Regulation of strawberry growth and development

Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.

Digestion capasity, nutrient digestibilities and physico-chemical conditions in the intestine influenced by the age of growing turkeys

Six experiments have been conducted to examine digestibility and feeding value of domestic Finnish fibre-rich cereals (barley and oats as compared to maize and wheat) and protein sources (rapeseed meal and cake, peas, faba beans, lupin seeds) for growing turkeys and to investigate effects of age of the birds (from 3 to 12 weeks of age) on digestion process and estimated nutrient digestibility and energy values. Besides, an objective of the study was to test applications of digestibility research methodology for turkeys. Total tract digestibility and apparent metabolizable energy (AME) was assayed in experimental cages using excreta collection, and a slaughter method was applied to sample small intestinal digesta for determination of apparent ileal crude protein digestibility (AICPD), jejuno-duodenal digesta viscosity and caecal volatile fatty acid (VFA) concentration. Digesta viscosity decreased and caecal VFA production increased with age of growing turkeys. Digesta retention times in the small intestine were generally longer in the older birds than in the younger ones. Crude fat digestibility and AME increased with age of growing turkeys, especially with viscous diets. AICPD seemed to decrease with age in most cases. Supplementation with β-gucanase-xylanase decreased viscosity, improved crude fat digestibility and metabolizable energy value and increased VFA production especially in barley-fed turkeys and especially in the young birds. Poor protein digestibility and low energy value of rapeseed meal and rapeseed cake decreased their feeding value for turkeys. In addition, a typical goitrogenic effect of rapeseed feeding was detected. Use of legume seeds as feed for growing turkeys is limited mostly by the low energy value in lupin seeds and the low ileal protein and amino acid digestibility in faba beans. Digestibility of fibre-rich protein sources was not improved with age of the turkeys. Euthanizing the turkeys for AICPD determination by carbon dioxide and bleeding led to lower digestibility values than mechanical stunning and cervical dislocation, suggesting inferiority of carbon dioxide stunning in experimental use. Comparison of AICPD and AME results obtained using different markers showed that considerable differences may occur, especially on total tract level, when acid-insoluble ash gave considerably lower AME values than titanium dioxide and chromic oxide.

Chemical characteristics and behaviour of sediment phosphorus in the northeastern Baltic Sea.

Chemical characteristics and behaviour of sediment phosphorus in the northeastern Baltic Sea Eutrophication is a severe environmental problem in the Baltic Sea, especially in the Gulf of Finland and the Archipelago Sea, and it is enhanced by the release of phosphorus (P) from bottom sediments. The release of P from sediment reserves largely depends on the occurrence of P in different chemical forms and on the prevailing conditions, especially on the presence of oxygen. This study examines the chemical character and the vertical distribution of sediment P in two shallow estuaries, in shallow coastal sediments overlain by oxic near-bottom water, and in poorly oxygenated open sea sediments in the northeastern Baltic Sea. The objective was to evaluate how much of the sediment P is buried and removed from the nutrient cycle, and how much of it is in forms that can be released from the sediment to the overlaying water over time. Relationships between the distribution of the different P forms and the chemical and physical properties of the sediment, sediment pore water, and near-bottom water were determined in order to examine the behaviour of P at the sediment-water interface. The results show that the chemical character of sediment P varied in the different areas. Generally, in the outer estuaries and in the organic-rich coastal areas in the eastern Gulf of Finland, the sediments were higher in P than the sediments in the poorly oxygenated open sea areas in the central and western Gulf. The estuary sediments that received erosion-transported material were characterised by P bound to hydrated oxides of iron and aluminum. Iron-bound P is sensitive to changes in redox-conditions, but part of it was buried in the estuaries, possibly because of high sedimentation rates and incomplete reduction of iron. The open sea sediments in the central and western Gulf of Finland were dominated by apatite-P, which was also abundant in the areas strongly affected by sediment transportation. The burial of sediment P was most effective in the areas rich in apatite-P, which is a relatively stable form of P in sediment. In the eastern Gulf of Finland, organic P forms predominated in the organic-rich sediments. A part of these P forms will be buried, while part will be degraded in the long term, releasing soluble P to the pore water. In the poorly oxygenated areas, iron compounds at the sediment surface are not able to retain P released during mineralisation of organic matter or reduction-induced dissolution of iron-compounds in deep sediment layers. However, in the shallow coastal areas overlain by oxic near-bottom water, the organic-rich surface sediment can also become temporarily reduced and release P from the sediment to the overlaying water. The considerable variation in the chemical composition of sediment P reserves in the northeastern Baltic Sea proved that it is an important factor and should be taken into account when evaluating the release of sediment P and the role of P reserves in bottom sediments in eutrophication.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Computational studies on new chemical species in the gas-phase and the solid-state.

