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  • Sjöblom, Solveig (Helsingin yliopisto, 2009)
    Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.
  • Furuhjelm, Johanna (Helsingin yliopisto, 2004)
  • Blomster, Tiina (Helsingin yliopisto, 2012)
    Life on earth is largely dependent on plants, which provide us energy and oxygen. Plants are sessile organisms adapted to their respective growth environments. However, these environmental conditions are rarely constant and changes in growth conditions require modifications in plant metabolism. Abiotic environmental factors may suddenly become limited or excessive, or a pathogen attack may cause biotic stress. Reactive oxygen species (ROS) are by-products of normal aerobic metabolism and their production is enhanced by biotic and abiotic stresses. ROS serve as signaling molecules, which regulate expression of stress-responsive genes together with other signaling pathways in order to achieve appropriate responses to the suboptimal environment. Human activities also modify the environment for instance by increasing levels of air pollutant ozone (O3) which is a ROS causing foliar damage in sensitive species and cultivars. Therefore, understanding the mechanisms governing plant stress tolerance is of increasing importance. Importantly for this work, O3 is known to cause production of apoplastic ROS in plants similar to other stresses and was therefore used here as a tool to study stress signaling. The Arabidopsis thaliana mutant radical-induced cell death1 (rcd1) harbors several stress phenotypes related to ROS signaling. In order to identify novel genes and signaling pathways regulating plant stress responses, RCD1 and its homologs were studied further. RCD1 belongs to a plant-specific SIMILAR TO RCD-ONE (SRO) gene family present in all land plants analyzed. The molecular function of SROs remains unknown as they have no poly(ADP-ribose) polymerase (PARP) activity and lack catalytic amino acids in the conserved PARP catalytic domain. However, SROs are able to interact with transcription factors via their C-terminal RST domain which suggests that they may regulate gene expression. In addition to altered rosette morphology, rcd1 has changes in gene expression in normal growth conditions which may partially be attributed to RCD1 protein-protein interactions. However, sro1 and sro5 plants are similar to wild-type Col-0 regarding their growth habitus and gene expression. A. thaliana paralogs RCD1 and SRO1 share unequal genetic redundancy: the rcd1 sro1 double mutant is stunted and SRO1 can partially complement rcd1. Transcriptomic analysis of apoplastic ROS-induced signaling triggered by O3 treatment revealed altered expression of thousands of genes in a time-dependent manner. In rcd1, this response was exaggerated, which may explain the triggering of programmed cell death in O3-treated rcd1. Transcription factor WRKY70 was identified as a positive regulator of cell death, putatively acting through altered balance of salicylic acid and jasmonic acid signaling. A transient decrease in auxin signaling together with altered expression of auxin-responsive transcripts by apoplastic ROS was observed. Decreased auxin signaling did not affect the extent of cell death caused by acute O3, but caused more severe morphological changes in chronically O3-treated plants. Altogether, these results suggest that auxin-ROS interaction modulates plant development under stress.
  • Chernenko, Anton (Helsingin yliopisto, 2012)
    Communication is probably one of the major means of life maintenance. Communication involves the use of signals, which can be visual, audial, olfactory etc. Organisms communicate in many different contexts, ranging from establishing own identity, foraging for food, finding a mate, protecting their territory, to more sophisticated ones such as engaging in social behaviour. Recognition is the action or process of recognizing or being recognized. Recognition based on olfactory cues is perhaps best known from many insect species and is mediated by a wide range of volatile compounds, e.g. esters or terpenes, or non-volatile compounds such as cuticular hydrocarbons. In insects cuticular hydrocarbons are often the main agents involved in species recognition, predator avoidance, fertility and dominance signalling, cues that help parasites intrude their host nests but also those that help hosts to fend off the parasites; and also as cues facilitating sexual selection. In social insects in addition to above mentioned, cuticular hydrocarbons are involved in coordinating division of labour and nest mate recognition. The aim of my thesis was to study recognition system underlining social behaviour in Formica ants such as recognition of con- and hetero-specifics, e.g. nestmates and social parasites, and their brood as well as to examine cuticular hydrocarbon profiles of males and females. The results show that queens of potential social parasites have a very low chance to invade host colonies of Formica ants and become fully integrated. Moreover, social parasitism pressure, beside from affecting how the parasites are discriminated against, may also have an effect on the host kin recognition system and lead to rejection errors of descendant brood. Thus selection mediated by temporary social parasitism may drive enhanced recognition abilities. Colony kin structure does not seem to have any effect on recognition system towards con-specific non-nestmate and nestmate queens as both low- and high-relatedness colonies were as stringent towards unrelated individuals, although colonies with presumably high genetic diversity also showed significantly greater chemical diversity based on chemical profiles of sexuals. Hence nest mate recognition entails more complex interactions between individual genotypes and colony recognition cue phenotype than previously assumed. This thesis provides important insights on host-parasite interactions and highlights the complex interactions between different selection regimes affecting recognition system.
