Mechanism of RNA Translocation by a Viral Packaging Motor

Molecular motors are proteins that convert chemical energy into mechanical work. The viral packaging ATPase P4 is a hexameric molecular motor that translocates RNA into preformed viral capsids. P4 belongs to the ubiquitous class of hexameric helicases. Although its structure is known, the mechanism of RNA translocation remains elusive. Here we present a detailed kinetic study of nucleotide binding, hydrolysis, and product release by P4. We propose a stochastic-sequential cooperative model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive. Further, we use hydrogen/deuterium exchange detected by high resolution mass spectrometry to visualize P4 conformational dynamics during the catalytic cycle. Concerted changes of exchange kinetics reveal a cooperative unit that dynamically links ATP binding sites and the central RNA binding channel. The cooperative unit is compatible with the structure-based model in which translocation is effected by conformational changes of a limited protein region. Deuterium labeling also discloses the transition state associated with RNA loading which proceeds via opening of the hexameric ring. Hydrogen/deuterium exchange is further used to delineate the interactions of the P4 hexamer with the viral procapsid. P4 associates with the procapsid via its C-terminal face. The interactions stabilize subunit interfaces within the hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading. Finally, we use single molecule techniques to characterize P4 translocation along RNA. While the P4 hexamer encloses RNA topologically within the central channel, it diffuses randomly along the RNA. In the presence of ATP, unidirectional net movement is discernible in addition to the stochastic motion. The diffusion is hindered by activation energy barriers that depend on the nucleotide binding state. The results suggest that P4 employs an electrostatic clutch instead of cycling through stable, discrete, RNA binding states during translocation. Conformational changes coupled to ATP hydrolysis modify the electrostatic potential inside the central channel, which in turn biases RNA motion in one direction. Implications of the P4 model for other hexameric molecular motors are discussed.

Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.

The Phraseology and Structure of Latin Building Inscriptions in Roman North Africa

This study analyses the diction of Latin building inscriptions. Despite its importance, this topic has rarely been discussed before: the most substantial contribution on the subject is a short dissertation by Klaus Gast (1965) that focuses on 100 inscriptions dating mostly from the Republican period. Marietta Horster (2001) also touched upon this theme in her thesis on imperial building inscriptions. I have collected my source material in North Africa because more Latin building inscriptions dating from the Imperial period have survived there than in any other area of the Roman Empire. By means of a thorough and independent survey, I have assembled all relevant African Latin building inscriptions datable to the Roman period (between 146 BC and AD 425), 1002 texts, into a corpus. These inscriptions are all fully edited in Appendix 1; Appendix 2 contains references to earlier editions. To facilitate search operations, both are also available in electronic form. They are downloadable from the address http://www.helsinki.fi/hum/kla/htm/jatkoopinnot.htm. Chapter one is an introduction dealing with the nature of building inscriptions as source material. Chapter two offers a statistical overview of the material. The following main section of the work falls into five chapters, each of which analyses one main part of a building inscription. An average building inscription can be divided into five parts: the starting phrase opens the inscription (a dedication to gods, for example), the subject part identifies the builder, the object part describes the constructed or repaired building, the predicate part records the building activity and the supplement part offers additional information on the project (it can specify the funding, for instance). These chapters are systematic and chronological and their purpose is to register and interpret the phrases used, to analyse reasons for their use and for their popularity among the different groups of builders. Chapter eight, which follows the main section of the work, creates a typology of building inscriptions based on their structure. It also presents the most frequently attested types of building inscriptions. The conclusion describes, on a general level, how the diction of building inscriptions developed during the period of study and how this striking development resulted from socio-economic changes that took place in Romano-African society during Antiquity. This study shows that the phraseology of building inscriptions had a clear correlation both with the type of builder and with the date of carving. Private builders tended to accentuate their participation (especially its financial side) in the project; honouring the emperor received more emphasis in the building inscriptions set up by communities; the texts produced by the army were concise. The chronological development is so clear that it enables stylistic dating. At the beginning of the imperial period the phrases were clear, concrete, formal and stereotyped but by Late Antiquity they have become vague, subjective, flexible, varied and even rhetorically or poetically coloured.

