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  • Marttila, Minttu (Helsingin yliopisto, 2014)
    We collected all mutations in TPM2 and TPM3 genes hitherto found to cause congenital myopathies, to perform genotype-phenotype correlations, and to increase our understanding of the pathogenetic mechanisms of congenital myopathies caused by mutations in the tropomyosin and nebulin genes. Nemaline myopathy (NM), a rare, genetic muscle disorder defined on the basis of muscle dysfunction and the presence of structural abnormalities in the muscle fibres (i.e. nemaline bodies), is caused by mutations in ten genes known to date: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin 2 (CFL2), KBTBD13, KLHL40, KLHL41 and leiomodin 3 (LMOD 3). Tropomyosin controls muscle contraction by inhibiting the actin myosin interaction in a calcium-sensitive manner. Mutations in tropomyosin genes may cause NM, cap myopathy, congenital fibre-type disproportion, distal arthrogryposes and Escobar syndrome. We correlated the clinical picture of these diseases to novel and previously published mutations to the TPM2 (30 mutations) and TPM3 (20 mutations) genes. Mutations in TPM2 and TPM3 caused an increased Ca2+ sensitivity, resulting in a hypercontractile molecular phenotype. We studied the pathogenetic mechanisms to which five disease-causing mutations in β-tropomyosin (p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro) lead. We showed that four of the mutations cause changes in the affinity for actin leading to muscle weakness in patients, while two mutations show defective Ca2+ activation of contractility. Nebulin (NEB) is a giant 600 900-kDa filamentous protein in thin filament. We produced four wild-type nebulin super-repeats and five corresponding mutation constructs (p.Glu2431Lys, p.Ser4665Ile, p.Thr5681Pro, p.Arg2478_Asp2512del and p.Val3681_Asn3686del) in the study. The mutations were identified in patients with NM or distal myopathy. We performed F-actin and tropomyosin-binding experiments for the nebulin fragments. Our results demonstrate actin nebulin interactions and, for the first time, tropomyosin nebulin interactions in vitro, and show that the interactions are altered by disease-causing mutations. This suggests that an abnormal interaction between aberrant thin filament proteins is a pathogenetic mechanism in NM and related disorders.
  • Al-Hello, Haider (Helsingin yliopisto, 2012)
    Enteroviruses (EVs) are small non-enveloped RNA viruses forming a large group of different serotypes. EVs belong to the family Picornaviridae. The primary replication site of an enterovirus is typically the epithelium of the respiratory tract and the gastrointestinal mucosa. Virus replication in the gastrointestinal mucosa may continue, often asymptomatically, for several weeks occasionally causing viremia. During the viremia the virus spreads through the lymphatic system and circulation. Organ-specific symptoms rise after viral replication in the secondary target tissues. Occasionally, cellular adaptation is required for a virus to initiate replication in the secondary target tissue(s). Adaptation is linked to mutation(s) which may lead to alteration in cellular tropism, e.g., recognition of new surface receptor molecules or other host cell constituents essential for virus entry and replication. However, the critical step may also occur later during in the interaction of the host cell and the replicating virus. In the present study, genetic changes responsible for altered phenotypic features were sought using two strains of Human enterovirus B (HEV-B) species. Firstly, a laboratory isolate of coxsackievirus B5 (CV-B5), strain DS, was passaged 15 times in mouse pancreas in vivo, which resulted in a diabetogenic mouse pancreas passaged virus strain (MPP). The concept of diabetogenic means the ability of the MPP strain to replicate, cause insulitis and dysregulation of the glucose metabolism in the mouse pancreas in vivo. The interaction between the MPP virus strain and insulin producing β-cells was further studied in cell culture using a mouse-derived insulinoma cell line, MIN-6 cells, as an experimental model. The replication of the MPP virus strain was clearly slower in the MIN-6 cells compared to the other tested cell lines. After three days of incubation, extensive replication of MPP was evident in MIN-6 cells and resulted in a MIN-6 cell-adapted virus strain (MCA). Secondly, the ability of the D207 virus strain, isolated from a type 1 diabetic patient, to replicate in a primary human β-cell culture was tested. D207 was initially serotyped as coxsackievirus A9 (CV-A9) in a virus-specific neutralization assay. The D207 virus strain was found to cause cytolysis in the primary human β-cells and, simultaneously, severe functional damage of the surviving β-cells. The genomes of the four virus strains DS, MPP, MCA and D207 were cloned and sequenced. The sequence comparison of three CV-B5 strains (DS, MPP, and MCA) revealed only limited changes, three capsid and two non-structural (NS) amino acid substitutions between MPP and DS, and two capsid and six NS amino acid substitutions between MCA and MPP. In order to determine which of the amino acid substitutions were responsible for the changed phenotype in vivo and in vitro, full-length infectious clones were constructed from the MPP virus and its parental DS virus. By using reverse mutagenesis and chimeric viruses (MPP/DS and DS/MPP), it was shown that a change from MPP to the MCA phenotype in MIN-6 cells was mediated by only a single amino acid at position 94 in VP1, while the in vivo adaptation of the DS virus strain to the inflammation-inducing MPP virus strain may require multiple genetic determinants in the virus capsid and probably also in the NS proteins. Sequence analyses of D207 revealed that the virus belonged to a genogroup D of E-11, but was also neutralized with monotypic antisera to CV-A9. The isolate D207 was found to be closely related to a specific E-11 strains known to cause uveitis. Uveitis-causing E-11 strains were also found to be well neutralized with both CV-A9- and E-11-specific antisera. In a further study, a wide range of E-11 isolates were included to test the observed dual neutralizibility among isolates belonging to the D genogroup. Five of the six studied strains belonging to genogroup D were also neutralized with antisera against coxsackievirus A9 Griggs. The peptide scanning technique was utilized to identify antigenic regions of the capsid proteins of the D207 strain responsible for the observed dual neutralization. Several regions in the capsid of D207 were found to cross-react with an antiserum raised against CV-A9. However, epitopes responsible for the cross-neutralization remained unidentified. In conclusion, these studies indicate that the specific location of mutation may affect the phenotype of an enterovirus more than the overall quantity of changes. In the experimental settings, radical changes in the viral phenotypic features occurred only after a few amino acid substitutions. The majority of the studied viruses in the genogroup D of E-11 maintained exceptional phenotypic property, the cross-neutralization with CV-A9 specific antiserum, despite their genetic divergence.
