Molecular studies on LIM-domain protein CRP1

Various intrinsic and external factors are constantly attacking the cells causing damage to DNA and to other cellular structures. Cells in turn have evolved with different kinds of mechanisms to protect against the attacks and to repair the damage. Ultraviolet radiation (UVR) is one of the major environmental genotoxic carcinogens that causes inflammation, mutations, immunosuppression, accelerated aging of the skin and skin cancers. Epidermis is the outermost layer of the skin consisting mostly of keratinocytes, whose primary function is to protect the skin against e.g. UV radiation. LIM domain proteins are a group of proteins involved in regulation of cell growth, damage signalling, cell fate determination and signal transduction. Despite their two zinc fingers, LIM domains do not bind to DNA, but rather mediate protein-protein interactions and function as modular protein binding interfaces. We initially identified CSRP1 as UVR-regulated transcript by using expression profiling. Here we have further studied the regulation and function of CRP1, a representative of cysteine rich protein- family consisting of two LIM domains. We find that CRP1 is increased by UVR in primary human keratinocytes and in normal human skin fibroblasts. Ectopic expression of CRP1 protected the cells against UVR and provided a survival advantage, whereas silencing of CRP1 rendered the cells more photosensitive. Actinic keratosis is a premalignant lesion of skin caused by excess exposure to sunlight and sunburn, which may lead to formation of squamous cell carcinoma. The expression of CRP1 was increased in basal keratinocytes of Actinic keratosis patient specimens suggesting that CRP1 may be increased by constant exposure to UVR and may provide survival advantage for the cells also in vivo. In squamous cell carcinoma, CRP1 was only expressed in the fibroblasts surrounding the tumour. Moreover, we found that ectopic expression of CRP1 suppresses cell proliferation. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine that regulates several functions in cell including growth, apoptosis and differentiation, and plays important roles in pathological disorders like cancer and fibrosis. We found that TGFbeta-signalling pathway regulates CRP1 at protein, but not at transcriptional level. The increase was mediated both through Smad and non-Smad signalling pathways involving MAPK/p38. Furthermore, we found that TGFbeta-mediated increase in CRP1 was associated with myofibroblast differentiation, and that CRP1 was significantly more expressed in idiopathic pulmonary fibrosis as compared to normal lung specimens. Since cell contractility is a distinct feature of myofibroblasts, and CRP1 is associated with actin cytoskeleton, we studied the role of CRP1 in cell contractility. CRP1 was found to localize to stress fibres that mediate contractility and to mediate myofibroblast contraction. These studies identify CRP1 as a stress responsive and cytokine regulated cytoskeletal protein that participates in pathological processes involved in fibrotic diseases and cancer.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Integrated climatic impacts of forestry and fibre-based packaging

The purpose of this study was to examine the integrated climatic impacts of forestry and the use fibre-based packaging materials. The responsible use of forest resources plays an integral role in mitigating climate change. Forests offer three generic mitigation strategies; conservation, sequestration and substitution. By conserving carbon reservoirs, increasing the carbon sequestration in the forest or substituting fossil fuel intensive materials and energy, it is possible to lower the amount of carbon in the atmosphere through the use of forest resources. The Finnish forest industry consumed some 78 million m3 of wood in 2009, while total of 2.4 million tons of different packaging materials were consumed that same year in Finland. Nearly half of the domestically consumed packaging materials were wood-based. Globally the world packaging material market is valued worth annually some €400 billion, of which the fibre-based packaging materials account for 40 %. The methodology and the theoretical framework of this study are based on a stand-level, steady-state analysis of forestry and wood yields. The forest stand data used for this study were obtained from Metla, and consisted of 14 forest stands located in Southern and Central Finland. The forest growth and wood yields were first optimized with the help of Stand Management Assistant software, and then simulated in Motti for forest carbon pools. The basic idea was to examine the climatic impacts of fibre-based packaging material production and consumption through different forest management and end-use scenarios. Economically optimal forest management practices were chosen as the baseline (1) for the study. In the alternative scenarios, the amount of fibre-based packaging material on the market decreased from the baseline. The reduced pulpwood demand (RPD) scenario (2) follows economically optimal management practices under reduced pulpwood price conditions, while the sawlog scenario (3) also changed the product mix from packaging to sawnwood products. The energy scenario (4) examines the impacts of pulpwood demand shift from packaging to energy use. The final scenario follows the silvicultural guidelines developed by the Forestry Development Centre Tapio (5). The baseline forest and forest product carbon pools and the avoided emissions from wood use were compared to those under alternative forest management regimes and end-use scenarios. The comparison of the climatic impacts between scenarios gave an insight into the sustainability of fibre-based packaging materials, and the impacts of decreased material supply and substitution. The results show that the use of wood for fibre-based packaging purposes is favorable, when considering climate change mitigation aspects of forestry and wood use. Fibre-based packaging materials efficiently displace fossil carbon emissions by substituting more energy intensive materials, and they delay biogenic carbon re-emissions to the atmosphere for several months up to years. The RPD and the sawlog scenarios both fared well in the scenario comparison. These scenarios produced relatively more sawnwood, which can displace high amounts of emissions and has high carbon storing potential due to the long lifecycle. The results indicate the possibility that win-win scenarios exist by shifting production from pulpwood to sawlogs; on some of the stands in the RPD and sawlog scenarios, both carbon pools and avoided emissions increased from the baseline simultaneously. On the opposite, the shift from packaging material to energy use caused the carbon pools and the avoided emissions to diminish from the baseline. Hence the use of virgin fibres for energy purposes, rather than forest industry feedstock biomass, should be critically judged if optional to each other. Managing the stands according to the silvicultural guidelines developed by the Forestry Development Centre Tapio provided the least climatic benefits, showing considerably lower carbon pools and avoided emissions. This seems interesting and worth noting, as the guidelines are the current basis for the forest management practices in Finland.