  • Kansikas, Minttu (Helsingin yliopisto, 2014)
    The most common inherited cancer syndrome, Lynch syndrome (LS), is caused by a defected post-replicative DNA mismatch repair (MMR) pathway. Human MMR is initiated by the binding of a heterodimeric mismatch recognition factor MutSα (MSH2+MSH6) or MutSβ (MSH2+MSH3), followed by the assembly of the repairosome by MutLα (MLH1+PMS2). In addition to sharing a common heterodimerisation protein, the roles of MutSα and MutSβ have been suggested to overlap in the repair of small insertion deletion loops. A germline mutation in MLH1, MSH2, MSH6 or PMS2 is most commonly the cause of LS. Mutation carriers have a significantly increased risk for colon and endometrial cancers in particular, although tumorigenesis is thought to only commence upon losing the healthy allele. The clinical importance of identifying LS patients is reflected by the significantly increased cancer risk of LS mutation carriers and the effectiveness of LS associated cancer surveillance. The clinical diagnosis of LS relies on tumour pathological analyses, the identification of an MMR gene variation by mutation analysis and the subsequent pathogenicity assessment of the variation. However, the clinical significance of non-truncating genetic alterations can be difficult to interpret as they are associated to a variety of clinical phenotypes, ranging from the lack of adverse effects to a highly increased cancer risk. Due to the unknown functional significance of such variants, functional assessment is required for their pathogenicity assessment. Hence, biological tools used to assess the pathogenicity of MMR gene variations can be central to identifying LS patients. The aim of the studies in this thesis was to understand how MMR proteins and MMR gene alterations affect the MMR mechanism and contribute to LS. The role and significance of the wild type and variant MMR proteins were analysed in a homologous human MMR system by an in vitro MMR assay. Three different substrate molecules consisting of a GT mismatch, a single or a two nucleotide loop were used to study the substrate specificities and MMR efficiencies of the MutS protein complexes. Even though MutSβ does not participate in mononucleotide loop repair, it was shown to exceed MutSα in dinucleotide loop repair indicating that dinucleotide microsatellite instability in the absence of mononucleotide instability is indicative of MSH3 defects. Furthermore, the introduction of a dinucleotide loop substrate to our assay allowed the novel pathogenicity assessment of an MSH3 variation. Functional variant pathogenicity assessment of MMR gene variations linked to atypical clinical features also confirmed the pathogenicity of two novel MSH6 variations and one biallelic MLH1 variation. The compound contribution of MMR gene VUS pairs, as found in LS cancer patients, was assessed and a subtle compound effect of two MSH2 variations that appear MMR proficient when assayed individually was shown. Also, the application of MMR gene variants with known molecular effects to verify a variation pathogenicity assessment model helped describe the model appropriate for MLH1 and MSH2 variations. Finally, we established MMR gene specific knockdown cell lines to investigate the effect of reduced MMR gene expression on the MMR efficiency. The knockdown clones retaining 50% of MSH2 or MSH6 mRNA expression demonstrated significantly reduced in vitro MMR efficiencies whilst a decrease was also detectable in MLH1 knockdown extracts. The knowledge of the gene specific mRNA expression levels that can be detected as MMR deficient, presents the opportunity to develop the assay to recognise LS from non-cancerous cells.
