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  • Seppä, Laura (Helsingin yliopisto, 2005)
  • Erkkilä, Krista (Helsingin yliopisto, 2002)
  • Jänne, Marja (Helsingin yliopisto, 2000)
  • Liesmaa, Inka (Helsingin yliopisto, 2010)
    Accumulating evidence show that kinins, notably bradykinin (BK) and kallidin, have cardioprotective effects. To these include reduction of left ventricular hypertrophy (LVH) and progression of heart failure. The effects are mediated through two G protein-coupled receptors- bradykinin type-2 receptor (BK-2R) and bradykinin type -1 receptor (BK-1R). The widely accepted cardioprotective effects of BK-receptors relate to triggering the production and release of vasodilating nitric oxide (NO) by endothelial cells. They also exert anti-proliferative effects on fibroblasts and anti-hypertrophic effects on myocytes, and thus may play an essential role in the cardioprotective response to myocardial injury. The role for BK-1Rs in HF is based on experimental animal models, where the receptors have been linked to cardioprotective- but also to cardiotoxic -effects. The BK-1Rs are induced under inflammatory and ischemic conditions, shown in animal models; no previous reports, concerning BK-1Rs in human heart failure, have been presented. The expression of BK-2Rs is down-regulated in human end-stage heart failure. Present results showed that, in these patients, the BK-1Rs were up-regulated, suggesting that also BK-1Rs are involved in the pathogenesis of human heart failure. The receptors were localized mainly in the endothelium of intramyocardial coronary vessels, and correlated with the increased TNF-α expression in the myocardial coronary vessels. Moreover, in cultured endothelial cells, TNF-α was a potent trigger of BK-1Rs. These results suggest that cytokines may be responsible for the up-regulation of BK-1Rs in human heart failure. A linear relationship between BK-2R mRNA and protein expression in normal and failing human left ventricles implies that the BK-2Rs are regulated on the transcriptional level, at least in human myocardium. The expression of BK-2Rs correlated positively with age in normal and dilated hearts (IDC). The results suggest that human hearts adapts to age-related changes, by up-regulating the expression of cardioprotective BK-2Rs. Also, in the BK-2R promoter polymorphism -58 T/C, the C-allele was accumulated in cardiomyopathy patients which may partially explain the reduced number of BK-2Rs. Statins reduce the level of plasma cholesterol, but also exert several non-cholesterol-dependent effects. These effects were studied in human coronary arterial endothelial cells (hCAEC) and incubation with lovastatin induced both BK-1 and BK-2Rs in a time and concentration-dependent way. The induced BK-2Rs were functionally active, thus NO production and cGMP signaling was increased. Induction was abrogated by mevalonate, a direct HMG-CoA metabolite. Lovastatin is known to inhibit Rho activation, and by a selective RhoA kinase inhibitor (Y27632), a similar induction of BK-2R expression as with lovastatin. Interestingly a COX-2-inhibitor (NS398) inhibited this lovastatin-induction of BK-2Rs, suggesting that COX-2 inhibitors may affect the endothelial BK-2Rs, in a negative fashion. Hypoxia is a common denominator in HF but also in other cardiovascular diseases. An induction of BK-2Rs in mild hypoxic conditions was shown in cultured hCAECs, which was abolished by a specific BK-2R inhibitor Icatibant. These receptors were functionally active, thus BK increased and Icatibant inhibited the production of NO. In rat myocardium the expression of BK-2R was increased in the endothelium of vessels, forming at the border zone, between the scar tissue and the healthy myocardium. Moreover, in in vitro wound-healing assay, endothelial cells were cultured under hypoxic conditions and BK significantly increased the migration of these cells and as Icatibant inhibited it. These results show, that mild hypoxia triggers a temporal expression of functionally active BK-2Rs in human and rat endothelial cells, supporting a role for BK-2Rs, in hypoxia induced angiogenesis. Our and previous results show, that BK-Rs have an impact on the cardiovascular diseases. In humans, at the end stage of heart failure, the BK-2Rs are down-regulated and BK-1Rs induced. Whether the up-regulation of BK-1Rs, is a compensatory mechanism against the down-regulation of BK-2Rs, or merely reflects the end point of heart failure, remains to bee seen. In a clinical point of view, the up-regulation of BK-2Rs, under hypoxic conditions or statin treatment, suggests that, the induction of BK-2Rs is protective in cardiovascular pathologies and those treatments activating BK-2Rs, might give additional tools in treating heart failure.