  • Ollila, Saara (Helsingfors universitet, 2008)
    Hereditary nonpolyposis colorectal cancer (HNPCC) is a hereditary cancer syndrome, which associates with high penetrance of early onset colorectal and endometrial tumours. Susceptibility for HNPCC is dominantly inherited with germline defects in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. A truncating mutation in one of these genes leads to deficient MMR, predisposing the mutation carriers to HNPCC, but a nontruncating mutation can either be neutral or lead to increased cancer risk and HNPCC. The correct determination of the pathogenicity of a found mutation is very important, as the verification of the causative mutation enables genetic counselling and surveillance of mutation carriers. This has been shown to lead to significantly lowered mortality. MSH2 is the second most commonly mutated HNPCC susceptibility gene and defects in it account for 39% of all identified HNPCC mutations. The aim of this work was to gather functional evidence on the pathogenicity of patient-derived nontruncating MSH2 variants. The proteins corresponding to the original genetic variants were expressed and purified. The expression level, MMR efficiency, interaction with MSH6, mismatch binding, and mismatch release capabilities of the protein variants were studied. The results of the functional assays were compared to the clinical characteristics of the mutation carriers. 12 of the studied 18 mutations were found to exhibit severe defects in the functional assays, supporting the hypothesis that these mutations were the underlying cause of the cancer phenotype in mutation carriers. 2 mutations reduced but did not abolish the function of the protein, leaving their pathogenicity status inconclusive. 4 mutations showed no or only a minor defect in the assays, suggesting nonpathogenicity. The functional defects were mediated through different mechanisms. The majority of the MMR-deficient mutations which were located in the amino-terminal domains of MSH2 demonstrated defects in the protein expression level. Most of the carboxy-terminal mutations, situated in the ATPase domain, had an impact on the ability of the protein to bind or release mismatched DNA. When comparing the biochemical data to the tumour phenotype, a significant correlation between the functional deficiency in vitro and lack of expression of the corresponding protein in the tumour was observed. The analyses demonstrated that the location of the mutation affects the biochemistry of MMR, but may also have an effect on the phenotype of MSH2 mutation carriers. This study significantly contributed to the knowledge of MSH2-associated HNPCC tumorigenesis, especially facilitating the diagnostics and counselling of the associated families.
