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  • 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.
  • Paukku, Kirsi (Helsingin yliopisto, 2003)
  • Falck, Sandra (Helsingin yliopisto, 2004)
  • Verbeeren, Jens (Helsingin yliopisto, 2016)
    The protein coding information in our genome is located on genes which are very often interrupted by non-coding regions called introns. For proper gene expression, introns must be removed accurately and the remaining protein coding parts, the exons, must be rejoined. This reaction, termed splicing, is carried out by an enormous macromolecular machine called the spliceosome, and is one of the most crucial steps in gene expression. Two different intron types have been identified in eukaryotes, each removed by their own dedicated spliceosome; the U2-type (or major) introns, which constitute the majority of introns, and the U12-type (or minor) introns, of which ca. 700-800 have been identified in the human genome. The presence of a second type of intron and spliceosome has always been enigmatic. However, studies investigating U12-type intron removal have provided us with an important clue; it appears that U12-type introns are spliced less efficiently than U2-type introns. This suggests that their removal could be rate-limiting for the expression of the genes that harbor these introns, and it also offers the intriguing possibility that the activity of the minor spliceosome could be altered in response to changing cellular conditions. These implications could offer a valuable explanation for the extraordinary conservation of the U12-type introns and the components that catalyze their excision. There is currently not much known about the regulation of the minor spliceosome and this study aimed to address this issue. I have investigated the characteristics of a negative feedback loop that regulates the expression level of two essential and unique protein components of the minor spliceosome, the U11-48K and the U11/U12-65K proteins. In the genes that encode these proteins, an ultraconserved sequence element can be found which consists of a tandem repeat of U12-type 5ʹ splice sites. We uncovered that binding of U11/U12 di-snRNPs on these elements leads to alternative splicing where an mRNA isoform is produced that is targeted for degradation or nuclear retention. The presence of such enhancer elements is conserved from plants to animals, highlighting an extreme selection pressure for this regulatory mechanism. I further investigated the role of the U11-35K protein, another protein uniquely associated with the minor spliceosome, in alternative splicing, and the functional requirements for enhancer binding. Furthermore, I uncovered the molecular mechanism by which the level of translational-competent U11/U12-65K mRNA is downregulated through U11/U12 di-snRNP enhancer binding.
  • Thomas-Crusells, Judith (Helsingin yliopisto, 2004)
  • Hautaniemi, Maria (Helsingin yliopisto, 2012)
    Parapoxviruses (PPVs) of the family Poxviridae are zoonotic viruses which cause contagious pustular skin infections of sheep, goats and cattle worldwide. Cases of contagious pustular stomatitis in Finnish reindeer have been reported for many years. The aims of this study were to establish specific and rapid detection methods for the causative agent of the disease and characterise the viruses circulating in Finland. The causative agent of reindeer pustular stomatitis was originally considered to be PPV Orf virus (ORFV). PCR methods amplifying different regions of the PPV genomes were developed to analyse clinical samples obtained from outbreaks of the disease in reindeer and later from viruses isolated from the disease of sheep and cattle in Finland. Subsequent phylogenetic analysis of the partial gene sequences indicated that reindeer virus from the early outbreaks is most closely related to ORFV whereas the PPV strains from the 1999-2000 outbreak is most closely related to the cattle PPV Pseudocowpox virus (PCPV). Furthermore, the phylogenetic analyses of Finnish reindeer, bovine and sheep isolates indicated that the viruses causing the disease in reindeer are very closely related to the PCPVs and ORFVs infecting Finnish cattle and sheep, respectively. Since the initial classification of the viruses causing disease in Finnish reindeer relied solely on the partial sequence analysis of two conserved PPV genes, the genome of PCPV-like reindeer isolate (F00.120R) was sequenced and analysed together with that of a reference strain of PCPV (VR634). The genomes of F00.120R and VR634 viruses were found to consist of a central core region of conserved genes, flanked by more variable terminal regions as seen in other poxvirus genomes. F00.120R was found to have four, possibly fragmented, genes at the left terminus and another near the central region of the genome that are not present in ORFV or Bovine papular stomatitis virus (BPSV; another PPV) genomes. In addition, the F00.120R genome was found to lack six genes seen near the right genome terminus of other PPVs. The protein coding contents and G+C profile of the genomes, as well as gene order and predicted protein homologies indicated that F00.120R is an isolate of PCPV and that PCPV is correctly classified as a member of the genus Parapoxvirus. These results expand the host range of PCPV to reindeer. The observed six gene deletion at the right terminus of the F00.120R genome was further investigated. The results showed that a 5431 bp sequence containing genes 116-121 was likely to have been deleted from the F00.120R genome prior to the 7th cell culture passage. These findings conclude that the genome of reindeer PCPV is 140 kbp in length and has 137 genes.
  • Kukkonen, Sami (Helsingin yliopisto, 2004)
  • Byholm, Patrik (Helsingin yliopisto, 2003)
  • Latva-Karjanmaa, Tarja (Helsingin yliopisto, 2006)
    The European aspen (Populus tremula) is a keystone species for biodiversity in boreal forests. However, the future of aspen may be threatened, because large aspens have mostly been removed from managed forests, whereas regeneration and the long-term persistence of mature trees are subjects of concern in protected areas. Aspen is a pioneer tree, and it can reproduce both sexually by seed and asexually by root suckers. Through asexual reproduction aspen forms clones, groups of genetically identical trees (ramets). In my thesis, I have studied the structure of aspen populations in terms of number, size, clonal and demographic properties. Additionally, I have investigated the emergence and survival of seedlings as well as the seed quantity and quality in crosses between the European and hybrid aspen. To study the regeneration and population structure, mature aspens were recorded in old-growth and managed forests in eastern Finland based on a large-scale inventory (11 400 ha). In addition, small aspen trees were surveyed on sample plots. Clonal structure was investigated both by morphological characters and by DNA-based markers (microsatellites). Seedling emergence and survival was studied with two sowing experiments. With crosses between European and hybrid aspens we wanted to study whether elevated temperatures due to climate change would benefit the different crosses of European and hybrid aspen unequally and thus affect the gene flow between the two species. The average volumes of mature aspen were 5.3 m3/ha in continuous old-growth, and 0.8 m3/ha in managed forests. Results indicate also that large aspen trees in managed forests are a legacy of the past less intensively managed forest landscapes. Long-term persistence of aspen in protected areas can only be secured by restoration measures creating sufficiently large gaps for regeneration. More emphasis should be given to sparing aspens in thinnings and to retaining of mature aspens in regeneration cutting in managed forests. Aspen was found to be spatially aggregated in the landscape. This could be explained by site type, disturbance history and / or limitations in seed dispersal. Clonal structure does not explain the spatial aggregation, since average size of the clones was only 2.3 ramets, and most clones (70 %) consisted of just one ramet. The small size of the clones suggests that most of them are relatively young. Therefore, sexual reproduction may be more common than has previously been thought. Seedling emergence was most successful in mineral soil especially, when the site had been burned. Only few seedlings occurred on humus. Survival of the seedlings was low, and strongly dependent on moisture, but also on seedbed conditions. The seeds were found to maintain their germinability longer than has earlier been thought to be possible. Interspecific crosses produced more seeds with higher quality than intraspecific crosses. When temperature was elevated, germination of hybrid aspen seeds increased more than seeds from P. tremula x P. tremula crosses. These results suggest that hybrid aspen may have a significant genetic impact on the European aspen, and this effect may become strengthened by climate warming.
  • Siitonen, Paula (Helsingin yliopisto, 2003)
  • Nieminen, Tiina Maileena (Helsingin yliopisto, 2005)