  • Kallio-Kokko, Hannimari (Helsingin yliopisto, 2000)
  • Schmidt, Michel (Helsingin yliopisto, 2000)
  • Simola, Mari (Helsingin yliopisto, 2010)
    All organisms have evolved mechanisms to acquire thermotolerance. A moderately high temperature activates heat shock genes and triggers thermotolerance towards otherwise lethal high temperature. The focus of this work is the recovery mechanisms ensuring survival of Saccharomyces cerevisiae yeast cells after thermal insult. Yeast cells, first preconditioned at 37˚C, can survive a short thermal insult at 48-50˚C and are able to refold heat-denatured proteins when allowed to recover at physiological temperature 24˚C. The cytoplasmic chaperone Hsp104 is required for the acquisition of thermotolerance and dissolving protein aggregates in the cytosol with the assistance of disaccharide trehalose. In the present study, Hsp104 and trehalose were shown to be required for conformational repair of heat-denatured secretory proteins in the endoplasmic reticulum. A reporter protein was first accumulated in the lumen of endoplasmic reticulum and heat-denatured by thermal insult, and then failed to be repaired to enzymatically active and secretion-competent conformation in the absence of Hsp104 or trehalose. The efficient transport of a glycoprotein CPY, accumulated in the endoplasmic reticulum, to the vacuole after thermal insult also needed the presence of Hsp104 and trehalose. However, proteins synthesized after thermal insult at physiological temperature were secreted with similar kinetics both in the absence and in the presence of Hsp104 or trehalose, demonstrating that the secretion machinery itself was functional. As both Hsp104 and trehalose are cytosolic, a cross-talk between cytosolic and luminal chaperone machineries across the endoplasmic reticulum membrane appears to take place. Global expression profiles, obtained with the DNA microarray technique, revealed that the gene expression was shut down during thermal insult and the majority of transcripts were destroyed. However, the transcripts of small cytosolic chaperones Hsp12 and Hsp26 survived. The first genes induced during recovery were related to refolding of denatured proteins and resumption of de novo protein synthesis. Transcription factors Spt3p and Med3p appeared to be essential for acquisition of full thermotolerance. The transcription factor Hac1p was found to be subject to delayed up-regulation at mRNA level and this up-regulation was diminished or delayed in the absence of Spt3p or Med3p. Consequently, production of the chaperone BiP/Kar2p, a target gene of Hac1p, was diminished and delayed in Δspt3 and Δmed3 deletion strains. The refolding of heat-denatured secretory protein CPY to a transport-competent conformation was retarded, and a heat-denatured reporter enzyme failed to be effectively reactivated in the cytoplasm of the deletion strains.
