Characterization of Leaf-Type Ferredoxin-NADP+ Oxidoreductase (FNR) Isoforms in Arabidopsis thaliana

Life on earth is based on sunlight, which is captured in chemical form by photosynthetic reactions. In the chloroplasts of plants, light reactions of photosynthesis take place at thylakoid membranes, whereas carbon assimilation reactions occur in the soluble stroma. The products of linear electron transfer (LET), highly-energetic ATP molecules, and reducing power in the form of NADPH molecules, are further used in the fixation of inorganic CO2 molecules into organic sugars. Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes the last of the light reactions by transferring electrons from ferredoxin (FD) to NADP+. In addition to LET, FNR has been suggested to play a role in cyclic electron transfer (CET), which produces ATP without the accumulation of reducing equivalents. CET is proposed to occur via two putative routes, the PGR5- route and the NDH-route. In this thesis, the leaf-type FNR (LFNR) isoforms LFNR1 and LFNR2 of a model organism, Arabidopsis thaliana, were characterized. The physiological roles of LFNRs were investigated using single and double mutant plants. The viability of the single mutants indicates functionality of both isoforms, with neither appearing to play a specific role in CET. The more severe phenotype of low-temperature adapted fnr2 plants compared to both wild-type (WT) and fnr1 plants suggests a specific role for LFNR2 under unfavorable growth conditions. The more severe phenotype of the fnr1 x fnr2 (F1 generation) plants compared to single mutants reflects down-regulated photosynthetic capacity, whereas slightly higher excitation pressure indicates mild over-excitation of electron transfer chain (ETC). However, induction of CET and various photoprotective mechanisms enable adaptation of fnr1 x fnr2 plants to scarcity of LFNR. The fnr1 fnr2 plants (F2 generation), without detectable levels of LFNR, were viable only under heterotrophic conditions. Moreover, drought stress induced acceleration of the rate of P700 + re-reduction in darkness was accompanied by a concomitant up-regulation of the PGR5-route specific components, PGR5 and PGRL1, demonstrating the induction of CET via the PGR5-route. The up-regulation of relative transcriptional expression of the FD1 gene indicates that the FD1 isoform may have a specific function in CET, while no such role could be defined for either of the LFNR isoforms. Both the membrane-bound and soluble LFNR1 and LFNR2 each appear as two distinct spots after 2D-PAGE with different isoelectric points (pIs), indicating the existence of post-translational modifications (PTMs) which do not determine the membrane attachment of LFNR. The possibility of phosphorylation and glycosylation PTMs were excluded, but all four LFNR forms were shown to contain acetylated lysine residues as well as alternative N-termini. N-terminal acetylation was shown to shift the pI of both LFNRs to be more acidic. In addition, all four LFNR forms were demonstrated to interact both with FD1 and FD2 in vitro

Improving the calculation of rate of stratification in APROS

APROS (Advanced Process Simulation Environment) is a computer simulation program developed to simulate thermal hydraulic processes in nuclear and conventional power plants. Earlier research at VTT Technological Research Centre of Finland had found the current version of APROS to produce inaccurate simulation results for a certain case of loop seal clearing. The objective of this Master’s thesis is to find and implement an alternative method for calculating the rate of stratification in APROS, which was found to be the reason for the inaccuracies. Brief literature study was performed and a promising candidate for the new method was found. The new method was implemented into APROS and tested against experiments and simulations from two test facilities and the current version of APROS. Simulation results with the new version were partially conflicting; in some cases the new method was more accurate than the current version, in some the current method was better. Overall, the new method can be assessed as an improvement.