Structure informationnelle et constructions du kabyle : Etude de trois types de phrase dans le cadre de la grammaire constructionnelle

Information structure and Kabyle constructions Three sentence types in the Construction Grammar framework The study examines three Kabyle sentence types and their variants. These sentence types have been chosen because they code the same state of affairs but have different syntactic structures. The sentence types are Dislocated sentence, Cleft sentence, and Canonical sentence. I argue first that a proper description of these sentence types should include information structure and, second, that a description which takes into account information structure is possible in the Construction Grammar framework. The study thus constitutes a testing ground for Construction Grammar for its applicability to a less known language. It constitutes a testing ground notably because the differentiation between the three types of sentences cannot be done without information structure categories and, consequently, these categories must be integrated also in the grammatical description. The information structure analysis is based on the model outlined by Knud Lambrecht. In that model, information structure is considered as a component of sentence grammar that assures the pragmatically correct sentence forms. The work starts by an examination of the three sentence types and the analyses that have been done in André Martinet s functional grammar framework. This introduces the sentence types chosen as the object of study and discusses the difficulties related to their analysis. After a presentation of the state of the art, including earlier and more recent models, the principles and notions of Construction Grammar and of Lambrecht s model are introduced and explicated. The information structure analysis is presented in three chapters, each treating one of the three sentence types. The analyses are based on spoken language data and elicitation. Prosody is included in the study when a syntactic structure seems to code two different focus structures. In such cases, it is pertinent to investigate whether these are coded by prosody. The final chapter presents the constructions that have been established and the problems encountered in analysing them. It also discusses the impact of the study on the theories used and on the theory of syntax in general.

Grammar and disciplinary culture : a corpus-based study

The present study provides a usage-based account of how three grammatical structures, declarative content clauses, interrogative content clause and as-predicative constructions, are used in academic research articles. These structures may be used in both knowledge claims and citations, and they often express evaluative meanings. Using the methodology of quantitative corpus linguistics, I investigate how the culture of the academic discipline influences the way in which these constructions are used in research articles. The study compares the rates of occurrence of these grammatical structures and investigates their co-occurrence patterns in articles representing four different disciplines (medicine, physics, law, and literary criticism). The analysis is based on a purpose-built 2-million-word corpus, which has been part-of-speech tagged. The analysis demonstrates that the use of these grammatical structures varies between disciplines, and further shows that the differences observed in the corpus data are linked with differences in the nature of knowledge and the patterns of enquiry. The constructions in focus tend to be more frequently used in the soft disciplines, law and literary criticism, where their co-occurrence patterns are also more varied. This reflects both the greater variety of topics discussed in these disciplines, and the higher frequency of references to statements made by other researchers. Knowledge-building in the soft fields normally requires a careful contextualisation of the arguments, giving rise to statements reporting earlier research employing the constructions in focus. In contrast, knowledgebuilding in the hard fields is typically a cumulative process, based on agreed-upon methods of analysis. This characteristic is reflected in the structure and contents of research reports, which offer fewer opportunities for using these constructions.

Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

The structure of Norway spruce (Picea abies [L.] Karst.) stems in relation to wood properties of sawn timber