  • Kiialainen, Anna (Helsingin yliopisto, 2007)
    PATHOGENIC MECHANISMS OF PLOSL Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease, is a recessively inherited disease of brain and bone. PLOSL manifests as early-onset progressive dementia and bone fractures. Mutations in the TYROBP (DAP12) and TREM2 genes have been identified as the primary cause of PLOSL. DAP12 and TREM2 encode important signalling molecules in cells of the innate immune system. The mechanism by which loss-of-function of the DAP12/TREM2 signalling complex leads to PLOSL is currently unknown. The aim of this thesis work was to gain insight into the pathogenic mechanisms behind PLOSL. To first identify the central nervous system (CNS) cell types that express both Dap12 and Trem2, the expression patterns of Dap12 and Trem2 in mouse CNS were analyzed. Dap12 and Trem2 expression was seen from embryonic stage to adulthood and microglial cells and oligodendrocytes were identified as the major Dap12/Trem2 producing cells of the CNS. To subsequently identify the pathways and biological processes associated with DAP12/TREM2 mediated signalling in human cells, genome wide transcript analysis of in vitro differentiated dendritic cells (DCs) of PLOSL patients representing functional knockouts of either DAP12 or TREM2 was performed. Both DAP12 and TREM2 deficient cells differentiated into DCs and responded to pathogenic stimuli. However, the DCs showed morphological differences compared to control cells due to defects in the actin filaments. Transcript profiles of the patient DCs showed differential expression of genes involved in immune response and for genes earlier associated with other disorders of the CNS as well as genes involved in the remodeling of bone, linking the findings with the tissue phenotype of PLOSL patients. To analyze the effect of Dap12 deficiency in the CNS, genome wide expression analysis of Dap12 deficient mouse brain and Dap12 deficient microglia as well as functional analysis of Dap12 deficient microglia was performed. Regulation of several pathways involved in synaptic function and transcripts coding for the myelin components was seen in Dap12 knockout mice. Decreased migration, morphological changes and shortened lifespan of the Dap12 knockout microglia was further observed. Taken together, this thesis work showed that both Dap12 and Trem2 are expressed by CNS microglia and that Dap12 deficiency results in functional defects of these cells. Lack of Dap12 in the CNS also leads to synaptic abnormalities even before pathological changes are seen in the tissue level.This work further showed that loss-of-function of DAP12 or TREM2 leads to changes in morphology and gene expression in human dendritic cells. These data underline the functional diversity of the molecules of the innate immune system and implies their significant contribution also in demyelinating CNS disorders, including those resulting in dementia.
  • Kariola, Tarja (Helsingin yliopisto, 2006)
    Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.
  • Raffaello, Tommaso (Helsingin yliopisto, 2013)
    The basidiomycete white-rot fungus Heterobasidion annosum sensu lato (s.l.) is a species complex comprising five species considered to be the most economically important pathogens of conifer trees in the northern hemisphere. The infection of new wood substrate is mediated by basidiospores, which land on the stump surface of a felled tree. After spore germination, the fungal mycelia actively colonise the stump and spread to new healthy trees by root-to-root contact. To start a new infection cycle, H. annosum s.l. must counteract the adverse environmental factors (abiotic stresses) at the stump surface. Moreover, active wood degradation requires the ability to detoxify the high levels of fungistatic and fungitoxic compounds (such as phenolics) that naturally accumulate in the tree wood tissue as a defence against pathogen attack. The availability of the genome sequence of the H. irregulare species allowed us to investigate the conservation of the intracellular pathways that are responsible for the abiotic stress response and cellular adaptation and proliferation. Using Saccharomyces cerevisiae as a model organism in which many of these pathways have been well characterised, we annotated all the conserved components of the mitogen activated protein kinase (MAPK) pathways in H. irregulare, namely, those involving the pheromone FUS3/KSS1, the high osmolarity gene HOG1, the cell integrity gene MPK1, calcium/calcineurin signalling, and the cAMP pathway. To better understand the H. annosum sensu stricto (s.s.) adaptation during abiotic stress and wood degradation, we investigated the general transcriptional profiles under several abiotic stresses (osmotic, oxidative, temperature, and nutrient starvation) and during growth on different pine woody materials (pine bark, sapwood, and heartwood). The results for abiotic stresses indicated the activation of genes involved in signalling (for example, protein kinase and transcription factors during starvation) but also genes involved in toxic substance detoxification and membrane transporters (cytochrome P450 and Major Facilitator Superfamily, MFS-1, respectively, in cold stress). During saprotrophic growth on different pine wood materials, a dramatic induction of several glycosyl hydrolase (GH) genes was observed. Some of these genes (for example, GH61) were specifically induced, mainly in pine heartwood, while others demonstrated less tissue specificity and were generally expressed during saprotrophic growth in all woody materials. During saprotrophic growth on pine lignocellulose material, several genes involved in lignin degradation, such as multi-copper oxidases (MCOs) and oxidoreductases, were also strongly induced. The central MAPK of one of the pathways involved in adaptation to abiotic stress (the HOG pathway) was further characterised. The H. annosum s.s. HaHOG1 gene was cloned and functionally studied to investigate its role in osmotic and oxidative stress response in this fungus. The HaHOG1 gene restored the function of the homologous HOG1, and the protein translocated to the cell nucleus under osmotic conditions in the S. cerevisiae heterologous host. Furthermore, HaHog1p was strongly phosphorylated in the presence of high concentrations of NaCl, KCl, and H2O2. These results suggest that the HOG pathway is activated when H. annosum s.s. is challenged with osmotic and oxidative stressors. This study sheds light on some adaptive mechanisms that characterise the growth of H. annosum s.s. under several conditions. Finally, this work provides new data at the transcriptome level to help identify genes that are activated during wood degradation and response to abiotic stresses.
