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  • Viitanen, Tero (Helsingin yliopisto, 2010)
    Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.
  • Uusisaari, Marylka (Helsingin yliopisto, 2003)
  • Lindfors, Päivi (Helsingin yliopisto, 2006)
    Glial cell line-derived neurotrophic factor (GDNF) family ligands: GDNF, neurturin, persephin and artemin, signal through a receptor tyrosine kinase Ret by binding first to a co-receptor (GFRα1-4) that is attached to the plasma membrane. The GDNF family factors can support the survival of various peripheral and central neuronal populations and have important functions also outside the nervous system, especially in kidney development. Activating mutations in the RET gene cause tumours in neuroendocrine cells, whereas inactivating mutations in RET are found in patients with Hirschsprung s disease (HSCR) characterized by loss of ganglionic cells along the intestine. The aim of this study was to examine the in vivo functions of neurturin receptor GFRα2 and persephin receptor GFRα4 using knockout (KO) mice. Mice lacking GFRα2 grow poorly after weaning and have deficits in parasympathetic and enteric innervation. This study shows that impaired secretion of the salivary glands and exocrine pancreas contribute to growth retardation in GFRα2-KO mice. These mice have a reduced number of intrapancreatic neurons and decreased cholinergic innervation of the exocrine pancreas as well as reduced excitatory fibres in the myenteric plexus of the small intestine. This study also demonstrates that GFRα2-mediated Ret signalling is required for target innervation and maintenance of soma size of sympathetic cholinergic neurons and sensory nociceptive IB4-binding neurons. Furthermore, lack of GFRα2 in mice results in deficient perception of temperatures above and below thermoneutrality and in attenuated inflammatory pain response. GFRα4 is co-expressed with Ret predominantly in calcitonin-producing thyroid C-cells in the mouse. In this study GFRα4-deficient mice were generated. The mice show no gross developmental deficits and have a normal number of C-cells. However, young but not adult mice lacking GFRα4 have a lower production of calcitonin in thyroid tissue and consequently, an increased bone formation rate. Thus, GFRα4/Ret signalling may regulate calcitonin production. In conclusion, this study reveals that GFRα2/Ret signalling is crucial for the development and function of specific components of the peripheral nervous system and that GFRα4-mediated Ret signalling is required for controlling transmitter synthesis in thyroid C-cells.
  • Bespalov, Maxim (Helsingin yliopisto, 2009)
    Neurotrophic factors play essential role in the development and functioning of the nervous system and other organs. Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands (GFLs) are of particular interest because they promote the survival of dopaminergic neurons in vitro, in Parkinson s disease animal models and in patients. GDNF is also a potent survival factor for the central motoneurons and thus is considered as a potential lead for the treatment of amyotrophic lateral sclerosis. The survival promoting receptor complex for GFLs consists of a ligand-specific co-receptor, GFRα and a signal transducing module, receptor tyrosine kinase RET. At least GDNF and persephin, a GFL, have established functions outside central nervous system. GDNF is crucial for enteric nervous system and kidney development as well as for spermatogenesis. Persephin controls calcitonin secretion. Communication between cells often occurs in the extracellular matrix (ECM), a meshwork, which is secreted and deposited by the cells and is mainly composed of fibrillar proteins and polymerized sugars. We evaluated the relationship between GFLs and extracellular matrix components and demonstrated that three GFLs - GDNF, neurturin and artemin bind heparan sulfates with nanomolar affinities. The fourth member of the family - persephin binds these polysaccharides thousand times less tightly. GDNF, neurturin and artemin also bind with high affinity to heparan sulfate proteoglycan (HSPG) isolated from the nervous system, syndecan-3. GDNF signals through HSPGs, evoking Src family kinase activation. This signaling induces cell spreading, hippocampal neurite outgrowth in vitro and cellular migration. Specifically, GDNF signaling through syndecan-3 is important for embryonic cortical neuron migration. Syndecan-3-deficient mice, similarly to mice lacking GDNF, have less GABAergic neurons in their cortex, as compared to the wild-type mice. This fact provides indirect evidence that GDNF interaction with syndecan-3 is important for cortical brain development. Noteworthy, in non-neuronal tissues GFLs may signal via other syndecans. We also present the structural model for a GDNF co-receptor, GFRα1. The X-ray structure of the GFRα1 domain 3 was solved with 1.8 Å resolution, revealing a new protein fold. Later we also solved the structure of the truncated GFRα1 in the complex with GDNF and this model was confirmed by site-directed mutagenesis. In summary, our work contributed to the structural characterization of GFRα-based receptor complex and revealed a new receptor for GDNF, neurturin and artemin the HSPG syndecan-3. This information is critically important for the development of GFRα/RET agonists for the treatment of neurodegenerative diseases.