An important challenge in forest industry is to get the appropriate raw material out from the forests to the wood processing industry. Growth and stem reconstruction simulators are therefore increasingly integrated in industrial conversion simulators, for linking the properties of wooden products to the three-dimensional structure of stems and their growing conditions. Static simulators predict the wood properties from stem dimensions at the end of a growth simulation period, whereas in dynamic approaches, the structural components, e.g. branches, are incremented along with the growth processes. The dynamic approach can be applied to stem reconstruction by predicting the three-dimensional stem structure from external tree variables (i.e. age, height) as a result of growth to the current state. In this study, a dynamic growth simulator, PipeQual, and a stem reconstruction simulator, RetroSTEM, are adapted to Norway spruce (Picea abies [L.] Karst.) to predict the three-dimensional structure of stems (tapers, branchiness, wood basic density) over time such that both simulators can be integrated in a sawing simulator. The parameterisation of the PipeQual and RetroSTEM simulators for Norway spruce relied on the theoretically based description of tree structure developing in the growth process and following certain conservative structural regularities while allowing for plasticity in the crown development. The crown expressed both regularity and plasticity in its development, as the vertical foliage density peaked regularly at about 5 m from the stem apex, varying below that with tree age and dominance position (Study I). Conservative stem structure was characterized in terms of (1) the pipe ratios between foliage mass and branch and stem cross-sectional areas at crown base, (2) the allometric relationship between foliage mass and crown length, (3) mean branch length relative to crown length and (4) form coefficients in branches and stem (Study II). The pipe ratio between branch and stem cross-sectional area at crown base, and mean branch length relative to the crown length may differ in trees before and after canopy closure, but the variation should be further analysed in stands of different ages and densities with varying site fertilities and climates. The predictions of the PipeQual and RetroSTEM simulators were evaluated by comparing the simulated values to measured ones (Study III, IV). Both simulators predicted stem taper and branch diameter at the individual tree level with a small bias. RetroSTEM predictions of wood density were accurate. For focusing on even more accurate predictions of stem diameters and branchiness along the stem, both simulators should be further improved by revising the following aspects in the simulators: the relationship between foliage and stem sapwood area in the upper stem, the error source in branch sizes, the crown base development and the height growth models in RetroSTEM. In Study V, the RetroSTEM simulator was integrated in the InnoSIM sawing simulator, and according to the pilot simulations, this turned out to be an efficient tool for readily producing stand scale information about stem sizes and structure when approximating the available assortments of wood products.

A capability-based view of organisational renewal : Combining opportunity- and advantage-seeking growth in large, established European and North American wood-industry companies

The purpose of this study was to extend understanding of how large firms pursuing sustained and profitable growth manage organisational renewal. A multiple-case study was conducted in 27 North American and European wood-industry companies, of which 11 were chosen for closer study. The study combined the organisational-capabilities approach to strategic management with corporate-entrepreneurship thinking. It charted the further development of an identification and classification system for capabilities comprising three dimensions: (i) the dynamism between firm-specific and industry-significant capabilities, (ii) hierarchies of capabilities and capability portfolios, and (iii) their internal structure. Capability building was analysed in the context of the organisational design, the technological systems and the type of resource-bundling process (creating new vs. entrenching existing capabilities). The thesis describes the current capability portfolios and the organisational changes in the case companies. It also clarifies the mechanisms through which companies can influence the balance between knowledge search and the efficiency of knowledge transfer and integration in their daily business activities, and consequently the diversity of their capability portfolio and the breadth and novelty of their product/service range. The largest wood-industry companies of today must develop a seemingly dual strategic focus: they have to combine leading-edge, innovative solutions with cost-efficient, large-scale production. The use of modern technology in production was no longer a primary source of competitiveness in the case companies, but rather belonged to the portfolio of basic capabilities. Knowledge and information management had become an industry imperative, on a par with cost effectiveness. Yet, during the period of this research, the case companies were better in supporting growth in volume of the existing activity than growth through new economic activities. Customer-driven, incremental innovation was preferred over firm-driven innovation through experimentation. The three main constraints on organisational renewal were the lack of slack resources, the aim for lean, centralised designs, and the inward-bound communication climate.

Population structure of Pyrenophora teres, the causal agent of net blotch of barley