  • Vuorenmaa, Jussi (Helsingin yliopisto, 2007)
    The present work provides a regional-scale assessment of the changes in acidifying deposition in Finland over the past 30 years and the current pattern in the recovery of acid-sensitive lakes from acidification in relation to changes in sulphate deposition. This information is needed for documenting the ecosystem benefits of costly emission reduction policies and further actions in air pollution policy. The development of sulphate deposition in Finland reflects that of European SO2 emissions. Before the 1990s, reductions in sulphur emissions in Europe had been relatively small and sulphate deposition showed no consistent trends. Due to emission reduction measures that were then taken, sulphate deposition started to clearly decline from the late 1980s. The bulk deposition of sulphate has declined 40-60% in most parts of the country during 1990-2003. The decline in sulphate deposition exceeded the decline of base cation deposition, which resulted in a decrease in acidity and acidifying potential of deposition over the 1990s. Nitrogen deposition also decreased since the late 1980s, but less than that of sulphate, and levelling off during the 1990s. Sulphate concentrations in all types of small lakes throughout Finland have declined from the early 1990s. The relative decrease in lake sulphate concentrations (average 40-50%) during 1990-2003 was rather similar to the decline in sulphate deposition, indicating a direct response to the reduction in deposition. There are presently no indications of elevated nitrate concentrations in forested headwater lakes. Base cation concentrations are still declining in many lakes, especially in south Finland, but to a lesser extent than sulphate allowing buffering capacity (alkalinity) to increase, being significant in 60% of the study lakes. Chemical recovery is resulting in biological recovery with populations of acid-sensitive fish species increasing. The recovery has been strongest in lakes in which sulphate has been the major acidifying agent, and recovery has been the strongest and most consistent in lakes in south Finland. The recovery of lakes in central Finland and north Finland is not as widespread and strong as observed in south. Many catchments, particularly in central Finland, have a high proportion of peatlands and therefore high TOC concentrations in lakes, and runoff-induced surges of organic acids have been an important confounding factor suppressing the recovery of pH and alkalinity in these lakes. Chemical recovery is progressing even in the most acidified lakes, but the buffering capacity of many lakes is still low and still sensitive to acidic input. Further reduction in sulphur emissions are needed for the alkalinity to increase in the acidified lakes. Increasing total organic carbon (TOC) concentrations are indicated in small forest lakes in Finland. The trends appear to be related to decreasing sulphate deposition and improved acid-base status of the soil, and the rise in TOC is integral to recovery from acidification. A new challenge is climate change with potential trends in temperature, precipitation and runoff, which are expected to affect future chemical and biological recovery from acidification. The potential impact on the mobilization and leaching of organic acids may become particularly important in Finnish conditions. Long-term environmental monitoring has evidently shown the success of international emission abatement strategies. The importance and value of integrated monitoring approach including physical, chemical and biological variables is clearly indicated, and continuous environmental monitoring is needed as a scientific basis for further actions in air pollution policy.
  • Ribacka, Camilla (Helsingin yliopisto, 2007)
    The respiratory chain is found in the inner mitochondrial membrane of higher organisms and in the plasma membrane of many bacteria. It consists of several membrane-spanning enzymes, which conserve the energy that is liberated from the degradation of food molecules as an electrochemical proton gradient across the membrane. The proton gradient can later be utilized by the cell for different energy requiring processes, e.g. ATP production, cellular motion or active transport of ions. The difference in proton concentration between the two sides of the membrane is a result of the translocation of protons by the enzymes of the respiratory chain, from the negatively charged (N-side) to the positively charged side (P-side) of the lipid bilayer, against the proton concentration gradient. The endergonic proton transfer is driven by the flow of electrons through the enzymes of the respiratory chain, from low redox-potential electron donors to acceptors of higher potential, and ultimately to oxygen. Cytochrome c oxidase is the last enzyme in the respiratory chain and catalyzes the reduction of dioxygen to water. The redox reaction is coupled to proton transport across the membrane by a yet unresolved mechanism. Cytochrome c oxidase has two proton-conducting pathways through which protons are taken up to the interior part of the enzyme from the N-side of the membrane. The K-pathway transfers merely substrate protons, which are consumed in the process of water formation at the catalytic site. The D-pathway transfers both substrate protons and protons that are pumped to the P-side of the membrane. This thesis focuses on the role of two conserved amino acids in proton translocation by cytochrome c oxidase, glutamate 278 and tryptophan 164. Glu278 is located at the end of the D-pathway and is thought to constitute the branching point for substrate and pumped protons. In this work, it was shown that although Glu278 has an important role in the proton transfer mechanism, its presence is not an obligatory requirement. Alternative structural solutions in the area around Glu278, much like the ones present in some distantly related heme-copper oxidases, could in the absence of Glu278 support the formation of a long hydrogen-bonded water chain through which proton transfer from the D-pathway to the catalytic site is possible. The other studied amino acid, Trp164, is hydrogen bonded to the ∆-propionate of heme a3 of the catalytic site. Mutation of this amino acid showed that it may be involved in regulation of proton access to a proton acceptor, a pump site, from which the proton later is expelled to the P-side of the membrane. The ion pair that is formed by the ∆-propionate of heme a3 and arginine 473 is likely to form a gate-like structure, which regulates proton mobility to the P-side of the membrane. The same gate may also be part of an exit path through which water molecules produced at the catalytically active site are removed towards the external side of the membrane. Time-resolved optical and electrometrical experiments with the Trp164 to phenylalanine mutant revealed a so far undetected step in the proton pumping mechanism. During the A to PR transition of the catalytic cycle, a proton is transferred from Glu278 to the pump site, located somewhere in the vicinity of the ∆-propionate of heme a3. A mechanism for proton pumping by cytochrome c oxidase is proposed on the basis of the presented results and the mechanism is discussed in relation to some relevant experimental data. A common proton pumping mechanism for all members of the heme-copper oxidase family is moreover considered.
