTBP-4S promoter, TGF-β did not induce its transcriptional activity. LTBP-3 promoter activity and mRNA expression instead were stimulated by TGF-β1 in osteosarcoma cells. It was found that the stimulatory effect of TGF-β was mediated by Smad and Erk MAPK signaling pathways. The current work explored the ECM targeting of LTBP-4S and identified binding partners of this protein. It was found that the N-terminal end of LTBP-4S possesses fibronectin (FN) binding sites which are critical for its ECM targeting. FN deficient fibroblasts incorporated LTBP-4S into their ECM only after addition of exogenous FN. Furthermore, LTBP-4S was found to have heparin binding regions, of which the C-terminal binding site mediated fibroblast adhesion. Soluble heparin prevented the ECM association of LTBP-4S in fibroblast cultures. In the current work it was observed that there are significant differences in the secretion, processing and ECM targeting of LTBP-4S and -4L. Interestingly, it was observed that most of the secreted LTBP-4L was associated with latent TGF-β1, whereas LTBP-4S was mainly secreted as a free form from CHO cells. This thesis provides information on transcriptional regulation of LTBP-3 and -4 genes, which is required for the deeper understanding of their tissue-specific functions. Further, the current work elucidates the structural variability of LTBPs, which appears to have impact on secretion and ECM targeting of TGF-β. These findings may advance understanding the abnormal activation of TGF-β which is associated with connective tissue disorders and cancer.

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.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Lignocellulose degradation and humus modification by the fungus Paecilomyces inflatus

Composting is the biological conversion of solid organic waste into usable end products such as fertilizers, substrates for mushroom production and biogas. Although composts are highly variable in their bulk composition, composting material is generally based on lignocellulose compounds derived from agricultural, forestry, fruit and vegetable processing, household and municipal wastes. Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore lignocellulose degradation is essential for maintaining the global carbon cycle. In compost, the active component involved in the biodegradation and conversion processes is the resident microbial population, among which microfungi play a very important role. In composting pile the warm, humid, and aerobic environment provides the optimal conditions for their development. Microfungi use many carbon sources, including lignocellulosic polymers and can survive in extreme conditions. Typically microfungi are responsible for compost maturation. In order to improve the composting process, more information is needed about the microbial degradation process. Better knowledge on the lignocellulose degradation by microfungi could be used to optimize the composting process. Thus, this thesis focused on lignocellulose and humic compounds degradation by a microfungus Paecilomyces inflatus, which belongs to a flora of common microbial compost, soil and decaying plant remains. It is a very common species in Europe, North America and Asia. The lignocellulose and humic compounds degradation was studied using several methods including measurements of carbon release from 14C-labelled compounds, such as synthetic lignin (dehydrogenative polymer, DHP) and humic acids, as well as by determination of fibre composition using chemical detergents and sulphuric acid. Spectrophotometric enzyme assays were conducted to detect extracellular lignocellulose-degrading hydrolytic and oxidative enzymes. Paecilomyces inflatus secreted clearly extracellular laccase to the culture media. Laccase was involved in the degradation process of lignin and humic acids. In compost P. inflatus mineralised 6-10% of 14C-labelled DHP into carbon dioxide. About 15% of labelled DHP was converted into water-soluble compounds. Also humic acids were partly mineralised and converted into water-soluble material, such as low-molecular mass fulvic acid-like compounds. Although laccase activity in aromatics-rich compost media clearly is connected with the degradation process of lignin and lignin-like compounds, it may preferentially effect the polymerisation and/or detoxification of such aromatic compounds. P. inflatus can degrade lignin and carbohydrates also while growing in straw and in wood. The cellulolytic enzyme system includes endoglucanase and β-glucosidase. In P. inflatus the secretion of these enzymes was stimulated by low-molecular-weight aromatics, such as soil humic acid and veratric acid. When strains of P. inflatus from different ecophysiological origins were compared, indications were found that specific adaptation strategies needed for lignocellulosics degradation may operate in P. inflatus. The degradative features of these microfungi are on relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not established. The results of this study may help to understand, control and better design the process of plant polymer conversion in compost environment, with a special emphasis on the role of ubiquitous microfungi.