  • Laine, Mikael (Helsingin yliopisto, 2010)
    Sjögren s syndrome (SS) is a common autoimmune disease affecting the lacrimal and salivary glands. SS is characterized by a considerable female predominance and a late age of onset, commonly at the time of adreno- and menopause. The levels of the androgen prohormone dehydroepiandrosterone-sulphate (DHEA-S) in the serum are lower in patients with SS than in age- and sex-matched healthy control subjects. The eventual systemic effects of low androgen levels in SS are not currently well understood. Basement membranes (BM) are specialized layers of extracellular matrix and are composed of laminin (LM) and type IV collagen matrix networks. BMs deliver messages to epithelial cells via cellular LM-receptors including integrins (Int) and Lutheran blood group antigen (Lu). The composition of BMs and distribution of LM-receptors in labial salivary glands (LSGs) of normal healthy controls and patients with SS was assessed. LMs have complex and highly regulated distribution in LSGs. LMs seem to have specific tasks in the dynamic regulation of acinar cell function. LM-111 is important for the normal acinar cell differentiation and its expression is diminished in SS. Also LM-211 and -411 seem to have some acinar specific functional tasks in LSGs. LM-311, -332 and -511 seem to have more general structure maintaining and supporting roles in LSGs and are relatively intact also in SS. Ints α3β1, α6β1, α6β4 and Lu seem to supply structural basis for the firm attachment of epithelial cells to the BM in LSGs. The expression of Ints α1β1 and α2β1 differed clearly from other LM-receptors in that they were found almost exclusively around the acini and intercalated duct cells in salivons suggesting some type of acinar cell compartment-specific or dominant function. Expression of these integrins was lower in SS compared to healthy controls suggesting that the LM-111 and -211-to-Int α1β1 and α2β1 interactions are defective in SS and are crucial to the maintenance of the acini in LSGs. DHEA/DHEA-S concentration in serum and locally in saliva of patients with SS seems to have effects on the salivary glands. These effects were first detected using the androgen-dependent CRISP-3 protein, the production and secretion of which were clearly diminished in SS. This might be due to the impaired function of the intracrine DHEA prohormone metabolizing machinery, which fails to successfully convert DHEA into its active metabolites in LSGs. The progenitor epithelial cells from the intercalated ductal area of LSGs migrate to the acinar compartment and then undergo a phenotype change into secretory acinar cells. This migration and phenotype change seem to be regulated by the LM-111-to-Int α1β1/Int α2β1 interactions. Lack of these interactions could be one factor limiting the normal remodelling process. Androgens are effective stimulators of Int α1β1 and α2β1 expression in physiologic concentrations. Addition of DHEA to the culture medium had effective stimulating effect on the Int α1β1 and α2β1 expression and its effect may be deficient in the LSGs of patients with SS.
  • Savinko, Terhi (Helsingin yliopisto, 2013)
    Atopic dermatitis (AD) is a common pruritic skin disease with prevalence rates up to 20 % in children and 3 % in adults. Skin barrier defects combined with modified immune responses of the innate and adaptive immune system activate complex pathophysiological pathways that are involved in the development of this disease. AD is characterized by acute flare-ups as well as chronic eczematous pruritic skin lesions and dry skin. It is crucial to clarify the mechanisms underlying AD in order to devise mechanism-based therapeutic approaches. However, the immunological mechanisms participating in AD are far from being completely understood. This thesis investigates mechanisms believed to be involved in atopic skin inflammation by utilizing an AD-like experimental animal model as well as human patients. The AD-like mouse model was also used to examine the model's suitability for evaluating topical medications for treating AD. In addition, this thesis investigates some of the mechanisms in the so-called atopic march. Results highlight new molecular mechanisms involved in AD and the atopic march. Microbial superantigen, derived from Staphylococcus aureus exacerbates the allergen-induced skin inflammation mostly by a mixed Th1/Th2 type inflammation in the presence of both CD8+ and CD4+ T cells and elevated IgE concentrations. This kind of severe inflammation, induced by allergen and superantigen in the murine skin, was declined with topical corticosteroid and calcineurin inhibitor. According to these results, this AD model is both reproducible and suitable for testing novel treatment options in AD. Finally, a recently characterized Th2-promoting cytokine, IL-33, and its receptor, ST2, were investigated in murine models of AD and allergic asthma as well as in human AD and in different cell models. The results obtained from ST2-/- mice suggest that the IL-33/ST2 pathway can regulate innate immune responses and CD8+ T cell mediated responses in the skin and in lung tissue. However, ST2 appeared to be dispensable for the development of Th2 response in the sensitized skin, whereas it was the main inducer of Th2 cytokines in asthmatic airways. Together, these results obtained from the murine model of AD and from the skin of patients with AD reveal new molecular mechanisms involved in AD.
