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  • Tohmola, Niina (Helsingin yliopisto, 2015)
    Metabolites are low molecular weight compounds participating in different functions of cellular systems. Metabolites can be used as diagnostic biomarkers for numerous diseases. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a powerful tool in quantification of metabolites from various sample matrices. Good sensitivity and specificity are the main benefits of the technique. Mass spectrometry is commonly used in industry, drug research and clinical diagnostics. Extensive validation of newly developed analytical methods will construct the basis to a reliable assay, and it is significant especially when analysing e.g. patient samples. The aim of this study was to develop quantitative assays for metabolites from biological samples for biomedical research and clinical diagnostics. We designed and constructed an on-line high performance liquid chromatography (HPLC) equipment and validated an assay for direct quantification of extracellular metabolites from cell cultivation. Automated sampling for LC-MS/MS analysis of intracellular metabolites was connected to the on-line system. The on-line analysis improves the methodology and shortens the time of analysis. Furthermore, a frequent sampling data can provide valuable information about physiological indications in various cell cultivations. On-line HPLC is suitable for various biotechnological applications because of its ability to monitor and collect data during cell cultivation. We developed and validated LC-MS/MS assays for neuroendocrine tumor (NET) biomarkers 5-hydroxyindole acetic acid (5-HIAA) and vanillylmandelic acid (VMA) from human serum. Generally, urinary HPLC assays are used for the determination of NET markers. HPLC assays have certain limitations and 24-h urine collection is laborious. Our LC-MS/MS assays are specific, fast and well suited for diagnostics of NETs. Furthermore, guidelines for urine collection advise to refrain from serotonin-containing foods for three days before sample collection. We showed that such a diet restriction before serum 5-HIAA assay is not necessary. Instead, one day serotonin-free diet before sampling is sufficient because the half-life of 5-HIAA in circulation was found to be 1.3 hours. All assays developed during this study were sensitive and had a wide linear range. Our serum 5-HIAA LC-MS/MS assay is routinely used for the analysis of NET patient samples at the Helsinki University Central Hospital Laboratory, HUSLAB. Serum VMA LC-MS/MS assay will be in routine use in the HUSLAB in near future. Furthermore, On-line HPLC Ltd, (Helsinki, Finland) has commercialized the on-line HPLC equipment developed in this study.
  • Demianova, Zuzana (Helsingin yliopisto, 2009)
    The basic goal of a proteomic microchip is to achieve efficient and sensitive high throughput protein analyses, automatically carrying out several measurements in parallel. A protein microchip would either detect a single protein or a large set of proteins for diagnostic purposes, basic proteome or functional analysis. Such analyses would include e.g. interactomics, general protein expression studies, detecting structural alterations or secondary modifications. Visualization of the results may occur by simple immunoreactions, general or specific labelling, or mass spectrometry. For this purpose we have manufactured chip-based proteome analysis devices that utilize the classical polymer gel electrophoresis technology to run one and two-dimensional gel electrophoresis separations of proteins in just a smaller size. In total, we manufactured three functional prototypes of which one performed a miniaturized one-dimensional gel electrophoresis (1-DE) separation, the second and third preformed two-dimensional gel electrophoresis (2-DE) separations. These microchips were successfully used to separate and characterize a set of predefined standard proteins, cell and tissue samples. Also, the miniaturized 2-DE (ComPress-2DE) chip presents a novel way of combining the 1st and 2nd dimensional separations, thus avoiding manual handling of the gels, eliminate cross-contamination, and make analyses faster and repeatability better. They all showed the advantages of miniaturization over the commercial devices; such as fast analysis, low sample- and reagent consumption, high sensitivity, high repeatability and inexpensive performance. All these instruments have the potential to be fully automated due to their easy-to-use set-up.