Spring barley is the most important crop in Finland based on cultivated land area. Net blotch, a disease caused by Pyrenophora teres Drech., is the most damaging disease of barley in Finland. The pressure to improve the economics and efficiency of agriculture has increased the need for more efficient plant protection methods. Development of durable host-plant resistance to net blotch is a promising possibility. However, deployment of disease resistant crops could initiate selection pressure on the pathogen (P. teres) population. The aim of this study was to understand the population biology of P. teres and to estimate the evolutionary potential of P. teres under selective pressure following deployment of resistance genes and application of fungicides. The study included mainly Finnish P. teres isolates. Population samples from Russia and Australia were also included. Using AFLP markers substantial genotypic variation in P. teres populations was identified. Differences among isolates were least within Finnish fields and significantly higher in Krasnodar, Russia. Genetic differentiation was identified among populations from northern Europe and from Australia, and between the two forms P. teres f. teres (PTT, net form of net blotch) and P. teres f. maculata (PTM, spot form of net blotch) in Australia. Differentiation among populations was also identified based on virulence between Finnish and Russian populations, and based on prochloraz (fungicide) tolerance in the Häme region in Finland. Surprisingly only PTT was recovered from Finland and Russia although both forms were earlier equally common in Finland. The reason for the shift in occurrence of forms in Finland remained uncertain. Both forms were found within several fields in Australia. Sexual reproduction of P. teres was supported by recover of both mating types in equal ratio in those areas although the prevalence of sexual mating seems to be less in Finland than in Australia. Population from Krasnodar was an exception since only one mating type was found in there. Based on the substantial high genotypic variation in Krasnodar it was suggested go represent an old P. teres population, whereas the Australian samples were suggested to represent newer populations. In conclusion, P. teres populations are differentiated at several levels. Human assistance in dispersal of P. teres on infected barley seed is obvious and decreases the differentiation among populations. This can increase the plant protection problems caused by this pathogen. P. teres is capable of sexual reproduction in several areas but the prevalence varies. Based on these findings it is apparent that P. teres has the potential to pose more serious problems in barley cultivation if plant protection is neglected. Therefore, good agricultural practices, including crop rotation and the use of healthy seed, are recommended.

Properties of intramuscular connective tissue in pork and poultry with reference to weakening of structure

The most common connective tissue research in meat science has been conducted on the properties of intramuscular connective tissue (IMCT) in connection with eating quality of meat. From the chemical and physical properties of meat, researchers have concluded that meat from animals younger than physiological maturity is the most tender. In pork and poultry, different challenges have been raised: the structure of cooked meat has weakened. In extreme cases raw porcine M. semimembranosus (SM) and in most turkey M. pectoralis superficialis (PS) can be peeled off in strips along the perimysium which surrounds the muscle fibre bundles (destructured meat), and when cooked, the slices disintegrate. Raw chicken meat is generally very soft and when cooked, it can even be mushy. The overall aim of this thesis was to study the thermal properties of IMCT in porcine SM in order to see if these properties were in association with destructured meat in pork and to characterise IMCT in poultry PS. First a 'baseline' study to characterise the thermal stability of IMCT in light coloured (SM and M. longissimus dorsi in pigs and PS in poultry) and dark coloured (M. infraspinatus in pigs and a combination of M. quadriceps femoris and M. iliotibialis lateralis in poultry) muscles was necessary. Thereafter, it was investigated whether the properties of muscle fibres differed in destructured and normal porcine muscles. Collagen content and also solubility of dark coloured muscles were higher than in light coloured muscles in pork and poultry. Collagen solubility was especially high in chicken muscles, approx. 30 %, in comparison to porcine and turkey muscles. However, collagen content and solubility were similar in destructured and normal porcine SM muscles. Thermal shrinkage of IMCT occurred at approximately 65 °C in pork and poultry. It occurred at lower temperature in light coloured muscles than in dark coloured muscles, although the difference was not always significant. The onset and peak temperatures of thermal shrinkage of IMCT were lower in destructured than in normal SM muscles, when the IMCT from SM muscles exhibiting ten lowest and ten highest ultimate pH values were investigated (onset: 59.4 °C vs. 60.7 °C, peak: 64.9 °C vs. 65.7 °C). As the destructured meat was paler than normal meat, the PSE (pale, soft, exudative) phenomenon could not be ruled out. The muscle fibre cross sectional area (CSA), the number of capillaries per muscle fibre CSA and per fibre and sarcomere length were similar in destructured and normal SM muscles. Drip loss was clearly higher in destructured than in normal SM muscles. In conclusion, collagen content and solubility and thermal shrinkage temperature vary between porcine and poultry muscles. One feature in the IMCT could not be directly associated with weakening of the meat structure. Poultry breast meat is very homogenous within the species.