  • Niittymäki, Jaana (Helsingin yliopisto, 2007)
    GDP-L-fucose: synthesis and role in inflammation The migration of leukocytes from intravascular locations to extravascular sites is essential to the immune responses. The initial attachment of leukocytes to the endothelium and the rolling step of the leukocyte extravasation cascade are mediated by selectins, a family of cell adhesion molecules on cell surfaces. Selectins are able to recognize glycoproteins and glycolipids containing the tetrasaccharide sialyl Lewis x (sLex, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc). Several glycosyltransferases are involved in the biosynthesis of sLex, fucosyltransferase VII (Fuc-TVII) being the last enzyme to modify the sLex structure. Fuc-TVII transfers L-fucose from GDP-L-fucose to sialylated N-acetyllactosamine. GDP-L-fucose is synthesized in the cytosol via two different metabolic pathways. The major, constitutively active de novo pathway involves conversion of GDP-α-D-mannose to GDP-β-L-fucose. In the alternative salvage pathway, L-fucokinase synthesizes from free fucose L-fucose-1-phosphate, which is further converted to GDP-L-fucose by GDP-L-fucose pyrophosphorylase. GDP-L-fucose is translocated from the cytosol to Golgi for fucosylation via the GDP-fucose transporter. This thesis involved the study of the synthesis of GDP-L-fucose via the salvage pathway: cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. The gene expression levels of these enzymes were found to be relatively high in various tissues; the mRNA levels were highest in brain, ovary and testis. This study also describes molecular cloning of rat fucosyltransferase VII (FUT7) and its expression as a functional enzyme. Gene expression levels of GDP-L-fucose synthesizing enzymes, GDP-fucose transporter and FUT7 were determined in inflamed tissues as well as cancer cells. Our results revealed a clear upregulation of the enzymes involved in the synthesis of GDP-L-fucose via de novo pathway, GDP-fucose transporter and FUT7 in inflamed tissues and in cancer cells. On the contrary, the GDP-L-fucose salvage pathway was found to be irrelevant in inflammation and in tumorigenesis. Furthermore, our results indicated the transcriptional coregulation of Golgi transporters involved in the synthesis of sulfo sLex, i.e. CMP-sialic acid, GDP-fucose and 3 phosphoadenosine 5 -phosphosulfate transporters, in inflammation.
  • Björklund, Mikael (Helsingin yliopisto, 2004)
  • Greco, Dario (Helsingin yliopisto, 2009)
    The time of the large sequencing projects has enabled unprecedented possibilities of investigating more complex aspects of living organisms. Among the high-throughput technologies based on the genomic sequences, the DNA microarrays are widely used for many purposes, including the measurement of the relative quantity of the messenger RNAs. However, the reliability of microarrays has been strongly doubted as robust analysis of the complex microarray output data has been developed only after the technology had already been spread in the community. An objective of this study consisted of increasing the performance of microarrays, and was measured by the successful validation of the results by independent techniques. To this end, emphasis has been given to the possibility of selecting candidate genes with remarkable biological significance within specific experimental design. Along with literature evidence, the re-annotation of the probes and model-based normalization algorithms were found to be beneficial when analyzing Affymetrix GeneChip data. Typically, the analysis of microarrays aims at selecting genes whose expression is significantly different in different conditions followed by grouping them in functional categories, enabling a biological interpretation of the results. Another approach investigates the global differences in the expression of functionally related groups of genes. Here, this technique has been effective in discovering patterns related to temporal changes during infection of human cells. Another aspect explored in this thesis is related to the possibility of combining independent gene expression data for creating a catalog of genes that are selectively expressed in healthy human tissues. Not all the genes present in human cells are active; some involved in basic activities (named housekeeping genes) are expressed ubiquitously. Other genes (named tissue-selective genes) provide more specific functions and they are expressed preferably in certain cell types or tissues. Defining the tissue-selective genes is also important as these genes can cause disease with phenotype in the tissues where they are expressed. The hypothesis that gene expression could be used as a measure of the relatedness of the tissues has been also proved. Microarray experiments provide long lists of candidate genes that are often difficult to interpret and prioritize. Extending the power of microarray results is possible by inferring the relationships of genes under certain conditions. Gene transcription is constantly regulated by the coordinated binding of proteins, named transcription factors, to specific portions of the its promoter sequence. In this study, the analysis of promoters from groups of candidate genes has been utilized for predicting gene networks and highlighting modules of transcription factors playing a central role in the regulation of their transcription. Specific modules have been found regulating the expression of genes selectively expressed in the hippocampus, an area of the brain having a central role in the Major Depression Disorder. Similarly, gene networks derived from microarray results have elucidated aspects of the development of the mesencephalon, another region of the brain involved in Parkinson Disease.