There is intense activity in the area of theoretical chemistry of gold. It is now possible to predict new molecular species, and more recently, solids by combining relativistic methodology with isoelectronic thinking. In this thesis we predict a series of solid sheet-type crystals for Group-11 cyanides, MCN (M=Cu, Ag, Au), and Group-2 and 12 carbides MC2 (M=Be-Ba, Zn-Hg). The idea of sheets is then extended to nanostrips which can be bent to nanorings. The bending energies and deformation frequencies can be systematized by treating these molecules as an elastic bodies. In these species Au atoms act as an 'intermolecular glue'. Further suggested molecular species are the new uncongested aurocarbons, and the neutral Au_nHg_m clusters. Many of the suggested species are expected to be stabilized by aurophilic interactions. We also estimate the MP2 basis-set limit of the aurophilicity for the model compounds [ClAuPH_3]_2 and [P(AuPH_3)_4]^+. Beside investigating the size of the basis-set applied, our research confirms that the 19-VE TZVP+2f level, used a decade ago, already produced 74 % of the present aurophilic attraction energy for the [ClAuPH_3]_2 dimer. Likewise we verify the preferred C4v structure for the [P(AuPH_3)_4]^+ cation at the MP2 level. We also perform the first calculation on model aurophilic systems using the SCS-MP2 method and compare the results to high-accuracy CCSD(T) ones. The recently obtained high-resolution microwave spectra on MCN molecules (M=Cu, Ag, Au) provide an excellent testing ground for quantum chemistry. MP2 or CCSD(T) calculations, correlating all 19 valence electrons of Au and including BSSE and SO corrections, are able to give bond lengths to 0.6 pm, or better. Our calculated vibrational frequencies are expected to be better than the currently available experimental estimates. Qualitative evidence for multiple Au-C bonding in triatomic AuCN is also found.

Molecular Interactions Chlorin Assemblies and NMR Separations

Photosynthesis is a chemical process in which the energy of the light quanta is transformed into chemical energy. Chlorophyll (Chl) molecules play a key role in photosynthesis; they function in the antennae systems and in the photosynthetic reaction center where the primary charge separation (CS) takes place. Bio-inspired mimicry of the CS is an essential unit in dye-sensitized solar cells. Aim of this study was to design and develop electron donor-acceptor (EDA) pairs from Chls and fullerenes (C60) or carbon nanotubes (CNT). The supramolecular approach was chosen, as long synthetic sequences required by the covalent approach lead to long reaction schemes and low yields. Here, a π-interaction between soluble CNTs and Chl was used in EDA construction. Also, a beta-face selective two-point bound Chl-C60 EDA was introduced. In addition, the photophysical properties of the supramolecular EDA dyads were analyzed. In organic chemistry, nuclear magnetic resonance (NMR) spectroscopy is the most vital analytical technique in use. Multi-dimensional NMR experiments have enabled a structural analysis of complex natural products and proteins. However, in mixture analysis NMR is still facing difficulties. In many cases overlapping signals can t be resolved even with the help of multi-dimensional experiments. In this work, an NMR tool based on simple host-guest chemistry between analytes and macromolecules was developed. Diffusion ordered NMR spectroscopy (DOSY) measures the mobilities of compounds in an NMR sample. In a liquid state NMR sample, each of the analytes has a characteristic diffusion coefficient, which is proportional to the size of the analyte. With normal DOSY experiment, provided that the diffusion coefficients of the analytes differ enough, individual spectra of analytes can be extracted. When similar sized analytes differ chemically, an additive can be introduced into the sample. Since macromolecules in a liquid state NMR sample can be considered practically stationary, even faint supramolecular interaction can change the diffusion coefficient of the analyte sufficiently for a successful resolution in DOSY. In this thesis, polyvinylpyrrolidone and polyethyleneglycol enhanced DOSY NMR techniques, which enable mixture analysis of similar in size but chemically differing natural products, are introduced.