  • Kajander, Kajsa (Helsingin yliopisto, 2008)
    Gastrointestinal symptoms and impaired quality of life caused by irritable bowel syndrome (IBS) affect up to 20% of the adult population worldwide. The exact aetiology and pathophysiology of IBS are incompletely understood. Clinical studies suggest that supplementation with certain probiotics may be beneficial in IBS, but there is not enough evidence to make general recommendations. The aim of this thesis was to investigate microbiota- and mucosa-associated pathophysiological factors of IBS, and to evaluate the long-term effects of multispecies probiotic supplementation on symptoms, quality of life, intestinal microbiota and systemic inflammatory markers in IBS. The intestinal microbiota composition in IBS patients and healthy control subjects was analysed by quantitative polymerase chain reaction (qPCR). Significantly lower counts for the Clostridium coccoides and the Bifidobacterium catenulatum groups were found in IBS compared to controls. Quantitative differences also appeared in subgroup analysis based on the predominant bowel habit: diarrhoea patients harboured significantly lower numbers of Lactobacillus spp. than the constipation-predominant patients, while higher counts for Veillonella spp. were detected in constipation-predominant patients compared to healthy controls. Analysis of mucosal biopsies by a metabolomic approach revealed multiple differences between patients and controls. The most prominent finding was an upregulation of specific lipid species, principally lysophosphatidylcholines and ceramides, in IBS. The effects of multispecies probiotic supplementation with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium breve Bb99 or Bifidobacterium animalis subsp. lactis Bb12 was evaluated in two, randomised, double-blind, placebo-controlled trials. Compared to placebo, the probiotic supplementation significantly reduced the total symptoms of IBS. No effects on bowel habit were seen. Health-related quality of life (HRQOL) is reduced in patients with IBS in comparison with the Finnish population on the whole. The probiotic supplementation improved one IBS-specific domain of quality of life (bowel symptoms), whereas no other effects on HRQOL were seen. The probiotics had no major effects on the predominant microbiota as measured by qPCR, but a microarray-based analysis suggested that the probiotic consumption stabilised the microbiota. No effects on serum sensitive-CRP or cytokines were detected. In conclusion, alterations in the microbiota composition and in the mucosal metabolite profile are potential pathophysiological factors of IBS. Multispecies probiotic supplementation alleviates the gastrointestinal symptoms of IBS, and improves the bowel symptoms domain of HRQOL. Probiotic supplementation in IBS is associated with a stabilisation of microbiota, but it does not influence systemic inflammatory markers.
  • Kuusniemi, Arvi-Matti (Helsingin yliopisto, 2007)
    Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disease which is highly enriched in the Finnish population. It is caused by mutations in the NPHS1 gene encoding for nephrin, which is a major component of the glomerular filtration barrier in the kidney. Patients with NPHS1 have heavy proteinuria and nephrotic syndrome (NS) from birth and develop renal fibrosis in early childhood. Renal transplantation (TX) is the only curative treatment for NPHS1. These patients form the largest group of pediatric kidney transplant children in our country. The NPHS1 kidneys are removed in infancy and they serve as an excellent human material for studies of the pathophysiology of proteinuric kidney diseases. Sustained proteinuria is a major factor leading to end-stage renal failure and understanding this process is crucial for nephrology. In this study we investigated the glomerular and tubulointerstitial changes that occur in the NPHS1 kidneys during infancy as well as the expression of nephrin in non-renal tissues. We also studied the pathology and management of recurrent proteinuria in kidney grafts transplanted to NPHS1 children. Severe renal lesions evolved in patients with NPHS1 during the first months of life. Glomerular sclerosis developed through progressive mesangial sclerosis, and capillary obliteration was an early consequence of this process. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. Few inflammatory cells were detected in the mesangial area. The glomerular epithelial cells (podocytes) showed severe ultrastructural changes and hypertrophy. Podocyte proliferation and apoptosis were rare, but moderate amounts of podocytes were detached and ended up in the urine. The results showed that endocapillary lesions not extracapillary lesions, as generally believed were important for the sclerotic process in the NPHS1 glomeruli. In the tubulointerstitium, severe lesions developed in NPHS1 kidneys during infancy. Despite heavy proteinuria, tubular epithelial cells (TECs) did not show transition into myofibroblasts. The most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1). Interstitial inflammation and fibrosis were first detected in the paraglomerular areas and the most abundant inflammatory cells were monocytes/macrophages. Arteries and arterioles showed intimal hypertrophy, but the pericapillary microvasculature remained quite normal. However, excessive oxidative stress was evident in NPHS1 kidneys. The results indicated that TECs were relatively resistant to the heavy tubular protein load. Nephrin was at first thought to be podocyte specific, but some studies especially in experimental animals have suggested that nephrin might also be expressed in non-renal tissues such as pancreas and central nervous system. The knowledge of nephrin biology is important for the evaluation of nephrin related diseases. In our study, no significant amounts of nephrin protein or mRNA were detected in non-renal tissues of man and pig as studied by immunohistochemistry and in situ hybridization. The phenotype analysis of NPHS1 children, who totally lack nephrin, revealed no marked impairment in the neurological, testicular, or pancreatic function speaking against the idea that nephrin would play an important functional role outside the kidney. The NPHS1 kidneys do not express nephrin and antibodies against this major glomerular filter protein have been observed in NPHS1 children after renal TX most likely as an immune reaction against a novel antigen. These antibodies have been associated with the development of recurrent NS in the kidney graft of NPHS1 patients. In our study, a third of the NPHS1 patients homozygous for Fin-Major mutation developed recurrent NS in the transplanted graft. Re-transplantations were performed to patients who lost their graft due to recurrent NS and heavy proteinuria immediately developed in all cases. While 73% of the patients had detectable serum anti-nephrin antibodies, the kidney biopsy findings were minimal. Introduction of plasma exchange (PE) to the treatment of recurrent nephroses increased the remission rate from 54% to 89%. If remission was achieved, recurrent NS did not significantly deteriorate the long term graft function. In conclusion, the results show that the lack of nephrin in podocyte slit diaphragm in NPHS1 kidneys induces progressive mesangial expansion and glomerular capillary obliteration and inflicts interstitial fibrosis, inflammation, and oxidative stress with surprisingly little involvement of the TECs in this process. Nephrin appears to have no clinical significance outside the kidney. Development of antibodies against nephrin seems to be a major cause of recurrent NS in kidney grafts of NPHS1 patients and combined use of PE and cyclophosphamide markedly improved remission rates.