  • Tornberg, Janne (Helsingin yliopisto, 2006)
    The cation-Cl- cotransporter (CCC) family comprises of Na+-Cl- cotransporter (NCC), Na+-K+-2Cl- cotransporters (NKCC1-2), and four K+-Cl- cotransporters (KCC1-4). These proteins are involved in several physiological activities, such as cell volume regulation. In neuronal tissues, NKCC1 and KCC2 are important in determining the intracellular Cl- levels and hence the neuronal responses to inhibitory neurotransmitters GABA and glycine. One aim of the work was to elucidate the roles for CCC isoforms in the control of nervous system development. KCC2 mRNA was shown to be developmentally up-regulated and follow neuronal maturation, whereas NKCC1 and KCC4 transcripts were highly expressed in the proliferative zones of subcortical regions. KCC1 and KCC3 mRNA displayed low expression throughout the embryogenesis. These expression profiles suggest a role for CCC isoforms in maturation of synaptic responses and in the regulation of neuronal proliferation during embryogenesis. The major aim of this work was to study the biological consequences of KCC2-deficiency in the adult CNS, by generating transgenic mice retaining 15-20% of normal KCC2 levels. In addition, by using these mice as a tool for in vivo pharmacological analysis, we investigated the requirements for KCC2 in tonic versus phasic GABAA receptor-mediated inhibition. KCC2-deficient mice displayed normal reproduction and life span, but showed several behavioral abnormalities, including increased anxiety-like behavior, impaired performance in water maze, alterations in nociceptive processing, and increased seizure susceptibility. In contrast, the mice displayed apparently normal spontaneous locomotor activity and motor coordination. Pharmacological analysis of KCC2-deficient mice revealed reduced sensititivity to diazepam, but normal gaboxadol-induced sedation, neurosteroid hypnosis and alcohol-induced motor impairment. Electrophysiological recordings from CA1-CA3 subregions of the hippocampus showed that KCC2 deficiency affected the reversal potentials of both the phasic and tonic GABA currents, and that the tonic conductance was not affected. The results suggest that requirement for KCC2 in GABAergic neurotransmission may differ among several functional systems in the CNS, which is possibly due to the more critical role of KCC2 activity in phasic compared to tonic GABAergic inhibition.
  • Pykäläinen, Anette (Helsingin yliopisto, 2011)
    Biological membranes are tightly linked to the evolution of life, because they provide a way to concentrate molecules into partially closed compartments. The dynamic shaping of cellular membranes is essential for many physiological processes, including cell morphogenesis, motility, cytokinesis, endocytosis, and secretion. It is therefore essential to understand the structure of the membrane and recognize the players that directly sculpt the membrane and enable it to adopt different shapes. The actin cytoskeleton provides the force to push eukaryotic plasma membrane in order to form different protrusions or/and invaginations. It has now became evident that actin directly co-operates with many membrane sculptors, including BAR domain proteins, in these important events. However, the molecular mechanisms behind BAR domain function and the differences between the members of this large protein family remain largely unresolved. In this thesis, the structure and functions of the I-BAR domain family members IRSp53 and MIM were thoroughly analyzed. By using several methods such as electron microscopy and systematic mutagenesis, we showed that these I-BAR domain proteins bind to PI(4,5)P2-rich membranes, generate negative membrane curvature and are involved in the formation of plasma membrane protrusions in cells e.g. filopodia. Importantly, we characterized a novel member of the BAR-domain superfamily which we named Pinkbar. We revealed that Pinkbar is specifically expressed in kidney and epithelial cells, and it localizes to Rab13-positive vesicles in intestinal epithelial cells. Remarkably, we learned that the I-BAR domain of Pinkbar does not generate membrane curvature but instead stabilizes planar membranes. Based on structural, mutagenesis and biochemical work we present a model for the mechanism of the novel membrane deforming activity of Pinkbar. Collectively, this work describes the mechanism by which I-BAR domain proteins deform membranes and provides new information about the biological roles of these proteins. Intriguingly, this work also gives evidence that significant functional plasticity exists within the I-BAR domain family. I-BAR proteins can either generate negative membrane curvature or stabilize planar membrane sheets, depending on the specific structural properties of their I-BAR domains. The results presented in this thesis expand our knowledge on membrane sculpting mechanisms and shows for the first time how flat membranes can be generated in cells.