  • Lipponen, Katriina (Helsingin yliopisto, 2014)
    This doctoral thesis focuses on the development of miniaturized biosensing systems for the study of biomolecular interactions. Acoustic biosensor quartz crystal microbalance (QCM), partial filling affinity capillary electrophoresis (PF-ACE), and open-tubular capillary electrochromatography (OT-CEC) were developed to allow study of interactions between glycosaminoglycans and lipoproteins, which are responsible for the accumulation of low density lipoprotein (LDL) on the arterial wall. The first step was to develop suitable coating methods for the immobilization of selected glycosaminoglycans on chip and capillary surfaces and to develop a neutral capillary surface for PF-ACE studies. Evaluation of different coating procedures demonstrated the significance of the procedure for the successful outcome of biological interaction studies. With suitable platforms available for the instrumental techniques in question, studies of interactions between glycosaminoglycans and lipoprotein particles were carried out to evaluate the strength of the binding processes. Affinity constants, retention factors, and reduced mobilities were measured. Since the commonly used approaches typically allow only the strongest binding site of the system to be determined, the scope of the investigation was broadened by introducing adsorption energy distribution (AED) calculations in the processing of QCM and PF-ACE data. Finally, molecular dynamics (MD) simulations were used as a supportive tool to visualize and study interactions at the atomic level. In addition, microscale thermophoresis, a relatively new technique, was used to complement and support some of the experimental studies. The results obtained by QCM methods, PF-ACE, OT-CEC, and MD simulations were in good agreement. Even minor changes in the binding process could be visualized by exploiting AED calculations in the data processing step. The main advantages of all three experimental methods were low sample consumption, relatively fast analysis times, and the possibility to evaluate the heterogeneity of the interactions. The findings of the work demonstrate the great potential of the developed biosensing systems for biomolecular interaction and biomimicking studies. Of particular interest is the availability of information on heterogeneous reactions when AED calculations are applied in the interpretation of QCM and PF-ACE results.
  • Mäntylahti, Sampo (Helsingin yliopisto, 2014)
    In the field of bioscience there is an ongoing explosive growth in discovery and information. Novel means in biotechnology as well as in medicines are introduced at an unseen rate. One of the aspects contributing to this development is the increased understanding of protein function and structure. Proteins have a role in almost every biological process. The function and structure of proteins are linked. Recent studies have discovered that the understanding of the protein structure has been biased. Namely, the studies have unearthed a previously dismissed protein structure state: intrinsically disordered proteins (IDPs). In this highly dynamic state a protein is without a globular fold, but does not meet the requirements of a random coil either. Rapid transition between folds renders most of the established research techniques to be poor methods to study the IDPs. Nuclear magnetic resonance (NMR) is a spectroscopy method, which enables the study of molecules at atomic resolution. The technique is based upon manipulation of the nuclear spins in specifically produced sample under strong magnetic field. In this method, spins of the system generate quantum coherence state(s), which is utilized to obtain information about the system. NMR is suitable for studying samples in solid and liquid mediums, but in case of biomolecules, water solution is preferable as it resembles in vivo environment. Highly mobile structure and chemical composition of IDPs cause many established NMR experiments to fail. Development of NMR pulse sequences is an obvious approach to solve the problem. This thesis presents a number of NMR pulse sequences, which are designed to improve acquisition of information from highly mobile sections of proteins. The key aspect is to utilize H atom instead of HN in coherence transfer. Additional improvements include limited residue specific identification and novel coherence transfer pathways. Articles I, II, and III present triple resonance experiments, which correlate protein backbone atoms. Combination of the spectra enables full sequential assignment. Article IV introduces an improved pulse sequence for measuring J couplings between nitrogen and amide proton. The experiments were subjected to experimental verification. Comparisons were drawn between established pulse sequences. In both globular proteins and IDPs the results show improvement over established pulse sequences. The proposed sequences yielded improved assignment coverage, resolution and sensitivity enhancement.
  • Zhu, Lei (Helsingin yliopisto, 2009)
    Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer and second leading cause of cancer-related death of men in developed countries. Measurement of prostate specific antigen (PSA) is a very sensitive method for diagnosing and monitoring of prostate cancer (PCa), but the specificity needs improvement. Measurements of different molecular forms of PSA have been shown to improve differentiation between PCa and benign prostatic diseases. However, accurate measurement of some isoforms has not been achieved in previous assays. The aim of the present study was to develop new assays that reliably measure enzymatically active PSA, PSA-α1-chymotryposin (PSA-ACT) and PSA-α1-protease inhibitor (PSA-API), and to evaluate their diagnostic value. Double-label immunofluorometric assays using a novel monoclonal antibody (MAb) and another antibody to either free PSA (fPSA) or total PSA (tPSA) were developed and used to measure PSA-ACT and fPSA or tPSA at the same time. These assays provide enough sensitivity for measurement of PSA-ACT in sera with low PSA levels. The results obtained confirmed that proportion of PSA-ACT to tPSA (%PSA-ACT) was as useful as proportion of fPSA to tPSA (%fPSA) for discrimination between PCa and benign prostatic hyperplasia (BPH). We developed an immunoassay for detection of PSA-API based on proximity ligation, which improved assay sensitivity 10-fold compared with conventional assays. Our results confirmed previous findings that the PSA-API level is somewhat lower in men with than without PCa, and the combination of %fPSA and proportion of PSA-API to tPSA (%PSA-API) provides diagnostic improvement compared with either method alone. Assays based on this principle should be applicable to other immunoassays in which the nonspecific background is a problem. An immunopeptidometric sandwich assay (IPMA) was developed to measure the enzymatically active PSA. This assay showed high specificity, but sensitivity was not good enough for measurement of PSA concentrations in the gray zone, 2-10 µg/L, in which tPSA does not efficiently differentiate between PCa and BPH. We further developed a solid-phase proximity ligation immunoassay, which provided a 10-fold improvement in sensitivity. This proof of concept study shows that peptides reacting with proteins are potentially useful for sensitive and specific measurement of protein variants for which specific MAbs cannot be obtained.