Chemical mass closure and source-specific composition of atmospheric particles

It has been known for decades that particles can cause adverse health effects as they are deposited within the respiratory system. Atmospheric aerosol particles influence climate by scattering solar radiation but aerosol particles act also as the nuclei around which cloud droplets form. The principal objectives of this thesis were to investigate the chemical composition and the sources of fine particles in different environments (traffic, urban background, remote) as well as during some specific air pollution situations. Quantifying the climate and health effects of atmospheric aerosols is not possible without detailed information of the aerosol chemical composition. Aerosol measurements were carried out at nine sites in six countries (Finland, Germany, Czech, Netherlands, Greece and Italy). Several different instruments were used in order to measure both the particulate matter (PM) mass and its chemical composition. In the off-line measurements the samples were collected first on a substrate or filter and gravimetric and chemical analysis were conducted in the laboratory. In the on-line measurements the sampling and analysis were either a combined procedure or performed successively within the same instrument. Results from the impactor samples were analyzed by the statistical methods. This thesis comprises also a work where a method for the determination carbonaceous matter size distribution by using a multistage impactor was developed. It was found that the chemistry of PM has usually strong spatial, temporal and size-dependent variability. In the Finnish sites most of the fine PM consisted of organic matter. However, in Greece sulfate dominated the fine PM and in Italy nitrate made the largest contribution to the fine PM. Regarding the size-dependent chemical composition, organic components were likely to be enriched in smaller particles than inorganic ions. Data analysis showed that organic carbon (OC) had four major sources in Helsinki. Secondary production was the major source in Helsinki during spring, summer and fall, whereas in winter biomass combustion dominated OC. The significant impact of biomass combustion on OC concentrations was also observed in the measurements performed in Central Europe. In this thesis aerosol samples were collected mainly by the conventional filter and impactor methods which suffered from the long integration time. However, by filter and impactor measurements chemical mass closure was achieved accurately, and a simple filter sampling was found to be useful in order to explain the sources of PM on the seasonal basis. The online instruments gave additional information related to the temporal variations of the sources and the atmospheric mixing conditions.

Quantum Chemical Studies of Intermolecular Interactions

This thesis studies the intermolecular interactions in (i) boron-nitrogen based systems for hydrogen splitting and storage, (ii) endohedral complexes, A@C60, and (iii) aurophilic dimers. We first present an introduction of intermolecular interactions. The theoretical background is then described. The research results are summarized in the following sections. In the boron-nitrogen systems, the electrostatic interaction is found to be the leading contribution, as 'Coulomb Pays for Heitler and London' (CHL). For the endohedral complex, the intermolecular interaction is formulated by a one-center expansion of the Coulomb operator 1/rab. For the aurophilic attraction between two C2v monomers, a London-type formula was derived by fully accounting for the anisotropy and point-group symmetry of the monomers.

Autoxidation of conjugated linoleic acid methyl ester in the presence of α-tocopherol: the hydroperoxide pathway