Contested Lands: Land Disputes in Semi-Arid Parts of Northern Tanzania : Case Studies of the Loliondo and Sale Divisions

This study Contested Lands: Land disputes in semi-arid parts of northern Tanzania. Case Studies of the Loliondo and Sale Division in the Ngorongoro District concentrates on describing the specific land disputes which took place in the 1990s in the Loliondo and Sale Divisions of the Ngorongoro District in northern Tanzania. The study shows the territorial and historical transformation of territories and property and their relation to the land disputes of the 1990s'. It was assumed that land disputes have been firstly linked to changing spatiality due to the zoning policies of the State territoriality and, secondly, they can be related to the State control of property where the ownership of land property has been redefined through statutory laws. In the analysis of the land disputes issues such as use of territoriality, boundary construction and property claims, in geographical space, are highlighted. Generally, from the 1980s onwards, increases in human population within both Divisions have put pressure on land/resources. This has led to the increased control of land/resource, to the construction of boundaries and finally to formalized land rights on village lands of the Loliondo Division. The land disputes have thus been linked to the use of legal power and to the re-creation of the boundary (informal or formal) either by the Maasai or the Sonjo on the Loliondo and Sale village lands. In Loliondo Division land disputes have been resource-based and related to multiple allocations of land or game resource concessions. Land disputes became clearly political and legal struggles with an ecological reference.Land disputes were stimulated when the common land/resource rights on village lands of the Maasai pastoralists became regulated and insecure. The analysis of past land disputes showed that space-place tensions on village lands can be presented as a platform on which spatial and property issues with complex power relations have been debated. The reduction of future land disputes will succeed only when/if local property rights to land and resources are acknowledged, especially in rural lands of the Tanzanian State.

An approach to palaeoseismicity in the Olkiluoto (sea) area during the early Holocene

Olkiluoto Island is situated in the northern Baltic Sea, near the southwestern coast of Finland, and is the proposed location of a spent nuclear fuel repository. This study examined Holocene palaeoseismicity in the Olkiluoto area and in the surrounding sea areas by computer simulations together with acoustic-seismic, sedimentological and dating methods. The most abundant rock type on the island is migmatic mica gneiss, intruded by tonalites, granodiorites and granites. The surrounding Baltic Sea seabed consists of Palaeoproterozoic crystalline bedrock, which is to a great extent covered by younger Mesoproterozoic sedimentary rocks. The area contains several ancient deep-seated fracture zones that divide it into bedrock blocks. The response of bedrock at the Olkiluoto site was modelled considering four future ice-age scenarios. Each scenario produced shear displacements of fractures with different times of occurrence and varying recovery rates. Generally, the larger the maximum ice load, the larger were the permanent shear displacements. For a basic case, the maximum shear displacements were a few centimetres at the proposed nuclear waste repository level, at proximately 500 m b.s.l. High-resolution, low-frequency echo-sounding was used to examine the Holocene submarine sedimentary structures and possible direct and indirect indicators of palaeoseismic activity in the northern Baltic Sea. Echo-sounding profiles of Holocene submarine sediments revealed slides and slumps, normal faults, debris flows and turbidite-type structures. The profiles also showed pockmarks and other structures related to gas or groundwater seepages, which might be related to fracture zone activation. Evidence of postglacial reactivation in the study area was derived from the spatial occurrence of some of the structures, especial the faults and the seepages, in the vicinity of some old bedrock fracture zones. Palaeoseismic event(s) (a single or several events) in the Olkiluoto area were dated and the palaeoenvironment was characterized using palaeomagnetic, biostratigraphical and lithostratigraphical methods, enhancing the reliability of the chronology. Combined lithostratigraphy, biostratigraphy and palaeomagnetic stratigraphy revealed an age estimation of 10 650 to 10 200 cal. years BP for the palaeoseismic event(s). All Holocene sediment faults in the northern Baltic Sea occur at the same stratigraphical level, the age of which is estimated at 10 700 cal. years BP (9500 radiocarbon years BP). Their movement is suggested to have been triggered by palaeoseismic event(s) when the Late Weichselian ice sheet was retreating from the site and bedrock stresses were released along the bedrock fracture zones. Since no younger or repeated traces of seismic events were found, it corroborates the suggestion that the major seismic activity occurred within a short time during and after the last deglaciation. The origin of the gas/groundwater seepages remains unclear. Their reflections in the echo-sounding profiles imply that part of the gas is derived from the organic-bearing Litorina and modern gyttja clays. However, at least some of the gas is derived from the bedrock. Additional information could be gained by pore water analysis from the pockmarks. Information on postglacial fault activation and possible gas and/or fluid discharges under high hydraulic heads has relevance in evaluating the safety assessment of a planned spent nuclear fuel repository in the region.