  • Malinen, Melina (Helsingin yliopisto, 2014)
    New organotypic liver cell cultures are needed to predict the metabolism, excretion, and safety of chemical compounds. Liver cell models are particularly important since the liver largely regulates the ultimate fate of compounds in the body. Approximately 70% of the drugs administered to the body are metabolized or excreted by the liver. Animal models, cell cultures, and cell-free assays are the most common liver models. However, animal models and animal cells do not represent humans due to the interspecies differences in drug metabolizing enzymes and transporters. Instead, the most common cell-free methods, microsomes, are appropriate for drug metabolism studies, but the lack of drug transporters and transcription machinery prevents the complete evaluation of compounds. Primary human hepatocytes are capable of both drug metabolism and drug transport, and are, therefore, considered the gold standard to assess metabolism and toxicity of compounds in vitro. Primary hepatocytes, however, suffer limited availability, high functional variability, and difficulty with maintaining differentiated phenotypes and functions in cell cultures. Therefore, continuous human liver cell lines, such as HepG2 and HepaRG, have been widely used to evaluate drugs and chemicals even though they have defects in their biotransformation functions. The advantages of cell lines are their good availability, easy maintenance, and inducible drug metabolism. Generally, these cells are cultured in a two-dimensional (2D) manner that deviates from the physiological morphology and functions of the hepatocytes. The flattened 2D phenotype leads to reduced polarization and loss of important signaling pathways; this is likely to be a major reason for the failure in the prediction of drug metabolism, pharmacokinetics, and hepatotoxicity. It is believed that for more predictive in vitro models, the liver cells should be maintained in a three-dimensional (3D) microenvironment that allows reconstruction of polarization, and cell-cell and cell-extracellular matrix (ECM) contacts. The 3D cell cultures have been generated by different methods, such as cultures in matrices, scaffolds, bioreactors, and microfluidic platforms. Biomaterial hydrogels have demonstrated great potential for 2D liver cell culturing, but their potential to generate functional 3D liver cell cultures is largely unknown. The main goal of this thesis was to establish improved 3D liver cell cultures with biomaterial hydrogels. Particular attention was focused on the effects of 3D hydrogels on drug metabolism and excretion, cytoarchitecture, and cellular differentiation of HepG2 and HepaRG cell lines. As a starting point, we studied the suitability of wood-derived nanofibrillar cellulose (NFC) hydrogel as a cell culture matrix. NFC hydrogel has not been studied in cell culture before; however, as a novel, defined, animal-free, and abundantly available material, it evoked interest for testing. Herein, the wood-derived NFC was proven to own rheological and structural characters that allow 3D cell culture. Moreover, the NFC was compatible with the HepG2 and HepaRG cells, allowing for the formation of 3D multicellular aggregates with increased apicobasal polarity. When compared to commercial hydrogels, the NFC supported the albumin secretion, an indicator of hepatocellular synthetic function, from HepG2 and HepaRG cells as well or even better. These results demonstrate the potential of wood- derived NFC to function as an ECM analogue, and present the first HepaRG aggregate cultures. Next, the effect of the RAD16-I peptide hydrogel on the HepG2 cell line was investigated in more detail. Immunofluorescence staining and vectorial transport showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of canalicular efflux transporters, multidrug resistance-associated protein 2 (MRP2), and multidrug resistance protein 1 (MDR1), in the spherical HepG2 cell aggregates. The study clearly demonstrated that the peptide hydrogel increases the apicobasal polarity and appearance of bile canaliculi structures in HepG2 cell cultures. The plasticity of HepaRG liver cells was exploited to investigate the impact of 3D NFC and hyaluronan-gelatin (HG) hydrogel cultures on the phenotype of both undifferentiated HepaRG cells (early liver progenitors) and differentiated HepaRG cells (hepatocyte-like cells together with cholangiocyte-like cells). Based on the expression and activity of hepatic markers, drug metabolizing enzymes, and drug transporters, the 3D NFC and HG hydrogels promoted the differentiation of HepaRG liver progenitor cells when compared to the standard 2D technique. Instead, the 3D hydrogel cultures could not really improve the properties of differentiated HepaRG cells. In conclusion, these findings reveal the capability of the NFC, RAD16-I peptide, and HG hydrogels to improve the properties of HepG2 and HepaRG human liver cells. The new spheroid cultures of HepG2 and HepaRG cells may represent added value for pharmacokinetic and toxicity predictions, showing a liver-like cytoarchitecture and demonstrating applicability for drug metabolism and transport studies. Overall, the results deepen our knowledge of the 3D liver cell cultures.