  • Alakulppi, Noora (Helsingin yliopisto, 2008)
    Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.
  • Palo, Jukka (Helsingin yliopisto, 2003)
  • Manninen, Outi (Helsingin yliopisto, 2000)
  • Broberg, Martin (Helsingin yliopisto, 2015)
    The interactions between phytopathogenic bacteria and their host plants can be characterized as an intricate web of signals and appropriate responses. Phytopathogenic soft rot bacteria occur globally, causing disease in Solanum tuberosum (potato) and other tubular staple foods in both the field and storage. One widely studied soft rot bacterium is Pectobacterium wasabiae, which has been identified in Eutrema wasabi (wasabi) plants in Japan and in potatoes in Finland. Generally, the interactions between this type of bacterium and host plants are characterized by maceration of plant tissue, due to the actions of secreted plant cell wall degrading enzymes (PCWDE), and the induction of phytohormone dependent defenses in the plants. The maceration of plant tissue involves the release of pectic oligogalacturonides (OGs) from plant cell walls. OGs have been identified as important signaling compounds, inducing the expression of a variety of defense-related genes. As the bacterial infection advances, the bacteria coordinate the production of virulence factors by utilizing regulatory proteins that modulate the transcriptome. Transcriptomic analyses have been used extensively in past studies to identify regulatory networks and signaling pathways, and these studies have provided insights into the processes underlying plant-pathogen interactions. The novel scientific results of this dissertation are derived from a combination of transcriptomic, genomic, genetic, and phenotypic analyses. This study analyzed various aspects of plant-pathogen interactions. The central bacterial model used was P. wasabiae, and the model plant of interest was Arabidopsis thaliana. This study characterized the genome of P. wasabiae via sequencing and bioinformatics analysis. Various virulence associated genes and operons, such as two distinct type 6 secretion systems, were identified and annotated. The bacterium was found to in fact be more related to P. wasabiae than Pectobacterium carotovorum, which the strain originally had been named after. Furthermore, a combination of functional genetics and transcriptomic methods, such as reverse transcription quantitative PCR (RT-qPCR) and microarrays, were used to determine the regulons controlled by the proteins ExpA and RsmA in P. wasabiae. These two proteins have been identified as important for the virulence of several γ-proteobacterial pathogens. This study analyzed the regulons via the use of three mutants: expA, rsmA, and an expA rsmA double mutant (DM). Overlapping and independently regulated targets were identified between ExpA and RsmA. Phenotypic assays for motility, growth, PCWDE activity, and virulence confirmed the transcriptomic data for the mutant strains. Novel findings included reduction of swimming motility in agar medium for P. wasabiae expA and rsmA mutants. In addition, the DM exhibited enhanced virulence and fitness in planta compared to either single mutant. Via analysis of transcriptomic data, a subset of genes was identified as affected in expression by an expA mutation independently of the presence of rsmA. The relatively unexplored role of short OGs (with a degree of polymerization (DP) less than 10) in damage-associated molecular pattern (DAMP) signaling in A. thaliana was characterized in this study. Comparative gene expression profiling based on RNA sequencing and RT-qPCR was performed on RNA harvested from plants treated with short OGs or with a mock suspension. Phenotypic assays confirmed the gene expression data. In a meta-data analysis, the resulting RNA sequencing and RT-qPCR data were compared with gene expression data from previous studies, in which long OGs (DP more than 10) were used to treat plants. This work demonstrated that short and long OGs induce genes and genesets associated with pathogen defense and phytohormone signaling, whereas reducing plant growth and development. The transcriptomic data of this study suggests that plant treatment with a mixture of short or long OGs yields a more pronounced and varied modulation of global gene expression, compared to treatment with only trimeric OGs. The regulation of the virulence of P. wasabiae, and the DAMP signaling triggered by plant cell wall damage in A. thaliana, are elements of the interactions between the plant and pathogen. The studies presented in this dissertation provide novel information about these two biological processes and highlights their connection.