The white-rot fungi Phlebia radiata and Dichomitus squalens in wood-based cultures: expression of laccases, lignin peroxidases, and oxalate decarboxylase

Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.

Glycoalkaloid Content and Starch Structure in Solanum Species and Interspecific Somatic Potato Hybrids

When genome sections of wild Solanum species are bred into the cultivated potato (S. tuberosum L.) to obtain improved potato cultivars, the new cultivars must be evaluated for their beneficial and undesirable traits. Glycoalkaloids present in Solanum species are known for their toxic as well as for beneficial effects on mammals. On the other hand, glycoalkaloids in potato leaves provide natural protection against pests. Due to breeding, glycoalkaloid profile of the plant is affected. In addition, the starch properties in potato tubers can be affected as a result of breeding, because the crystalline properties are determined by the botanical source of the starch. Starch content and composition affect the texture of cooked and processed potatoes. In order to determine glycoalkaloid contents in Solanum species, simultaneous separation of glycoalkaloids and aglycones using reversed-phase high-performance liquid chromatography (HPLC) was developed. Clean-up of foliage samples was improved using a silica-based strong cation exchanger instead of octadecyl phases in solid-phase extraction. Glycoalkaloids alpha-solanine and alpha-chaconine were detected in potato tubers of cvs. Satu and Sini. The total glycoalkaloid concentration of non-peeled and immature tubers was at an acceptable level (under 20 mg/100 g of FW) in the cv. Satu, whereas concentration in cv. Sini was 23 mg/100 g FW. Solanum species (S. tuberosum, S. brevidens, S. acaule, and S. commersonii) and interspecific somatic hybrids (brd + tbr, acl + tbr, cmm + tbr) were analyzed for their glycoalkaloid contents using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The concentrations in the tubers of the brd + tbr and acl + tbr hybrids remained under 20 mg/100 g FW. Glycoalkaloid concentration in the foliage of the Solanum species was between 110 mg and 890 mg/100 g FW. However, the concentration in the foliage of S. acaule was as low as 26 mg/100 g FW. The total concentrations of brd + tbr, acl + tbr, and cmm + tbr hybrid foliages were 88 mg, 180 mg, and 685 mg/100 g FW, respectively. Glycoalkaloids of both parental plants as well as new combinations of aglycones and saccharides were detected in somatic hybrids. The hybrids contained mainly spirosolanes, and glycoalkaloid structures having no 5,6-double bond in the aglycone. Based on these results, the glycoalkaloid profiles of the hybrids may represent a safer and more beneficial spectrum of glycoalkaloids than that found in currently cultivated varieties. Starch nanostructure of three different cultivars (Satu, Saturna, and Lady Rosetta), a wild species S. acaule, and interspecific somatic hybrids were examined by wide-angle and small-angle X-ray scattering (WAXS, SAXS). For the first time, the measurements were conducted on fresh potato tuber samples. Crystallinity of starch, average crystallite size, and lamellar distance were determined from the X-ray patterns. No differences in the starch nanostructure between the three different cultivars were detected. However, tuber immaturity was detected by X-ray scattering methods when large numbers of immature and mature samples were measured and the results were compared. The present study shows that no significant changes occurred in the nanostructures of starches resulting from hybridizations of potato cultivars.

Synthesis, Reactivity and Biological Activity of 17β-HSD1 Inhibitors Based on a Thieno[2,3-d]pryrimidin-4(3H)-one Core