  • Kestilä, Laura (Helsingin yliopisto, 2008)
    There is increasing evidence that the origins of poor adult health and health inequalities can be traced back to circumstances preceding current socioeconomic position and living conditions. The life-course approach to examining the determinants of health has emphasised that exposure to adverse social and economic circumstances in earlier life or concurrent adverse circumstances due to unfavourable living conditions in earlier life may lead to poor health, health-damaging behaviour, disease or even premature death in adulthood. There is, however, still a lack of knowledge about the contribution of social and economic circumstances in childhood and youth to adult health and health inequalities, and even less is known about how environmental and behavioural factors in adulthood mediate the effects of earlier adverse experiences. The main purpose of this study was to deepen our understanding of the development of poor health, health-damaging behaviours and health inequalities during the life-course. Its aim was to find out which factors in earlier and current circumstances determine health, the most detrimental indicators of health behaviour (smoking, heavy drinking and obesity as a proxy for the balance between nutrition and exercise), and educational health differences in young adults in Finland. Following the ideas of the social pathway theory, it was assumed that childhood environment affects adult health and its proximal determinants via different pathways, including educational, work and family careers. Early adulthood was studied as a significant phase of life when many behavioural patterns and living conditions relevant to health are established. In addition, socioeconomic health inequalities seem to emerge rapidly when moving into adulthood; they are very small or non-existent in childhood and adolescence, but very marked by early middle age. The data of this study were collected in 2000 2001 as part of the Health 2000 Survey (N = 9,922), a cross-sectional and nationally representative health interview and examination survey. The main subset of data used in this thesis was the one comprising the age group 18 29 years (N = 1,894), which included information collected by standardised structured computer-aided interviews and self-administered questionnaires. The survey had a very high participation rate at almost 90% for the core questions. According to the results of this study, childhood circumstances predict the health of young adults. Almost all the childhood adversities studied were found to be associated with poor self-rated health and psychological distress in early adulthood, although fewer associations were found with the somatic morbidity typical of young adults. These effects seemed to be more or less independent of the young adult s own education. Childhood circumstances also had a strong effect on smoking and heavy drinking, although current circumstances and education in particular, played a role in mediating this effect. Parental smoking and alcohol abuse had an influence on the corresponding behaviours of offspring. Childhood circumstances had a role in the development of obesity and, to a lesser extent, overweight, particularly in women. The findings support the notion that parental education has a strong effect on early adult obesity, even independently of the young adult s own educational level. There were marked educational differences in self-rated health in early adulthood: those in the lowest educational category were most likely to have average or poorer health. Childhood social circumstances seemed to explain a substantial part of these educational differences. In addition, daily smoking and heavy drinking contributed substantially to educational health differences. However, the contribution of childhood circumstances was largely shared with health behaviours adopted by early adulthood. Employment also shared the effects of childhood circumstances on educational health differences. The results indicate that childhood circumstances are important in determining health, health behaviour and health inequalities in early adulthood. Early recognition of childhood adversities followed by relevant support measures may play an important role in preventing the unfortunate pathways leading to the development of poor health, health-damaging behaviour and health inequalities. It is crucially important to recognise the needs of children living in adverse circumstances as well as children of substance abusing parents. In addition, single-parent families would benefit from support. Differences in health and health behaviours between different sub-groups of the population mean that we can expect to see ever greater health differences when today s generation of young adults grows older. This presents a formidable challenge to national health and social policy as well as health promotion. Young adults with no more than primary level education are at greatest risk of poor health. Preventive policies should emphasise the role of low educational level as a key determinant of health-damaging behaviours and poor health. Keywords: health, health behaviour, health inequalities, life-course, socioeconomic position, education, childhood circumstances, self-rated health, psychological distress, somatic morbidity, smoking, heavy drinking, BMI, early adulthood