  • Koivula, Teija (Helsingin yliopisto, 2011)
    Positron emission tomography (PET) is a molecular imaging technique that utilises radiopharmaceuticals (radiotracers) labelled with a positron-emitting radionuclide, such as fluorine-18 (18F). Development of a new radiotracer requires an appropriate radiosynthesis method: the most common of which with 18F is nucleophilic substitution with [18F]fluoride ion. The success of the labelling reaction is dependent on various factors such as the reactivity of [18F]fluoride, the structure of the target compound in addition to the chosen solvent. The overall radiosynthesis procedure must be optimised in terms of radiochemical yield and quality of the final product. Therefore, both quantitative and qualitative radioanalytical methods are essential in developing radiosynthesis methods. Furthermore, biological properties of the tracer candidate need to be evaluated by various pre-clinical studies in animal models. In this work, the feasibility of various nucleophilic 18F-fluorination strategies were studied and a labelling method for a novel radiotracer, N-3-[18F]fluoropropyl-2beta-carbomethoxy-3beta-4-fluorophenyl)nortropane ([18F]beta-CFT-FP), was optimised. The effect of solvent was studied by labelling a series of model compounds, 4-(R1-methyl)benzyl R2-benzoates. 18F-Fluorination reactions were carried out both in polar aprotic and protic solvents (tertiary alcohols). Assessment of the 18F-fluorinated products was studied by mass spectrometry (MS) in addition to conventional radiochromatographic methods, using radiosynthesis of 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) as a model reaction. Labelling of [18F]beta-CFT-FP was studied using two 18F-fluoroalkylation reagents, [18F]fluoropropyl bromide and [18F]fluoropropyl tosylate, as well as by direct 18F-fluorination of sulfonate ester precursor. Subsequently, the suitability of [18F]beta-CFT-FP for imaging dopamine transporter (DAT) was evaluated by determining its biodistribution in rats. The results showed that protic solvents can be useful co-solvents in aliphatic 18F-fluorinations, especially in the labelling of sulfonate esters. Aromatic 18F-fluorination was not promoted in tert-alcohols. Sensitivity of the ion trap MS was sufficient for the qualitative analysis of the 18F-labelled products; p-[18F]MPPF was identified from the isolated product fraction with a mass-to-charge (m/z) ratio of 435 (i.e. protonated molecule [M+H]+). [18F]beta-CFT-FP was produced most efficiently via [18F]fluoropropyl tosylate, leading to sufficient radiochemical yield and specific radioactivity for PET studies. The ex vivo studies in rats showed fast kinetics as well as the specific uptake of [18F]beta-CFT-FP to the DAT rich brain regions. Thus, it was concluded that [18F]beta-CFT-FP has potential as a radiotracer for imaging DAT by PET.
