Browsing by Subject "solu- ja molekyylibiologia"

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  • Kajanne, Risto (Helsingin yliopisto, 2009)
    Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.
  • Niku, Mikael (Helsingin yliopisto, 2007)
    Stem cells exist in most adult tissues. Some of these somatic stem cells may be more plastic than expected, not limited to generating new cells for the tissue of their origin. While such findings suggest a revolution in regenerative medicine, they remain controversial. Most stem cell research is based on transplantation experiments of isolated and often cultured cells in mice, and the results may be affected by the manipulation of the animals and the transplanted cells. Here, naturally chimeric twin cattle were used to investigate the differentiation potential of stem cells in a non-manipulated large mammal. Due to conjoined placental circulations, blood of the bovine twins is mixed for most of the fetal period, and circulating stem cells are effectively exchanged. We developed powerful methods for tracing their progeny in various tissues of freemartin cattle, females born as a twin to a bull. While from 10% to 90% of the hematopoietic system in freemartins was donor-derived, donor contribution to non-hematopoietic tissues was in most cases minor. Thus, hematopoietic stem cells and other cell types circulating in fetal blood do not generate significant numbers of non-hematopoietic cells in the development, growth and physiological turnover of bovine tissues. However, they may be important in tissue repair and regeneration, as suggested by increasing numbers of donor-derived cells in newly forming granulation tissue. Chimeric cattle were also used to investigate the generation of bovine B lymphocytes, which occurs differently than in the commonly studied human and mouse, and is poorly understood. The results indicate that the ileal Peyer s patch determines the peripheral B cell pool in young cattle, and is likely responsible for the production of the preimmune antibody repertoire by post-rearrangement strategies. To facilitate a direct analysis of bovine stem cells, the first antibodies against bovine CD34 were generated. The CD34 glycoprotein is commonly used as a marker for hematopoietic progenitors and endothelial cells in the human and mouse. CD34 mRNA was found to be alternatively spliced and widely expressed in cattle tissues. Using the new antibody, the protein was detected in most blood vessel endothelia, primitive hematopoietic cells and some non-hematopoietic cell types.
  • Ilmarinen, Tanja (Helsingin yliopisto, 2007)
    Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.
  • Uusi-Rauva, Kristiina (2012)
    Neuronal ceroid lipofuscinoses (NCLs) are inherited severe neurodegenerative diseases of childhood. NCLs are characterised by progressive, selective neuronal death and lysosomal accumulation of autofluorescent storage material. Due to the poor knowledge on the functions of the proteins primarily defective in NCLs, the intracellular changes critical to the pathogenesis of NCLs are not known. In this thesis study, the primary functions of CLN3, a protein defective in classic juvenile onset form of NCLs (juvenile CLN3 disease), was studied in terms of its protein interactions. CLN3 was determined to interact with Na+, K+ ATPase and associated fodrin cytoskeleton and 78 kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP). Plasma membrane association and ouabain-induced endocytosis of Na+, K+ ATPase were found to be impaired in CLN3-deficient neurons. This suggests that CLN3 may play a role in the ouabain-regulated non-pumping functions of Na+, K+ ATPase, including intracellular signalling, apoptosis and calcium oscillations. Furthermore, putative structural changes in the fodrin cytoskeleton were observed in CLN3-deficient mouse brain sections and patient cells implying that fodrin-associated events in axonal and synaptic intracellular trafficking, synaptic transmission, and neuritogenesis may also be compromised in early stage of juvenile CLN3 disease. CLN3 was also found to interact with Hook1 involved in late endosomal/lysosomal maturation and with microtubular motor protein complexes, dynein-dynactin and kinesin-2-dynactin. CLN3 was shown to associate with the motor proteins most likely through direct interactions with Rab7 GTPase and its effector Rab7-interacting lysosomal protein (RILP). Rab7 and its effectors have been reported to regulate both minus and plus end-directed microtubular membrane trafficking. Interestingly, membrane trafficking was found to be affected in CLN3 deficiency demonstrated by abnormal intracellular position of late endosomes/lysosomes and unbalanced functional GTP/GDP cycle of Rab7 as well defects in the late endosomal targeting of endocytosed cargo and kinesin-dependent movement of late endosomes/lysosomes in CLN3-deficient cells. These results suggest that Rab7-guided motor protein functions in neurons, such as axonal retrograde trafficking of neurotrophins, neurite outgrowth and maturation, and transportation of neuronal autophagic vesicles could also be affected in CLN3 disease. This thesis work has provided important novel data on the functions of CLN3 and the primary intracellular defects possibly resulting in CLN3 disease. This study also contributes to the determination of the pathogenesis of other NCLs and general processes of neurodegeneration.