  • DeFaveri, Jacquelin (Helsingin yliopisto, 2013)
    Spatial differentiation in phenotypic traits is commonly observed in the wild, but both the proximate (cf. environmental vs. genetic) and ultimate (cf. adaptive vs. stochastic) causes underlying this differentiation often remain obscure. Studies focussed on the genetic basis of this differentiation can inform us about these issues, especially if the genetic variants under investigation can be linked with information about their functional role(s) and/or gauged against expectations derived from evolutionary null models. However, due to the difficulties in deciphering and studying the genetic basis of phenotypic variability and differentiation in quantitative traits especially in marine vertebrates the occurrence and scale of local adaptation in them is still poorly understood. Yet, identifying patterns of adaptive divergence and the ecological factors that have contributed to them is essential for understanding how natural selection can maintain local adaptation in the face of gene flow. In this thesis I used a genome-wide set of candidate gene-based microsatellite markers, in combination with quantitative genetic approaches, to explore the patterns of adaptive diversity and divergence among stickleback populations from a variety of habitats ranging from global to local geographic scales. Through comparisons of several independent, isolated pairs of marine and freshwater populations, I found that selection is acting on many genomic regions harbouring genes whose putative functions are related to a wide variety of physiological processes. I also found indications that adaptation to freshwater environments may have been achieved through different genetic pathways in different populations. Importantly, the design of my study was such that alternative demographic explanations for observed patterns could be excluded. Focussing on populations within the physically continuous, yet environmentally heterogeneous marine habitat, I further investigated whether selection is acting strongly enough to promote adaptive population structuring despite high gene flow. Signatures of selection were detected in several candidate genes, along with clear evidence for adaptive differentiation in a phenotypic trait (lateral plate number). Analysis of population structure with only these outlier loci uncovered a higher degree of differentiation than was evident in neutral loci, and in some cases, patterns of adaptive differentiation were correlated with environmental variables likely to act as selective agents in the marine environment (viz. salinity and temperature). Evidence for local adaptation among Baltic Sea sticklebacks was confirmed in a common garden experiment, which demonstrated a loss of fitness in populations native to low salinity regions when exposed to high salinity treatments. Overall, the results from this thesis point to the conclusion that adaptive genetic and phenotypic differentiation is common, even in continuous marine habitats lacking obvious physical barriers to dispersal and gene flow. These results are particularly noteworthy, firstly from the perspective that earlier studies conducted using neutral marker genes have largely overlooked the patterns and magnitude of divergence, and secondly due to the comprehensive geographic coverage of the investigations.
  • Haimila, Katri (Helsingin yliopisto, 2009)
    Co-stimulatory signals are essential for the activation of naïve T cells and productive immune response. Naïve T cells receive first, antigen-specific signal through T cell receptor. Co-stimulatory receptors provide the second signal which can be either activating or inhibitory. The balance between signals determines the outcome of an immune response. CD28 is crucial for T cell activation; whereas cytotoxic T lymphocyte associated antigen 4 (CTLA4) mediates critical inhibitory signal. Inducible co-stimulator (ICOS) augments cytokine expression and plays role in immunoglobulin class switching. Programmed cell death 1 (PDCD1) acts as negative regulator of T cell proliferation and cytokine responses. The co-stimulatory receptor pathways are potentially involved in self-tolerance and thus, they provide a promising therapeutic strategy for autoimmune diseases and transplantation. The genes encoding CD28, CTLA4 and ICOS are located adjacently in the chromosome region 2q33. The PDCD1 gene maps further, to the region 2q37. CTLA4 and PDCD1 are associated with the risk of a few autoimmune diseases. There is strong linkage disequilibrium (LD) on the 2q33 region; the whole gene of CD28 exists in its own LD block but CTLA4 and the 5' part of ICOS are within a same LD block. The 3' part of ICOS and PDCD1 are in their own separate LD blocks. Extended haplotypes covering the 2q33 region can be identified. This study focuses on immune related conditions like coeliac disease (CD) which is a chronic inflammatory disease with autoimmune features. Immunoglobulin