  • Mazanikov, Maxim (Helsingin yliopisto, 2013)
    Endoscopic retrograde cholangiopancreatography (ERCP) is the radiographic examination of the biliary and/or pancreatic ductus via endoscopically cannulated duodenal papilla.The method of choice for sedation of patients during ERCP is still to be defined.Self administration of propofol by the patients (patient-controlled sedation, PCS)might be one of the possibilities of this kind. Dexmedetomidine has not been previously evaluated for sedation of alcoholics during ERCP. Four prospective randomized control trials consisting of 293 ERCP patients were performed. PCS was compared with anesthesiologist administered sedation (AAS) using manually adjusted propofol infusion (I) and target-controlled infusion (TCI) (III). Remifentanil and alfentanil were compared in sedative mixture for PCS (II) and dexmedetomidine was evaluated for sedation of patients with chronic alcohol-ism (IV). Self-administration device was adjusted to deliver 1ml single bolus-dose of propofol or propo-fol-opioid mixture. Loading dose of 1 mcg kg-1 of dexmedetomidine was infused in 10 minutes before ERCP start thereafter maintenance infusion at the constant rate of 0.7 mcg kg-1 h-1 was continued un-til the end of procedure (IV). In control groups sedation was administered by anesthesiologist. Hy-poxemia, respiratory depression, hypotension, arrhythmia, and pulmonary aspiration were considered as sedation related adverse events (SRAE). Consumption of propofol was the main objective. Secondary objectives were success rate of PCS, SRAE, patient satisfaction with sedation, easiness of ERCP per-formance, and rapidity of the recovery. With the use of PCS propofol consumption was significantly lesser than with AAS. The success rate of PCS was 88 -100 %. Patients received PCS recovered faster than received AAS. Combined with propo-fol, remifentanil depressed spontaneous respiration and produced nausea more frequently than alfen-tanil. Increase of alfentanil concentration in sedative mixture from 0.04 mg ml-1 to 0.08 mg ml-1 did not provide any demonstrable benefit. The studied regimen of dexmedetomidine administration showed poor suitability for sedation of alcoholics during ERCP. Instead, PCS might be successful in such pa-tients. In conclusion, PCS with combination of propofol and alfentanil is recommended as a primary method of sedation during ERCP.
  • Kiljunen, Timo (Helsingin yliopisto, 2008)
    Diagnostic radiology represents the largest man-made contribution to population radiation doses in Europe. To be able to keep the diagnostic benefit versus radiation risk ratio as high as possible, it is important to understand the quantitative relationship between the patient radiation dose and the various factors which affect the dose, such as the scan parameters, scan mode, and patient size. Paediatric patients have a higher probability for late radiation effects, since longer life expectancy is combined with the higher radiation sensitivity of the developing organs. The experience with particular paediatric examinations may be very limited and paediatric acquisition protocols may not be optimised. The purpose of this thesis was to enhance and compare different dosimetric protocols, to promote the establishment of the paediatric diagnostic reference levels (DRLs), and to provide new data on patient doses for optimisation purposes in computed tomography (with new applications for dental imaging) and in paediatric radiography. Large variations in radiation exposure in paediatric skull, sinus, chest, pelvic and abdominal radiography examinations were discovered in patient dose surveys. There were variations between different hospitals and examination rooms, between different sized patients, and between imaging techniques; emphasising the need for harmonisation of the examination protocols. For computed tomography, a correction coefficient, which takes individual patient size into account in patient dosimetry, was created. The presented patient size correction method can be used for both adult and paediatric purposes. Dental cone beam CT scanners provided adequate image quality for dentomaxillofacial examinations while delivering considerably smaller effective doses to patient compared to the multi slice CT. However, large dose differences between cone beam CT scanners were not explained by differences in image quality, which indicated the lack of optimisation. For paediatric radiography, a graphical method was created for setting the diagnostic reference levels in chest examinations, and the DRLs were given as a function of patient projection thickness. Paediatric DRLs were also given for sinus radiography. The detailed information about the patient data, exposure parameters and procedures provided tools for reducing the patient doses in paediatric radiography. The mean tissue doses presented for paediatric radiography enabled future risk assessments to be done. The calculated effective doses can be used for comparing different diagnostic procedures, as well as for comparing the use of similar technologies and procedures in different hospitals and countries.