  • Tuovinen, Heli (Helsingin yliopisto, 2009)
    The role of the immune system is to protect an organism against pathogens while maintaining tolerance against self. T cells are an essential component of the immune system and they develop in the thymus. The AIRE (autoimmune regulator) gene product plays an important role in T cell development, as it promotes expression of peripheral tissue antigens in the thymus. Developing T cells, thymocytes, which recognize self-antigens with high affinity are deleted. However, this deletion process is not perfect and not all autoreactive T cells are destroyed. When the distinction between self and non-self fails, tolerance breaks and the immune system attacks the host s own tissues. This results in autoimmunity. Regulatory T cells contribute to the maintenance of self-tolerance. They can actively suppress the function of autoreactive cells. Several populations of cells with regulatory properties have been described, but the best characterized population is the natural regulatory T cells (Treg cells), which develop in the thymus and express the transcription factor FOXP3. The thymic development of Treg cells in humans is the subject of this thesis. Thymocytes at different developmental stages were analyzed using flow cytometry. The CD4-CD8- double-negative (DN) thymocytes are the earliest T cell precursors in the T cell lineage. My results show that the Treg cell marker FOXP3 is up-regulated already in a subset of these DN thymocytes. FOXP3+ cells were also found among the more mature CD4+CD8+ double-positive (DP) cells and among the CD4+ and CD8+ single-positive (SP) thymocytes. The different developmental stages of the FOXP3+ thymocytes were isolated and their gene expression examined by quantitative PCR. T cell receptor (TCR) repertoire analysis was used to compare these different thymocyte populations. My data show that in humans commitment to the Treg cell lineage is an early event and suggest that the development of Treg cells follows a linear developmental pathway, FOXP3+ DN precursors evolving through the DP stage to become mature CD4+ Treg cells. Most T cells have only one kind of TCR on their cell surface, but a small fraction of cells expresses two different TCRs. My results show that the expression of two different TCRs is enriched among Treg cells. Furthermore, both receptors were capable of transmitting signals when bound by a ligand. By extrapolating flow cytometric data, it was estimated that the majority of peripheral blood Treg cells are indeed dual-specific. The high frequency of dual-specific cells among human Treg cells suggests that dual-specificity has a role in directing these cells to the Treg cell lineage. It is known that both genetic predisposition and environmental factors influence the development of autoimmunity. It is also known that the dysfunction or absence of Treg cells leads to the development of autoimmune manifestations. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare monogenic autoimmune disease, caused by mutations in the AIRE gene. In the absence of AIRE gene product, deletion of self-specific T cells is presumably disturbed and autoreactive T cells escape to the periphery. I examined whether Treg cells are also affected in APECED. I found that the frequency of FOXP3+ Treg cells and the level of FOXP3 expression were significantly lower in APECED patients than in controls. Additionally, when studied in cell cultures, the suppressive capacity of the patients' Treg cells was impaired. Additionally, repertoire analysis showed that the TCR repertoire of Treg cells was altered. These results suggest that AIRE contributes to the development of Treg cells in humans and the selection of Treg cells is impaired in APECED patients. In conclusion, my thesis elucidates the developmental pathway of Treg cells in humans. The differentiation of Tregs begins early during thymic development and both the cells dual-specificity and AIRE probably affect the final commitment of Treg cells.
  • Kivilompolo, Maarit (Helsingin yliopisto, 2009)
    In this study, novel methodologies for the determination of antioxidative compounds in herbs and beverages were developed. Antioxidants are compounds that can reduce, delay or inhibit oxidative events. They are a part of the human defense system and are obtained through the diet. Antioxidants are naturally present in several types of foods, e.g. in fruits, beverages, vegetables and herbs. Antioxidants can also be added to foods during manufacturing to suppress lipid oxidation and formation of free radicals under conditions of cooking or storage and to reduce the concentration of free radicals in vivo after food ingestion. There is growing interest in natural antioxidants, and effective compounds have already been identified from antioxidant classes such as carotenoids, essential oils, flavonoids and phenolic acids. The wide variety of sample matrices and analytes presents quite a challenge for the development of analytical techniques. Growing demands have been placed on sample pretreatment. In this study, three novel extraction techniques, namely supercritical fluid extraction (SFE), pressurised hot water extraction (PHWE) and dynamic sonication-assisted extraction (DSAE) were studied. SFE was used for the extraction of lycopene from tomato skins and PHWE was used in the extraction of phenolic compounds from sage. DSAE was applied to the extraction of phenolic acids from Lamiaceae herbs. In the development of extraction methodologies, the main parameters of the extraction were studied and the recoveries were compared to those achieved by conventional extraction techniques. In addition, the stability of lycopene was also followed under different storage conditions. For the separation of the antioxidative compounds in the extracts, liquid chromatographic methods (LC) were utilised. Two novel LC techniques, namely ultra performance liquid chromatography (UPLC) and comprehensive two-dimensional liquid chromatography (LCxLC) were studied and compared with conventional high performance liquid chromatography (HPLC) for the separation of antioxidants in beverages and Lamiaceae herbs. In LCxLC, the selection of LC mode, column dimensions and flow rates were studied and optimised to obtain efficient separation of the target compounds. In addition, the separation powers of HPLC, UPLC, HPLCxHPLC and HPLCxUPLC were compared. To exploit the benefits of an integrated system, in which sample preparation and final separation are performed in a closed unit, dynamic sonication-assisted extraction was coupled on-line to a liquid chromatograph via a solid-phase trap. The increased sensitivity was utilised in the extraction of phenolic acids from Lamiaceae herbs. The results were compared to those of achieved by the LCxLC system.