A deficiency (IgAD) belongs to the group of primary antibody deficiencies characterised by reduced levels of immunoglobulins. IgAD co-occurs often with coeliac disease. Renal transplantation is needed in the end stage kidney diseases. Transplantation causes strong immune response which is tried to suppress with drugs. All these conditions are multifactorial with complex genetic background and multiple environmental factors affecting the outcome. We have screened ICOS for polymorphisms by sequencing the exon regions. We detected 11 new variants and determined their frequencies in Finnish population. We have measured linkage disequilibrium on the 2q33 region in Finnish as well as other European populations and observed conserved haplotypes. We analysed genetic association and linkage of the co-stimulatory receptor gene region aiming to study if it is a common risk locus for immune diseases. The 2q33 region was replicated to be linked to coeliac disease in Finnish population and CTLA4-ICOS haplotypes were found to be associated with CD and IgAD being the first non-HLA risk locus common for CD and immunodeficiencies. We also showed association between ICOS and the outcome of kidney transplantation. Our results suggest new evidence for CTLA4-ICOS gene region to be involved in susceptibility of coeliac disease. The earlier published contradictory association results can be explained by involvement of both CTLA4 and ICOS in disease susceptibility. The pattern of variants acting together rather than a single polymorphism may confer the disease risk. These genes may predispose also to immunodeficiencies as well as decreased graft survival and delayed graft function. Consequently, the present study indicates that like the well established HLA locus, the co-stimulatory receptor genes predispose to variety of immune disorders.
  • Jaatinen, Taina (Helsingin yliopisto, 2002)
  • Kukkonen, Mari (Helsingin yliopisto, 2013)
    Both tobacco smoke and asbestos fibers enter the body mainly by inhalation. In the lungs, they may evoke oxidative stress, alter the protease-antiprotease balance, induce innate and adaptive immune responses, and create persistent inflammation leading eventually to lung injury. The type and severity of lung injury induced by foreign compounds varies greatly between individuals, even with similar exposure history. These differences are believed to originate from the complex interplay between genetic, epigenetic, environmental, and life course factors. In this thesis, the roles of several genes encoding proteins involved in xenobiotic metabolism (EPHX1, GSTM1, GSTM3, GSTP1, GSTT1, and NAT2), protease-antiprotease balance (MMP1, MMP9, MMP12, SERPINE2, and TIMP2), innate immunity (NLRP3 and CARD8), and inflammation (TNF, TGFB1, and GC) were studied in the development of asbestos and tobacco smoke exposure related nonmalignant pleural and pulmonary changes, peripheral obstruction, and impairment of pulmonary diffusing capacity in two clinically and radiologically examined cohorts of Finnish construction workers. The results indicate that polymorphisms of xenobiotic metabolizing genes are potential modifiers of the risk of developing pleural and pulmonary changes related to asbestos and tobacco smoke exposures. The most convincing evidence came from GSTT1; the deletion of this gene was significantly associated with several types of changes in the whole study population and in both of the study cohorts separately. In addition, certain genotype in GSTM3 gene was associated with lowered pulmonary function. Furthermore, gene polymorphisms in metalloproteinase MMP9, metalloproteinase inhibitor TIMP2, and inflammatory cytokines TNF and TGFB1 were associated with different emphysema subtypes and/or lung function. Together with the finding that certain genotypes of the serine protease inhibitor gene SERPINE2 predispose to panlobular emphysema, they imply that polymorphisms of genes involved in protease-antiprotease balance likely contribute to the development of pulmonary emphysema and bronchial obstruction, and different molecular mechanisms may explain the development of different emphysema subtypes. The present results also indicate that genetic variation in innate immunity related genes might have an important role in coping with asbestos exposure. One finding supporting this view was the association between interstitial lung fibrosis and polymorphism of NLRP3, an essential component of the NLRP3 inflammasome complex. Polymorphism in another member of the complex, CARD8, was associated with the greatest thickness of pleural plaques. In conclusion, the results of this thesis work show that genetic variation is an important modifier of the risks for developing non-malignant lung diseases related to external exposures. The present findings may also help to clarify the molecular mechanisms behind these diseases, expediting their prevention and the development of more efficient therapies.