  • Toroi, Paula (Helsingin yliopisto, 2009)
    The methods for estimating patient exposure in x-ray imaging are based on the measurement of radiation incident on the patient. In digital imaging, the useful dose range of the detector is large and excessive doses may remain undetected. Therefore, real-time monitoring of radiation exposure is important. According to international recommendations, the measurement uncertainty should be lower than 7% (confidence level 95%). The kerma-area product (KAP) is a measurement quantity used for monitoring patient exposure to radiation. A field KAP meter is typically attached to an x-ray device, and it is important to recognize the effect of this measurement geometry on the response of the meter. In a tandem calibration method, introduced in this study, a field KAP meter is used in its clinical position and calibration is performed with a reference KAP meter. This method provides a practical way to calibrate field KAP meters. However, the reference KAP meters require comprehensive calibration. In the calibration laboratory it is recommended to use standard radiation qualities. These qualities do not entirely correspond to the large range of clinical radiation qualities. In this work, the energy dependence of the response of different KAP meter types was examined. According to our findings, the recommended accuracy in KAP measurements is difficult to achieve with conventional KAP meters because of their strong energy dependence. The energy dependence of the response of a novel large KAP meter was found out to be much lower than with a conventional KAP meter. The accuracy of the tandem method can be improved by using this meter type as a reference meter. A KAP meter cannot be used to determine the radiation exposure of patients in mammography, in which part of the radiation beam is always aimed directly at the detector without attenuation produced by the tissue. This work assessed whether pixel values from this detector area could be used to monitor the radiation beam incident on the patient. The results were congruent with the tube output calculation, which is the method generally used for this purpose. The recommended accuracy can be achieved with the studied method. New optimization of radiation qualities and dose level is needed when other detector types are introduced. In this work, the optimal selections were examined with one direct digital detector type. For this device, the use of radiation qualities with higher energies was recommended and appropriate image quality was achieved by increasing the low dose level of the system.
  • Vilo, Jaak (Helsingin yliopisto, 2002)
  • Junttila, Esa (Helsingin yliopisto, 2011)
    Reorganizing a dataset so that its hidden structure can be observed is useful in any data analysis task. For example, detecting a regularity in a dataset helps us to interpret the data, compress the data, and explain the processes behind the data. We study datasets that come in the form of binary matrices (tables with 0s and 1s). Our goal is to develop automatic methods that bring out certain patterns by permuting the rows and columns. We concentrate on the following patterns in binary matrices: consecutive-ones (C1P), simultaneous consecutive-ones (SC1P), nestedness, k-nestedness, and bandedness. These patterns reflect specific types of interplay and variation between the rows and columns, such as continuity and hierarchies. Furthermore, their combinatorial properties are interlinked, which helps us to develop the theory of binary matrices and efficient algorithms. Indeed, we can detect all these patterns in a binary matrix efficiently, that is, in polynomial time in the size of the matrix. Since real-world datasets often contain noise and errors, we rarely witness perfect patterns. Therefore we also need to assess how far an input matrix is from a pattern: we count the number of flips (from 0s to 1s or vice versa) needed to bring out the perfect pattern in the matrix. Unfortunately, for most patterns it is an NP-complete problem to find the minimum distance to a matrix that has the perfect pattern, which means that the existence of a polynomial-time algorithm is unlikely. To find patterns in datasets with noise, we need methods that are noise-tolerant and work in practical time with large datasets. The theory of binary matrices gives rise to robust heuristics that have good performance with synthetic data and discover easily interpretable structures in real-world datasets: dialectical variation in the spoken Finnish language, division of European locations by the hierarchies found in mammal occurrences, and co-occuring groups in network data. In addition to determining the distance from a dataset to a pattern, we need to determine whether the pattern is significant or a mere occurrence of a random chance. To this end, we use significance testing: we deem a dataset significant if it appears exceptional when compared to datasets generated from a certain null hypothesis. After detecting a significant pattern in a dataset, it is up to domain experts to interpret the results in the terms of the application.
  • Ruutu, Hanna (Helsingin yliopisto, 2007)
    This study examines the position and meaning of Classical mythological plots, themes and characters in the oeuvre of the Russian Modernist poet Marina Tsvetaeva (1892-1941). The material consists of lyric poems from the collection Posle Rossii (1928) and two longer lyrical tragedies, Ariadna (1924) and Fedra (1927). These works are examined in the context of Russian Modernism and Tsvetaeva s own poetic development, also taking into account the author s biography, namely, her correspondence with Boris Pasternak. Tsvetaeva s appropriations of the myths enter into a dialogue with the Classical tradition and with the earlier Russian and Western literary manifestations of the source material. Her Classical texts are inextricably linked with her own authorial myth, they are used to project both her ideas about poetry as well as the authored self of her poems. An important context for Tsvetaeva s application of the Classical myths is the concept of the Platonic ladder of Eros. This plot evokes the process of transcendence of the mortal subject into the immaterial realm and is applied by the author as an extended metaphor of the poet s birth. Emphasizing the dialectical movement between the earthly and the divine, Tsvetaeva s Classical personae foreground various positions of the individual between these two realms. By means of kaleidoscopic reformulations of similar metaphors and concepts, Tsvetaeva s mythological poems illustrate the poet s position between the material and the immaterial and the various consequences of this dichotomy on the creative mission. At the heart of Tsvetaeva s appropriation of the Sibyl, Phaedra, Eurydice and Ariadne is the tension between the body and disembodiment. The two lyrical tragedies develop the dichotomous worldview further, nevertheless emphasizing the dual perspective of the divine and the earthly realms: immaterial existence is often evaluated from a material perspective and vice versa. The Platonic subtext is central for Ariadna, focussing on Theseus development from an earthly hero to a spiritual one. Fedra concentrates on Phaedra s divinely induced physical passion, which is nevertheless evoked in a creative light.