  • Beletskaya/Santamala, Maria (2014)
    This study investigates the nature and economic effects of the contractual remedies and their role and specifics of application in the contemporary international trade, demonstrated on the example of standard construction contracts. Various remedies are examined within several legal orders across Civil and Common law traditions from the position of their convergence and utilization in the international contracting. Main focus of the research is on the economic efficiency of the contractual remedies as elements of integrated system, flexible and adjustable due to changes (internal and external to the parties’) in the circumstances after conclusion of the contract. Purpose of the study is to analyze possibilities for further harmonization of the contractual remedies’ regulation and adoption of their more efficient forms, developed within different jurisdictions and soft law codifications. These improved forms, as discussed in the work, are capable of enhancing economic efficiency of individual remedies and their productive incorporation in a cohesive remedial system, enabling contractual parties to divide various interests inside one contract and vary levels of their respective protection, exploiting suitable function of each remedy. First part is dedicated to studying definition of remedy, its functions and approach to it, developed within various contractual theories; it also briefly examines general differences between Common and Civil law systems, explaining dissimilarities in formation of remedies in various legal orders. In the second part I analyze specifics of each remedy in respective jurisdictions, including analysis of local statutory and case law, considering laws of France, Germany, UK, USA, and Finland and international codifications, underlying economic effects and drawbacks of every remedy. Remedies in question are: specific performance; damages’ compensation; termination of contract; liquidated damages and penalty; price reduction and performance withholding. Third chapter is devoted to history of development of the construction contract, combining elements of sale and service; application of each remedy in the standard construction contracts in their dynamic system and methods of improving economic efficiency. The research concludes that each remedy may simultaneously have various functions (preventive, restorative and corrective) regardless of compensatory or non-compensatory criterion, which currently becomes irrelevant. Remedial system adopted in the contract should be flexible and easy adjustable to the changes in the post-contractual circumstances. Following the ongoing process of normative convergence various legal orders should be harmonized; they should recognize autonomy of the parties to create own enforceable hierarchy of remedies within the contract and should ensure judicial intervention solely from the angle of safeguarding just and fair outcome. Study further determines that measures of enhancing economic efficiency of each remedy have been already developed in the practice and only need recognition and change of attitude in the respective national systems, widening perspectives of interpretation and qualification.
  • Liu, Yanbo (2011)
    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.
  • Paupitz, Johanna (2008)
    This is a qualitative research. The purpose of this study is to assess the influence of international development policies in the Third World using Brazilian water resources management policies as an example. The focus of the study is the Guarapiranga water basin and reservoir located in the Metropolitan Region of São Paulo, Brazil. The research demonstrates how international development agencies and ideologies influence the development of Brazilian water resources management. The objective is to see how local experts view development policies in water resources management and their impact in the region. The research reflects the criticism given in the general literature on contemporary development policies. The results demonstrate the problems associated with the Third World city and the interrelationships between environmental, social and economic development. The outcome also questions the government decentralisation and the redemocratisation processes brought by the 1988 Constitution in Brazil. The research also points out the need for further and more elaborate research on the topic. References: Finger, M. and Allrouche, J. 2002 Water Privatisation: Trans-National Corporations and the Re-regulation of the Water Industry. Spon Press, New York. Oman, C. P. and Wignaraja, G. 1991 The Postwar Evolution of Development Thinking. Macmillan in association with the OECD Development Centre, London Alasuutari, P. 1994 Laadullinen Tutkimus, 2nd edition. Vastapaino, Tampere.