  • Achim, Kaia (Helsingin yliopisto, 2010)
    Gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the vertebrate brain. In the midbrain, GABAergic neurons contribute to the regulation of locomotion, nociception, defensive behaviours, fear and anxiety, as well as sensing reward and addiction. Despite the clinical relevance of this group of neurons, the mechanisms regulating their development are largely unknown. In addition, their migration and connectivity patterns are poorly characterized. This study focuses on the molecular mechanisms specifying the GABAergic fate, and the developmental origins of midbrain GABAergic neurons. First, we have characterized the function of a zink-finger transcription factor Gata2. Using a tissue-specific mutagenesis in mouse midbrain and anteror hindbrain, we showed that Gata2 is a crucial determinant of the GABAergic fate in midbrain. In the absence of Gata2, no GABAergic neurons are produced from the otherwise competent midbrain neuroepithelium. Instead, the Gata2-mutant cells acquire a glutamatergic neuron phenotype. Ectopic expression of Gata2 was also sufficient to induce GABAergic in chicken midbrain. Second, we have analyzed the midbrain phenotype of mice mutant for a proneural gene Ascl1, and described the variable and region-dependent requirements for Ascl1 in the midbrain GABAergic neurogenesis. These studies also have implications on the origin of distinct anatomical and functional GABAergic subpopulations in midbrain. Third, we have identified unique developmental properties of GABAergic neurons that are associated with the midbrain dopaminergic nuclei, the substantia nigra pars reticulata (SNpr) and ventral tegmental area (VTA). Namely, the genetic regulation of GABAergic fate in these cells is distinct from the rest of midbrain. In accordance to this phenomenon, our detailed fate-mapping analyses indicated that the SNpr-VTA GABAergic neurons are generated outside midbrain, in the neuroepithelium of anterior hindbrain.
  • Mattila, Jaakko (Helsingin yliopisto, 2009)
    Forkhead box class O (FoxO) transcription factors are members of the forkhead box transcription factor superfamily, with orthologues in various species such as human, worm and fly. FoxO proteins are key regulators of growth, metabolism, stress resistance and, consequently, life span. FoxOs integrate signals from different pathways, e.g. the growth controlling Insulin-TOR signaling pathway and the stress induced JNK and Hippo signaling pathways. FoxO proteins have evolved to guide the cellular response to varying energy and stress conditions by inducing the expression of genes involved in the regulation of growth and metabolism. This work has aimed to deepen the understanding of how FoxO executes its biological functions. A particular emphasis has been laid to its role in growth control. Specifically, evidence is presented indicating that FoxO restricts tissue growth in a situation when TOR signaling is high. This finding can have implications in a human condition called Tuberous sclerosis, manifested by multiple benign tumors. Further, it is shown that FoxO directly binds to the promoter and regulates the expression of a Drosophila Adenylate cyclase gene, ac76e, which in turn modulates the fly s development and growth systemically. These results strengthen FoxOs position among central size regulators as it is able to operate at the level of individual cells as well as in the whole organism. Finally, an attempt to reveal the regulatory network upstream of FoxO has been carried out. Several putative FoxO activity regulators were identified in an RNAi screen of Drosophila kinases and phosphatases. The results underscore that FoxO is regulated through an elaborate network, ensuring the correct execution of key cellular processes in metabolism and response to stress. Overall, the evidence provided in this study strengthens our view of FoxO as a key integrator of growth and stress signals.