The autoxidation of conjugated linoleic acid (CLA) is poorly understood in spite of increasing interest in the beneficial biological properties of CLA and growing consumption of CLA-rich foods. In this thesis, the autoxidation reactions of the two major CLA isomers, 9-cis,11-trans-octadecadienoic acid and 10-trans,12-cis-octadecadienoic acid, are investigated. The results contribute to an understanding of the early stages of the autoxidation of CLA methyl ester, and provide for the first time a means of producing and separating intact CLA methyl ester hydroperoxides as well as basic knowledge on lipid hydroperoxides and their hydroxy derivatives. Conjugated diene allylic monohydroperoxides were discovered as primary autoxidation products formed during autoxidation of CLA methyl esters in the presence and absence of α-tocopherol. This established that one of the autoxidation pathways of CLA methyl ester is the hydroperoxide pathway. Hydroperoxides were produced from the two major CLA methyl esters by taking advantage of the effect of α-tocopherol to promote hydroperoxide formation. The hydroperoxides were analysed and separated first as methyl hydroxyoctadecadienoates and then as intact hydroperoxides by HPLC. The isolated products were characterized by UV, GC-MS, and NMR techniques. In the presence of a high amount of α-tocopherol, the autoxidation of CLA methyl ester yields six kinetically-controlled conjugated diene monohydroperoxides and is diastereoselective in favour of one particular geometric isomer as a pair of enantiomers. The primary autoxidation products produced from the two major CLA isomers include new positional isomers of conjugated diene monohydroperoxides, the 8-, 10-, 12-, and 14-hydroperoxyoctadecadienoates. Furthermore, two of these new positional isomers have an unusual structure for a cis,trans lipid hydroperoxide where the allylic methine carbon is adjacent to the cis instead of the usual trans double bond. The 1H and 13C NMR spectra of nine isomeric methyl hydroxyoctadecadienoates and of ten isomeric methyl hydroperoxyoctadecadienoates including the unusual cis,trans hydroperoxides, i.e. Me 8-OOH-9c,11t and Me 14-OOH-10t,12c, were fully assigned with the aid of 2D NMR spectroscopy. The assigned NMR data enabled determination of the effects of the hydroxyl and hydroperoxyl groups on the carbon chemical shifts of CLA isomers, identification of diagnostic signals, and determination of chemical shift differences of the olefinic resonances that may help with the assignment of structure to as yet unknown lipid hydroperoxides either as hydroxy derivatives or as intact hydroperoxides. A mechanism for the hydroperoxide pathway of CLA autoxidation in the presence of a high amount of α-tocopherol was proposed based on the characterized primary products, their relative distribution, and theoretical calculations. This is an important step forward in CLA research, where exact mechanisms for the autoxidation of CLA have not been presented before. Knowledge of these hydroperoxide formation steps is of crucial importance for understanding the subsequent steps and the different pathways of the autoxidation of CLA. Moreover, a deeper understanding of the autoxidation mechanisms is required for ensuring the safety of CLA-rich foods. Knowledge of CLA oxidation and how it differs from the oxidation of nonconjugated polyunsaturated fatty acids may also be the key to understanding the biological mechanisms of CLA activity.

Chemical and source characterisation of size-segregated urban air particulate matter

Recent epidemiological studies have shown a consistent association of the mass concentration of urban air thoracic (PM10) and fine (PM2.5) particles with mortality and morbidity among cardiorespiratory patients. However, the chemical characteristics of different particulate size ranges and the biological mechanisms responsible for these adverse health effects are not well known. The principal aims of this thesis were to validate a high volume cascade impactor (HVCI) for the collection of particulate matter for physicochemical and toxicological studies, and to make an in-depth chemical and source characterisation of samples collected during different pollution situations. The particulate samples were collected with the HVCI, virtual impactors and a Berner low pressure impactor in six European cities: Helsinki, Duisburg, Prague, Amsterdam, Barcelona and Athens. The samples were analysed for particle mass, common ions, total and water-soluble elements as well as elemental and organic carbon. Laboratory calibration and field comparisons indicated that the HVCI can provide a unique large capacity, high efficiency sampling of size-segregated aerosol particles. The cutoff sizes of the recommended HVCI configuration were 2.4, 0.9 and 0.2 μm. The HVCI mass concentrations were in a good agreement with the reference methods, but the chemical composition of especially the fine particulate samples showed some differences. This implies that the chemical characterization of the exposure variable in toxicological studies needs to be done from the same HVCI samples as used in cell and animal studies. The data from parallel, low volume reference samplers provide valuable additional information for chemical mass closure and source assessment. The major components of PM2.5 in the virtual impactor samples were carbonaceous compounds, secondary inorganic ions and sea salt, whereas those of coarse particles (PM2.5-10) were soil-derived compounds, carbonaceous compounds, sea salt and nitrate. The major and minor components together accounted for 77-106% and 77-96% of the gravimetrically-measured masses of fine and coarse particles, respectively. Relatively large differences between sampling campaigns were observed in the organic carbon content of the PM2.5 samples as well as the mineral composition of the PM2.5-10 samples. A source assessment based on chemical tracers suggested clear differences in the dominant sources (e.g. traffic, residential heating with solid fuels, metal industry plants, regional or long-range transport) between the sampling campaigns. In summary, the field campaigns exhibited different profiles with regard to particulate sources, size distribution and chemical composition, thus, providing a highly useful setup for toxicological studies on the size-segregated HVCI samples.

A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.