  • 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.
  • Suomalainen, Laura (Helsingin yliopisto, 2004)
  • 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.
  • Pihlajoki, Marjut (Helsingin yliopisto, 2014)
    The main steroidogenic organs, adrenal cortex and ovary, arise from a common pool of progenitors in the developing embryo. Similar signaling pathways regulate the differentiation, growth, and survival of cells in these tissues. Proper development of the adrenal cortex and ovary requires precise spatiotemporal control of gene expression and apoptosis; disruption of these processes may lead to congenital disorders or malignant transformation. Earlier in vitro studies demonstrated that transcription factor GATA6 regulates the expression of multiple steroidogenic genes in the adrenal cortex. To show that GATA6 is a crucial regulator of adrenocortical development and function in vivo, we generated a mouse model in which Gata6 is conditionally deleted in steroidogenic cells. These mice exhibited a complex adrenal phenotype that includes cortical thinning, blunted aldosterone production, lack of an X-zone, impaired apoptosis, and subcapsular cell hyperplasia. These results offer genetic proof that GATA6 regulates the differentiation of steroidogenic progenitors into adrenocortical cells. Ovarian granulosa cell tumors (GCTs), the most common sex-cord stromal tumors in women, are thought to be caused by aberrant granulosa cell apoptosis during folliculogenesis. A somatic missense mutation in transcription factor FOXL2 (402C→G) is present in vast majority of human GCTs. FOXL2 plays a key role in the development and function of normal granulosa cells. Wild type (wt) FOXL2 induces GCT cell apoptosis, while mutated FOXL2 is less effective. To clarify the molecular pathogenesis of GCTs, we investigated the impact of FOXL2 and two other factors implicated in granulosa cell function, GATA4 and SMAD3, on gene expression and cell viability in GCTs. We found that these factors physically interact and that GATA4 and SMAD3 synergistically induce CCND2 promoter transactivation, which is reduced by both wt and mutated FOXL2. Finally, we demonstrated that GATA4 and SMAD3 protect GCT cells from wt FOXL2 induced apoptosis without affecting the apoptosis induced by mutated FOXL2. These findings suggest that mutated FOXL2 disrupts the balance between growth and apoptosis in granulosa cells, leading to malignant transformation. The treatment of recurrent or metastatic GCTs is challenging, and biologically targeted treatment modalities are needed. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) activates the extrinsic apoptotic pathway. Interestingly, TRAIL is able to induce apoptosis in malignant cells without affecting normal cells. Vascular endothelial growth factor (VEGF) is the key regulator of both physiological and pathological angiogenesis. Cancer cells often express VEGF receptor, and an autocrine VEGF/VEGFR signaling loop exists in several types of cancer cells. We found that GCT cells express functional TRAIL and VEGF receptors, and that treatment with TRAIL and the VEGF-binding antibody (bevacizumab) induce GCT cell apoptosis. These findings establish a preclinical basis for targeting these two pathways in the GCT treatment.
  • 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.
  • Myöhänen, Heli (Helsingin yliopisto, 2003)
  • Paukku, Kirsi (Helsingin yliopisto, 2003)
  • Hytönen, Timo (Helsingin yliopisto, 2009)
    Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.
  • Falck, Sandra (Helsingin yliopisto, 2004)
  • Martelin, Eeva (Helsingin yliopisto, 2004)
  • Thomas-Crusells, Judith (Helsingin yliopisto, 2004)