  • Seppä, Laura (Helsingin yliopisto, 2005)
  • Jänne, Marja (Helsingin yliopisto, 2000)
  • Uotila, Liisa (Helsingin yliopisto, 2014)
    The adhesion molecules of blood cells are of great importance in the regulation of many of the most central processes of the human body, e.g. haematopoiesis, immune functions, haemostasis and wound healing, and the delivery of oxygen to the tissues. Leukocyte β2 integrins, VLA-4 integrin and members of the immunoglobulin superfamily like ICAMs (intercellular adhesion molecules) and VCAM (vascular cell adhesion molecule) are the most essential adhesion molecules of blood cells. The adhesion molecules on blood cells have many requirements that they need to fulfil in order to maintain a physiological system: they need to stay in an inactive, non-binding state for most of the time, and to be activated and thus become adhesive only when needed. In addition, they should specifically recognise their binding partners or ligands, as unnecessary binding could lead for example to clogging of the blood vessels, autoimmune diseases or allergic reactions. Still one important feature of blood cell adhesion is the ability to let go and release the adhesion, when the cell needs to move forward or continue patrolling the circulation. In my thesis work I have analysed the properties of leukocyte integrins and their ligands as well as the regulation of their interactions. We observed that the red cell adhesion molecule ICAM-4 can bind to CR4, a leukocyte integrin expressed on monocytes and macrophages, and that the I domain is the ICAM-4 binding site on leukocyte integrins (LFA-1, Mac-1 and CR4). We also characterised the phosphorylation of the cytoplasmic tail of CR4, and found that αX chain is phosphorylated on Ser1158, and that this phosphorylation is essential for CR4 inside-out activation, adhesion and phagocytosis but not for outside-in signaling initiated by CR4. Finally we analysed the regulation of VLA-4 mediated adhesion to VCAM-1 that is controlled by the β2 integrins. The findings of my studies show how leukocyte integrins are involved in numerous blood cell functions and that their functions are tightly regulated. Due to their multifold roles, they also offer attractive targets for therapeutic use. The specificity of phosphorylations or ligands may serve as distinctive factors between different integrins, even members of the same family.
  • Koivuniemi, Raili (Helsingin yliopisto, 2013)
    Neural progenitor cells (NPCs) are present in the developing and adult neuroepithelium of the brain and are regulated by internal and external signals that influence neurogenesis and tissue homeostasis. NPCs are multipotent tissue stem cells that can arouse all neural cell types, including neurons and glial cells. In culture, NPCs grow preferentially as cell aggregates called neurospheres. This suggests that interactions between cells are essential to regulate NPC behavior and development. Interactions between cells may be facilitated by cell surface-attached proteases and their inhibitors that play an important role in development and during tissue remodeling after injury. Neuroinflammation, an innate immune response of the nervous system, is part of many neurodegenerative diseases. Neuroinflammation involves activation of microglia and production of proinflammatory cytokines. Inflammation may have negative effects on NPCs and thus, agents that protect NPCs could serve as a therapeutic potential for neuronal injuries and neurodegenerative diseases by enabling local tissue repair in the brain. The aim of this thesis was to study the regulation of NPC development by membrane-associated proteins and the effects of inflammation on NPCs. Glucocorticoid hormone (GH) levels increase in inflammation and after stress. GHs have previously been shown to decrease NPC proliferation and neurogenesis. We have studied the effects of a synthetic GH dexamethasone on the cytosolic membrane-associated and anti-apoptotic protein BRUCE, and how BRUCE affects NPC behaviour. In addition, we have studied the secretion of cytokine interferon-gamma (IFN-gamma) after microglial activation and further the influence of IFN-gamma on NPCs. To address the role of cell surface-associated protease inhibitors during NPC development, we have studied the expression and function of Kunitz type serine protease inhibitors HAI-1 and HAI-2 in NPCs. The results show that dexamethasone enhances degradation of BRUCE by the ubiquitin-proteasome system (UPS), which leads to decreased NPC proliferation. NPC division was negatively affected also by IFN-gamma produced by microglial cells as well as protease inhibitors HAI-1 and HAI-2. Moreover, IFN-gamma induced NPC cell death that was rescued by a neuropeptide PACAP. In the developing NPCs, HAI-1 and HAI-2 expression was increased by bone morphogenetic protein-2 (BMP-2) and BMP-4, which inhibited NPC proliferation and increased glial cell differentiation partly in a HAI-dependent manner. This thesis provides knowledge about interplay between immune cells and NPCs as well as developmental signaling systems, including proteolytic pathways, that affect NPC behaviour. In NPCs, proteolytic pathways may be regulated by external signals, like cytokines, from the neighboring cells. Proteolysis is involved also in the UPS that regulates the cell cycle machinery and thus, cell division. This thesis also deals with NPC survival, which is of importance for stem cell therapies. Knowledge of reciprocal effects of IFN-gamma and PACAP on NPCs is relevant when designing treatment for brain inflammation and disease.