Breast cancer is the most common cancer in women in Western countries. In the early stages of development most breast cancers are hormone-dependent, and estrogens, especially estradiol, have a pivotal role in their development and progression. One approach to the treatment of hormone-dependent breast cancers is to block the formation of the active estrogens by inhibiting the action of the steroid metabolising enzymes. 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is a key enzyme in the biosynthesis of estradiol, the most potent female sex hormone. The 17beta-HSD1 enzyme catalyses the final step and converts estrone into the biologically active estradiol. Blocking 17beta-HSD1 activity with a specific enzyme inhibitor could provide a means to reduce circulating and tumour estradiol levels and thus promote tumour regression. In recent years 17beta-HSD1 has been recognised as an important drug target. Some inhibitors of 17beta-HSD1 have been reported, however, there are no inhibitors on the market nor have clinical trials been announced. The majority of known 17beta-HSD1 inhibitors are based on steroidal structures, while relatively little has been reported on non-steroidal inhibitors. As compared with 17beta-HSD1 inhibitors based on steroidal structures, non-steroidal compounds could have advantages of synthetic accessibility, drug-likeness, selectivity and non-estrogenicity. This study describes the synthesis of large group of novel 17beta-HSD1 inhibitors based on a non-steroidal thieno[2,3-d]pyrimidin-4(3H)-one core. An efficient synthesis route was developed for the lead compound and subsequently employed in the synthesis of thieno[2,3-d]pyrimidin-4(3H)-one based molecule library. The biological activities and binding of these inhibitors to 17beta-HSD1 and, finally, the quantitative structure activity relationship (QSAR) model are also reported. In this study, several potent and selective 17beta-HSD1 inhibitors without estrogenic activity were identified. This establishment of a novel class of inhibitors is a progressive achievement in 17beta-HSD1 inhibitor development. Furthermore, the 3D-QSAR model, constructed on the basis of this study, offers a powerful tool for future 17beta-HSD1 inhibitor development. As part of the fundamental science underpinning this research, the chemical reactivity of fused (di)cycloalkeno thieno[2,3-d]pyrimidin-4(3H)-ones with electrophilic reagents, i.e. Vilsmeier reagent and dimethylformamide dimethylacetal, was investigated. These findings resulted in a revision of the reaction mechanism of Vilsmeier haloformylation and further contributed to understanding the chemical reactivity of this compound class. This study revealed that the reactivity is dependent upon a stereoelectronic effect arising from different ring conformations.

Polymerization of Ethene and Branched α-olefins with Group IV Metal Complexes Bearing Nitrogen- and Oxygen-Based Ligands

The commodity plastics that are used in our everyday lives are based on polyolefin resins and they find wide variety of applications in several areas. Most of the production is carried out in catalyzed low pressure processes. As a consequence polymerization of ethene and α-olefins has been one of the focus areas for catalyst research both in industry and academia. Enormous amount of effort have been dedicated to fine tune the processes and to obtain better control of the polymerization and to produce tailored polymer structures The literature review of the thesis concentrates on the use of Group IV metal complexes as catalysts for polymerization of ethene and branched α-olefins. More precisely the review is focused on the use of complexes bearing [O,O] and [O,N] type ligands which have gained considerable interest. Effects of the ligand framework as well as mechanical and fluxional behaviour of the complexes are discussed. The experimental part consists mainly of development of new Group IV metal complexes bearing [O,O] and [O,N] ligands and their use as catalysts precursors in ethene polymerization. Part of the experimental work deals with usage of high-throughput techniques in tailoring properties of new polymer materials which are synthesized using Group IV complexes as catalysts. It is known that the by changing the steric and electronic properties of the ligand framework it is possible to fine tune the catalyst and to gain control over the polymerization reaction. This is why in this thesis the complex structures were designed so that the ligand frameworks could be fairly easily modified. All together 14 complexes were synthesised and used as catalysts in ethene polymerizations. It was found that the ligand framework did have an impact within the studied catalyst families. The activities of the catalysts were affected by the changes in complex structure and also effects on the produced polymers were observed: molecular weights and molecular weight distributions were depended on the used catalyst structure. Some catalysts also produced bi- or multi-modal polymers. During last decade high-throughput techniques developed in pharmaceutical industries have been adopted into polyolefin research in order to speed-up and optimize the catalyst candidates. These methods can now be regarded as established method suitable for both academia and industry alike. These high-throughput techniques were used in tailoring poly(4-methyl-1-pentene) polymers which were synthesized using Group IV metal complexes as catalysts. This work done in this thesis represents the first successful example where the high-throughput synthesis techniques are combined with high-throughput mechanical testing techniques to speed-up the discovery process for new polymer materials.