  • Raheem, Bamidele (Helsingin yliopisto, 2006)
    African indigenous foods have received limited research. Most of these indigenous foods are fermented and they form part of the rich nutritional culture of many groups in African countries. The industrialization and commercialisation of these indigenous African fermented foods should be preceded by a thorough scientific knowledge of their processing which can be vital in the elimination of hunger and poverty. This study highlighted emerging developments and the microbiology of cereal-based and cassava-based food products that constitute a major part of the human diet in most African countries. In addition, investigations were also carried out on the coagulant of the Calotropis procera plant used in traditional production of Nigerian Wara cheese and on the effects of adding a nisin producing Lactococcus lactis strain originating from human milk to Nigerian Wara cheese. Fermented cereal-based food such as ogi utilize popular African and readily available grains maize, millet or sorghum as substrates and is popular as a weaning diet in infants. In this study, the bulkiness caused by starch gelatinization was solved by amylase treatments in the investigation on cooked and fermented oat bran porridge. A similar treatment could reduce the viscosity of any cereal porridge. The properties of the Sodom apple leaves (Calotropis procera) extract in cheesemaking were studied. C. procera was affected by monovalent (K+ and Na+) and divalent (Mg2+ and Ca2+) cations during coagulation. The rennet strength of this coagulant was found to be 7 % compared to animal rennet at 35 °C. Increasing the incubation temperature to 70 °C increased the rennet strength 28-fold. The molecular weight of the partially purified protease was determined by SDS-PAGE and was confirmed by Zymography to be approximately 60 kilodaltons. The high proteolytic activity at 70 °C supported the suitability of the protease enzyme as a coagulant in future commercial production of Nigerian Wara cheese. It was also possible to extend the shelf life of Wara cheese by a nisin producing lactic acid bacteria Lactococcus lactis LAC309. The levels of nisin in both whey and curd fractions of Wara were investigated, results showed a 3 log reduction of toxicogenic Bacillus licheniformis spiked on Wara after 3 days. These studies are the first in Finland to promote the advancement of scientific knowledge in African foods. Recognizing these indigenous food products and an efficient transfer of technology from the developed countries to industrialize them are necessary towards a successful realization of the United Nations Millenium Development Program.
  • Honkonen, Ilja (Helsingin yliopisto, 2013)
    Currently the majority of space-based assets are located inside the Earth's magnetosphere where they must endure the effects of the near-Earth space environment, i.e. space weather, which is driven by the supersonic flow of plasma from the Sun. Space weather refers to the day-to-day changes in the temperature, magnetic field and other parameters of the near-Earth space, similarly to ordinary weather which refers to changes in the atmosphere above ground level. Space weather can also cause adverse effects on the ground, for example, by inducing large direct currents in power transmission systems. The performance of computers has been growing exponentially for many decades and as a result the importance of numerical modeling in science has also increased rapidly. Numerical modeling is especially important in space plasma physics because there are no in-situ observations of space plasmas outside of the heliosphere and it is not feasible to study all aspects of space plasmas in a terrestrial laboratory. With the increasing number of computational cores in supercomputers, the parallel performance of numerical models on distributed memory hardware is also becoming crucial. This thesis consists of an introduction, four peer reviewed articles and describes the process of developing numerical space environment/weather models and the use of such models to study the near-Earth space. A complete model development chain is presented starting from initial planning and design to distributed memory parallelization and optimization, and finally testing, verification and validation of numerical models. A grid library that provides good parallel scalability on distributed memory hardware and several novel features, the distributed cartesian cell-refinable grid (DCCRG), is designed and developed. DCCRG is presently used in two numerical space weather models being developed at the Finnish Meteorological Institute. The first global magnetospheric test particle simulation based on the Vlasov description of plasma is carried out using the Vlasiator model. The test shows that the Vlasov equation for plasma in six-dimensionsional phase space is solved correctly by Vlasiator, that results are obtained beyond those of the magnetohydrodynamic (MHD) description of plasma and that global magnetospheric simulations using a hybrid-Vlasov model are feasible on current hardware. For the first time four global magnetospheric models using the MHD description of plasma (BATS-R-US, GUMICS, OpenGGCM, LFM) are run with identical solar wind input and the results compared to observations in the ionosphere and outer magnetosphere. Based on the results of the global magnetospheric MHD model GUMICS a hypothesis is formulated for a new mechanism of plasmoid formation in the Earth's magnetotail.