  • Pekkinen, Minna (Helsingin yliopisto, 2008)
    Bone is a mineralized tissue that enables multiple mechanical and metabolic functions to be carried out in the skeleton. Bone contains distinct cell types: osteoblasts (bone-forming cells), osteocytes (mature osteoblast that embedded in mineralized bone matrix) and the osteoclasts (bone-resorbing cells). Remodelling of bone begins early in foetal life, and once the skeleton is fully formed in young adults, almost all of the metabolic activity is in this form. Bone is constantly destroyed or resorbed by osteoclasts and then replaced by osteoblasts. Many bone diseases, i.e. osteoporosis, also known as bone loss, typically reflect an imbalance in skeletal turnover. The cyclic adenosine monophosphate (cAMP) and the cyclic guanosine monophosphate (cGMP) are second messengers involved in a variety of cellular responses to such extracellular agents as hormones and neurotransmitters. In the hormonal regulation of bone metabolism, i.e. via parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) and prostaglandin E2 signal via cAMP. cAMP and cGMP are formed by adenylate and guanylate cyclases and are degraded by phosphodiesterases (PDEs). PDEs determine the amplitudes of cyclic nucleotide-mediated hormonal responses and modulate the duration of the signal. The activities of the PDEs are regulated by multiple inputs from other signalling systems and are crucial points of cross-talk between the pathways. Food-derived bioactive peptides are reported to express a variety of functions in vivo. The angiotensin-converting enzymes (ACEs) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. The bioactive peptides offer also a nutriceutical and a nutrigenomic aspect to bone cell biology. The aim of this study was to investigate the influence of PDEs and bioactive peptides on the activation and the differentiation of human osteoblast cells. The profile of PDEs in human osteoblast-like cells and the effect of glucocorticoids on the function of cAMP PDEs, were investigated at the mRNA and enzyme levels. The effects of PDEs on bone formation and osteoblast gene expression were determined with chemical inhibitors and siRNAs (short interfering RNAs). The influence of bioactive peptides on osteoblast gene expression and proliferation was studied at the mRNA and cellular levels. This work provides information on how PDEs are involved in the function and the differentiation of osteoblasts. The findings illustrate that gene-specific silencing with an RNA interference (RNAi) method is useful in inhibiting, the gene expression of specific PDEs and further, PDE7 inhibition upregulates several osteogenic genes and increases bALP activity and mineralization in human mesenchymal stem cells-derived osteoblasts. PDEs appear to be involved in a mechanism by which glucocorticoids affect cAMP signaling. This may provide a potential route in the formation of glucocorticoid-induced bone loss, involving the down-regulation of cAMP-PDE. PDEs may play an important role in the regulation of osteoblastic differentiation. Isoleucine-proline-proline (IPP), a bioactive peptide, possesses the potential to increase osteoblast proliferation, differentiation and signalling.