Microbial quality of hemp (Cannabis sativa L.) and flax (Linum usitatissimum L.) from plants to thermal insulation

Flax and hemp have traditionally been used mainly for textiles, but recently interest has also been focused on non-textile applications. Microbial quality throughout the whole processing chain of bast fibres has not previously been studied. This study concentrates on the microbial quality and possible microbial risks in the production chain of hemp and flax fibres and fibrous thermal insulations. In order to be able to utilize hemp and flax fibres, the bast fibres must be separated from the rest of the plant. Non-cellulosic components can be removed with various pretreatment processes, which are associated with a certain risk of microbial contamination. In this study enzymatic retting and steam explosion (STEX) were examined as pretreatment processes. On the basis of the results obtained in this study, the microbial contents on stalks of both plants studied increased at the end of the growing season and during the winter. However, by processing and mechanical separation it is possible to produce fibres containing less moulds and bacteria than the whole stem. Enzymatic treatment encouraged the growth of moulds in fibres. Steam explosion reduced the amount of moulds in fibres. Dry thermal treatment used in this study did not markedly reduce the amount of microbes. In this project an emission measurement chamber was developed which was suitable for measurements of emissions from both mat type and loose fill type insulations, and capable of interdisciplinary sampling. In this study, the highest amounts of fungal emissions were in the range of 10^3 10^5 cfu/m^3 from the flax and hemp insulations at 90% RH of air. The fungal emissions from stone wool, glass wool and recycled paper insulations were below 10^2 cfu/m^3 even at 90% RH. Equally low values were obtained from bast fibrous materials in lower humidities (at 30% and 80% RH of air). After drying of moulded insulations at 30% RH, the amounts of emitted moulds were in all cases higher compared to the emissions at 90% RH before drying. The most common fungi in bast fibres were Penicillium and Rhizopus. The widest variety of different fungi was in the untreated hemp and linseed fibres and in the commercial loose-fill flax insulation. Penicillium, Rhizopus and Paecilomyces were the most tolerant to steam explosion. According to the literature, the most common fungi in building materials and indoor air are Penicillium, Aspergillus and Cladosporium, which were all found in some of the bast fibre materials in this study. As organic materials, hemp and flax fibres contain high levels of nutrients for microbial growth. The amount of microbes can be controlled and somewhat decreased by the processing methods presented.

Genetic Diversity and Adaptation of Date Palm (Phoenix dactylifera L.)

Acquiring sufficient information on the genetic variation, genetic differentiation, and the ecological and genetic relationships among individuals and populations are essential for establishing guidelines on conservation and utilization of the genetic resources of a species, and more particularly when biotic and abiotic stresses are considered. The aim of this study was to assess the extent and pattern of genetic variation in date palm (Phoenix dacttylifera L) cultivars; the genetic diversity and structure in its populations occurring over geographical ranges; the variation in economically and botanically important traits of it and the variation in its drought adaptive traits, in conservation and utilization context. In this study, the genetic diversity and relationships among selected cultivars from Sudan and Morocco were assessed using microsatellite markers. Microsatellite markers were also used to investigate the genetic diversity within and among populations collected from different geographic locations in Sudan. In a separate investigation, fruits of cultivars selected from Sudan, involved morphological and chemical characterization, and morphological and DNA polymorphism of the mother trees were also investigated. Morphological and photosynthetic adjustments to water stress were studied in the five most important date palm cultivars in Sudan, namely, Gondaila, Barakawi, Bitamoda, Khateeb and Laggai; and the mechanism enhancing photosynthetic gas exchange in date palm under water stress was also investigated. Results showed a significant (p < 0.001, t-test) differentiation between Sudan and Morocco groups of cultivars. However, the major feature of all tested cultivars was the complete lack of clustering and the absence of cultivars representing specific clones. The results indicated high genetic as well as compositional and morphological diversity among cultivars; while, compositional and morphological traits were found to be characteristic features that strongly differentiate cultivars as well as phenotypes. High genetic diversity was observed also in different populations. Slight but significant (p < 0.01, AMOVA) divergence was observed for soft and dry types; however, the genetic divergence among populations was relatively weak. The results showed a complex genetic relationships between some of the tested populations especially when isolation by distance was considered. The results of the study also revealed that date palm cultivars and phenotypes possess specific direct or interaction effects due to water availability on a range of morphological and physiological traits. Soft and dry phenotypes responded differently to different levels of water stress, while the dry phenotype was more sensitive and conservative. The results indicated that date palm has high fixation capacity to photosynthetic CO2 supply with interaction effect to water availability, which can be considered as advantageous when coping with stresses that may arise with climate change. In conclusion, although a large amount of diversity exists among date palm germplasm, the findings in this study show that the role of biological nature of the tree, isolation by distance and environmental effects on structuring date palm genome was highly influenced by human impacts. Identity of date palm cultivars as developed and manipulated by date palm growers, in the absence of scientific breeding programmes, may continue to mainly depend on tree morphology and fruit characters. The pattern of genetic differentiation may cover specific morphological and physiological traits that contribute to adaptive mechanisms in each phenotype. These traits can be considered for further studies related to drought adaptation in date palm.

Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

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.

L-rhamnose-1-dehydrogenase gene and L-rhamnose catabolism in the yeast Pichia stipitis

The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

Jumping numbers of a simple complete ideal in a two-dimensional regular local ring

The multiplier ideals of an ideal in a regular local ring form a family of ideals parametrized by non-negative rational numbers. As the rational number increases the corresponding multiplier ideal remains unchanged until at some point it gets strictly smaller. A rational number where this kind of diminishing occurs is called a jumping number of the ideal. In this manuscript we shall give an explicit formula for the jumping numbers of a simple complete ideal in a two dimensional regular local ring. In particular, we obtain a formula for the jumping numbers of an analytically irreducible plane curve. We then show that the jumping numbers determine the equisingularity class of the curve.

Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

GDP-L-fucose: synthesis and role in inflammation

GDP-L-fucose: synthesis and role in inflammation The migration of leukocytes from intravascular locations to extravascular sites is essential to the immune responses. The initial attachment of leukocytes to the endothelium and the rolling step of the leukocyte extravasation cascade are mediated by selectins, a family of cell adhesion molecules on cell surfaces. Selectins are able to recognize glycoproteins and glycolipids containing the tetrasaccharide sialyl Lewis x (sLex, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc). Several glycosyltransferases are involved in the biosynthesis of sLex, fucosyltransferase VII (Fuc-TVII) being the last enzyme to modify the sLex structure. Fuc-TVII transfers L-fucose from GDP-L-fucose to sialylated N-acetyllactosamine. GDP-L-fucose is synthesized in the cytosol via two different metabolic pathways. The major, constitutively active de novo pathway involves conversion of GDP-α-D-mannose to GDP-β-L-fucose. In the alternative salvage pathway, L-fucokinase synthesizes from free fucose L-fucose-1-phosphate, which is further converted to GDP-L-fucose by GDP-L-fucose pyrophosphorylase. GDP-L-fucose is translocated from the cytosol to Golgi for fucosylation via the GDP-fucose transporter. This thesis involved the study of the synthesis of GDP-L-fucose via the salvage pathway: cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. The gene expression levels of these enzymes were found to be relatively high in various tissues; the mRNA levels were highest in brain, ovary and testis. This study also describes molecular cloning of rat fucosyltransferase VII (FUT7) and its expression as a functional enzyme. Gene expression levels of GDP-L-fucose synthesizing enzymes, GDP-fucose transporter and FUT7 were determined in inflamed tissues as well as cancer cells. Our results revealed a clear upregulation of the enzymes involved in the synthesis of GDP-L-fucose via de novo pathway, GDP-fucose transporter and FUT7 in inflamed tissues and in cancer cells. On the contrary, the GDP-L-fucose salvage pathway was found to be irrelevant in inflammation and in tumorigenesis. Furthermore, our results indicated the transcriptional coregulation of Golgi transporters involved in the synthesis of sulfo sLex, i.e. CMP-sialic acid, GDP-fucose and 3 phosphoadenosine 5 -phosphosulfate transporters, in inflammation.

The ecology of the mountain hare (Lepus timidus L.) in managed boreal forests: habitat associations at different forest scales

Aims of this thesis This study is part of a larger hare project in Finland, which provides answers to basic ecological questions regarding the mountain hare. This study of the ecology of the mountain hare focuses in particular on different levels of managed boreal forest. The feeding habits and intensity of mountain hares in winter are explored, and the connections between mountain hares versus the forest structure are also studied (e.g. habitat use and the importance of different forest layers for hares). The use of the environment by hares at the landscape level was examined (forest patch structures), and the home ranges of mountain hares were studied. Finally, the productivity and survival rate of mountain hare populations were also studied (discussion e.g. predator effects on hare populations). Conclusions Feeding intensity seemed to be highest in the spring-winter, when home ranges were also largest. Favourable food species are covered by snow in winter and the mobility of hares is highest during late winter. A shortage of suitable food species may be problematic for hares, especially during the winter period. In this study mountain hares preferred a dense shrub layer at local level and deciduous and mixed tree forest over coniferous forest at the landscape level. Food and shelter are vital for hares and the preference for particular habitats may also affect the population dynamics of the mountain hare. It would be possible to improve the quality of food and shelter or at least prevent the most negative habitat changes through forest management. At a local level it is also possible to add supplementary food for hares through the winter period. The intensive clearing of young sapling stands and especially the removal of deciduous shrubs and trees reduces the quality of habitats for the mountain hare. Mountain hares primarily live in forest habitat and it is possible that changes in the forest structure play a crucial role in mountain hare habitat preference. Ecological knowledge of the mountain hare is vital to create habitat structure more suitable for the species. More deciduous trees should be saved in managing forests and the mechanical clearing of the shrub layer should be done carefully.