Understanding the in vitro dissolution rate of glasses with respect to future clinical applications

Glass is a unique material with a long history. Several glass products are used daily in our everyday life, often unnoticed. Glass can be found not only in obvious applications such as tableware, windows, and light bulbs, but also in tennis rackets, windmill turbine blades, optical devices, and medical implants. The glasses used at present as implants are inorganic silica-based melt-derived compositions mainly for hard-tissue repair as bone graft substitute in dentistry and orthopedics. The degree of glass reactivity desired varies according to implantation situation and it is vital that the ion release from any glasses used in medical applications is controlled. Understanding the in vitro dissolution rate of glasses provides a first approximation of their behavior in vivo. Specific studies concerning dissolution properties of bioactive glasses have been relatively scarce and mostly concentrated to static condition studies. The motivation behind this work was to develop a simple and accurate method for quantifying the in vitro dissolution rate of highly different types of glass compositions with interest for future clinical applications. By combining information from various experimental conditions, a better knowledge of glass dissolution and the suitability of different glasses for different medical applications can be obtained. Thus, two traditional and one novel approach were utilized in this thesis to study glass dissolution. The chemical durability of silicate glasses was tested in water and TRIS-buffered solution at static and dynamic conditions. The traditional in vitro testing with a TRISbuffered solution under static conditions works well with bioactive or with readily dissolving glasses, and it is easy to follow the ion dissolution reactions. However, in the buffered solution no marked differences between the more durable glasses were observed. The hydrolytic resistance of the glasses was studied using the standard procedure ISO 719. The relative scale given by the standard failed to provide any relevant information when bioactive glasses were studied. However, the clear differences in the hydrolytic resistance values imply that the method could be used as a rapid test to get an overall idea of the biodegradability of glasses. The standard method combined with the ion concentration and pH measurements gives a better estimate of the hydrolytic resistance because of the high silicon amount released from a glass. A sensitive on-line analysis method utilizing inductively coupled plasma optical emission spectrometer and a flow-through micro-volume pH electrode was developed to study the initial dissolution of biocompatible glasses. This approach was found suitable for compositions within a large range of chemical durability. With this approach, the initial dissolution of all ions could be measured simultaneously and quantitatively, which gave a good overall idea of the initial dissolution rates for the individual ions and the dissolution mechanism. These types of results with glass dissolution were presented for the first time during the course of writing this thesis. Based on the initial dissolution patterns obtained with the novel approach using TRIS, the experimental glasses could be divided into four distinct categories. The initial dissolution patterns of glasses correlated well with the anticipated bioactivity. Moreover, the normalized surface-specific mass loss rates and the different in vivo models and the actual in vivo data correlated well. The results suggest that this type of approach can be used for prescreening the suitability of novel glass compositions for future clinical applications. Furthermore, the results shed light on the possible bioactivity of glasses. An additional goal in this thesis was to gain insight into the phase changes occurring during various heat treatments of glasses with three selected compositions. Engineering-type T-T-T curves for glasses 1-98 and 13-93 were stablished. The information gained is essential in manufacturing amorphous porous implants or for drawing of continuous fibers of the glasses. Although both glasses can be hot worked to amorphous products at carefully controlled conditions, 1-98 showed one magnitude greater nucleation and crystal growth rate than 13-93. Thus, 13-93 is better suited than 1-98 for working processes which require long residence times at high temperatures. It was also shown that amorphous and partially crystalline porous implants can be sintered from bioactive glass S53P4. Surface crystallization of S53P4, forming Na2O∙CaO∙2SiO2, was observed to start at 650°C. The secondary crystals of Na2Ca4(PO4)2SiO4, reported for the first time in this thesis, were detected at higher temperatures, from 850°C to 1000°C. The crystal phases formed affected the dissolution behavior of the implants in simulated body fluid. This study opens up new possibilities for using S53P4 to manufacture various structures, while tailoring their bioactivity by controlling the proportions of the different phases. The results obtained in this thesis give valuable additional information and tools to the state of the art for designing glasses with respect to future clinical applications. With the knowledge gained we can identify different dissolution patters and use this information to improve the tuning of glass compositions. In addition, the novel online analysis approach provides an excellent opportunity to further enhance our knowledge of glass behavior in simulated body conditions.