Beatific Enjoyment in Scholastic Philosophy and Theology: 1240-1335

This dissertation examines the concept of beatific enjoyment (fruitio beatifica) in scholastic theology and philosophy in the thirteenth and early fourteenth century. The aim of the study is to explain what is enjoyment and to show why scholastic thinkers were interested in discussing it. The dissertation consists of five chapters. The first chapter deals with Aurelius Augustine's distinction between enjoyment and use and the place of enjoyment in the framework of Augustine's view of the passions and the human will. The first chapter also focuses upon the importance of Peter Lombard's Sentences for the transmission of Augustine's treatment of enjoyment in scholastic thought as well as upon Lombard's understanding of enjoyment. The second chapter treats thirteenth-century conceptions of the object and psychology of enjoyment. Material for this chapter is provided by the writings - mostly Sentences commentaries - of Alexander of Hales, Albert the Great, Bonaventure, Thomas Aquinas, Peter of Tarentaise, Robert Kilwardby, William de la Mare, Giles of Rome, and Richard of Middleton. The third chapter inspects early fourteenth-century views of the object and psychology of enjoyment. The fourth chapter focuses upon discussions of the enjoyment of the Holy Trinity. The fifth chapter discusses the contingency of beatific enjoyment. The main writers studied in the third, fourth and fifth chapters are John Duns Scotus, Peter Aureoli, Durandus of Saint Pourçain, William of Ockham, Walter Chatton, Robert Holcot, and Adam Wodeham. Historians of medieval intellectual history have emphasized the significance of the concept of beatific enjoyment for understanding the character and aims of scholastic theology and philosophy. The concept of beatific enjoyment was developed by Augustine on the basis of the insight that only God can satisfy our heart's desire. The possibility of satisfying this desire requires a right ordering of the human mind and a detachment of the will from the relative goals of earthly existence. Augustine placed this insight at the very foundation of the notion of Christian learning and education in his treatise On Christian Doctrine. Following Augustine, the twelfth-century scholastic theologian Peter Lombard made the concept of enjoyment the first topic in his plan of systematic theology. The official inclusion of Lombard's Sentences in the curriculum of theological studies in the early universities stimulated vigorous discussions of enjoyment. Enjoyment was understood as a volition and was analyzed in relation to cognition and other psychic features such as rest and pleasure. This study shows that early fourteenth-century authors deepened the analysis of enjoyment by concentrating upon the relationship between enjoyment and mental pleasure, the relationship between cognition and volition, and the relationship between the will and the beatific object (i.e., the Holy Trinity). The study also demonstrates the way in which the idea of enjoyment was affected by changes in the method of theological analysis - the application of Aristotelian logic in a Trinitarian context and the shift from virtue ethics to normative ethics.