  • Serlachius, Martina (Helsingin yliopisto, 2007)
    Stanniocalcin-1 (STC-1) is a 56 kD homodimeric protein which was originally identified in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. STC-1 was considered unique to fish until the cloning of cDNA for human STC-1 in 1995 and mouse Stc-1 in 1996. STC-1 is conserved through evolution with human and salmon STC-1 sharing 60% identity and 80% similarity. The surprisingly high homology between mammalian and fish STC-1 and the protective actions of STC-1 in terminally differentiated neurons, originally reported by my colleagues, prompted me to further study the role of STC-1 in cell stress and differentiation. One purpose was to determine whether there is an inter-relationship between terminally differentiated cells and STC-1 expression. The study revealed an accumulation of STC-1 in mature megakaryocytes and adipocytes, i.e. postmitotic cells with limited or lost proliferative capacity. Still proliferating uninduced cells were negative for STC-1 mRNA and protein, whereas differentiating cells accumulated STC-1 in their cytoplasm. Interestingly, in liposarcomas the grade inversely correlated with STC-1 expression. Another aim was to study how STC-1 gene expression is regulated. Given that IL-6 is a cytokine with neuroprotective actions, by unknown mechanisms, we examined whether IL-6 regulates STC-1 gene expression. Treatment of human neural Paju cells with IL-6 induced a dose-dependent upregulation of STC-1 mRNA levels. This induction of STC-1 expression by IL-6 occurred mainly through the MAPK signaling pathway. Furthermore, I studied the role of IL-6-mediated STC-1 expression as a mechanism of cytoprotection conferred by hypoxic preconditioning (HOPC) in brain and heart. My findings show that Stc-1 was upregulated in brain after hypoxia treatment. In the brain of IL-6 deficient mice, however, no upregulation of Stc-1 expression was evident. After induced brain injury the STC-1 response in brains of IL-6 transgenic mice, with IL-6 overexpression in astroglial cells, was stronger than in brains of WT mice. These results indicate that IL-6-mediated expression of STC-1 is one molecular mechanism of HOPC-induced tolerance to brain ischemia. The protection conferred by HOPC in heart occurs during a bimodal time course comprising early and delayed preconditioning. Interestingly, my results showed that the expression of Stc-1 in heart was upregulated in a biphasic manner during HOPC. IL-6 deficient mice did not, however, show a similar biphasic manner of Stc-1 upregulation as did WT mice. Instead, only an early upregulation of Stc-1 expression was evident. The results suggest that the upregulation of Stc-1 during the delayed preconditioning is IL-6-dependent. The upregulated expression of Stc-1 during the early preconditioning, however, is only partly IL-6-dependent and possibly also directly mediated by HIF-1. These findings suggest that STC-1 is a pro-survival protein for terminally differentiated cells and that STC-1 expression may in fact be regulated by stress. In addition, I show that STC-1 gene upregulation, mediated in part by IL-6, is a new mechanism of protection conferred by HOPC in brain and heart. Because of its importance for fundamental biological processes, such as differentiation and cytoprotection, STC-1 may have therapeutic implications for management of stroke, neurodegenerative diseases, cancer, and obesity.