Variation in tracheid cross-sectional dimensions and wood viscoelasticity extent and control methods

Printing papers have been the main product of the Finnish paper industry. To improve properties and economy of printing papers, controlling of tracheid cross-sectional dimensions and wood viscoelasticity are examined in this study. Controlling is understood as any procedure which yields raw material classes with distinct properties and small internal variation. Tracheid cross-sectional dimensions, i.e., cell wall thickness and radial and tangential diameters can be controlled with methods such as sorting wood into pulpwood and sawmill chips, sorting of logs according to tree social status and fractionation of fibres. These control methods were analysed in this study with simulations, which were based on measured tracheid cross-sectional dimensions. A SilviScan device was used to measure the data set from five Norway spruce (Picea abies) and five Scots pine (Pinus sylvestris) trunks. The simulation results indicate that the sawmill chips and top pulpwood assortments have quite similar cross-sectional dimensions. Norway spruce and Scots pine are on average also relatively similar in their cross-sectional dimensions. The distributions of these species are somewhat different, but from a practical point of view, the differences are probably of minor importance. The controlling of tracheid cross-sectional dimensions can be done most efficiently with methods that can separate fibres into earlywood and latewood. Sorting of logs or partitioning of logs into juvenile and mature wood were markedly less efficient control methods than fractionation of fibres. Wood viscoelasticity affects energy consumption in mechanical pulping, and is thus an interesting control target when improving energy efficiency of the process. A literature study was made to evaluate the possibility of using viscoelasticity in controlling. The study indicates that there is considerable variation in viscoelastic properties within tree species, but unfortunately, the viscoelastic properties of important raw material lots such as top pulpwood or sawmill chips are not known. Viscoelastic properties of wood depend mainly on lignin, but also on microfibrillar angle, width of cellulose crystals and tracheid cross-sectional dimensions.

Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

Kidney Induction: control by Notch, Wnt and GDNF/Ret signalling

The permanent mammalian kidney (metanephros) develops as a result of complex reciprocal tissue interactions between a ureteric epithelium and the renal mesenchyme. The overall goal of the research in this thesis was to gain data that will eventually help in elucidating the formation of congenital renal malformations. The experiments in my thesis aimed to reveal the mechanisms by which Notch, Wnt and GDNF/Ret signalling pathways regulate the development of functional kidney. The function of Notch pathway was studied by a transgenic mouse model, where it was shown that overactivation of Notch signalling disturbs kidney development and alters the expression of Gdnf and Ret/GFRa1. This indicates that Notch signalling interplays with GDNF/Ret in the regulation of the primary ureteric budding and its subsequent branching. The data also suggested that strict spatio-temporal regulation of these two pathways is required for determination of ureteric tip-identity, which appeared to be crucial for the branch formation. The function of Wnt signalling in the ureteric morphogenesis was studied by in vivo and in vitro methods to show that a canonical pathway is required for ureteric branching. Stabilisation and deletion of the canonical pathway mediator, b-catenin specifically in the ureteric epithelium result in renal aplasia/hypodysplasia. These defects originate from severe blockage of ureteric branching due to the disrupted Ret signalling. Consequently, ureteric tip specific markers are lost and ureteric stalk identity is expanded throughout the whole epithelium. Thus, the data demonstrates that the Wnt/b-catenin pathway plays an essential role in the patterning and branching of the ureteric epithelium. A novel in vitro method was generated and utilised in nephron induction studies to reveal the mechanisms through which nephrogenesis is induced. Transient GSK3 inhibition results in stabilisation of b-catenin in the isolated renal mesenchyme, which efficiently triggers nephron formation. Also genetic stabilisation of b-catenin specifically in the mesenchyme results in spontaneous nephrogenesis. The results show that activation of the canonical Wnt pathway is sufficient to initiate nephrogenesis, and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes. Taken together, this thesis demonstrates Notch and Wnt signalling pathways as novel regulators of ureteric branching morphogenesis, and that activation of the canonical Wnt pathway is sufficient for nephron induction. The studies also indicate that the Notch and Wnt pathways cross-talk with GDNF/Ret signalling in the patterning of ureteric epithelium.

To reactivate or not to reactivate : Control of KSHV lytic replication is essential for apoptosis in response to p53 restoration

Kaposi's sarcoma herpesvirus (KSHV) is an oncogenic human virus and the causative agent of three human malignancies: Kaposi's sarcoma (KS), Multicentric Castleman's Disease (MCD), and primary effusion lymphoma (PEL). In tumors, KSHV establishes latent infection during which it produces no infectious particles. Latently infected cells can enter the lytic replication cycle, and upon provision of appropriate cellular signals, produce progeny virus. PEL, commonly described in patients with AIDS, represents a diffuse large-cell non-Hodgkin's lymphoma, with median survival time less than six months after diagnosis. As tumor suppressor gene TP53 mutations occur rarely in PEL, the aim of this thesis was to investigate whether non-genotoxic activation of the p53 pathway can eradicate malignant PEL cells. This thesis demonstrates that Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction, efficiently restored p53 function in PEL cells, leading to cell cycle arrest and massive apoptosis. Furthermore, we found that KSHV infection activated DNA damage signaling, rendering the cells more sensitive to p53-dependent cell death. We also showed in vivo the therapeutic potential of p53 restoration that led to regression of subcutaneous and intraperitoneal PEL tumor xenografts without adversely affecting normal cells. Importantly, we demonstrated that in a small subset of intraperitoneal PEL tumors, spontaneous induction of viral reactivation dramatically impaired Nutlin-3-induced p53-mediated apoptosis. Accordingly, we found that elevated KSHV lytic transcripts correlated with PEL tumor burden in animals and that inhibition of viral reactivation in vitro restored cytotoxic activity of a small-molecule inhibitor of the p53-MDM2 interaction. Latency provides a unique opportunity for KSHV to escape host immune surveillance and to establish persistent infections. However, to maintain viral reservoirs and spread to other hosts, KSHV must be reactivated from latency and enter into the lytic growth phase. We showed that phosphorylation of nucleolar phosphoprotein nucleophosmin (NPM) by viral cyclin-CDK6 is critical for establishment and maintenance of the KSHV latency. In short, this study provides evidence that the switch between latent phase and lytic replication is a critical step that determines the outcome of viral infection and the pathogenesis of KSHV-induced malignancies. Our data may thus contribute to development of novel targeted therapies for intervention and treatment of KSHV-associated cancers.

Impact of Tobacco Control Policy on Smoking and Exposure to Environmental Tobacco Smoke

The aim of the study was to evaluate the impact of the Finnish tobacco control measures for reduction of smoking. First, the trends and patterns in ever smoking among adult Finns in 1978 2001 as well as the associations of trends with the Tobacco Control Act in 1976 were examined. Secondly, the impact of the 1976 TCA on the proportion of ever daily smokers in different socioeconomic groups was studied. Thirdly, the impact of the 1995 TCAA on recent trends in the prevalence of daily smoking was evaluated by gender and employment status. Fourthly, the trends of exposure to environmental tobacco smoke (ETS) at workplaces and homes were investigated. The study is based on data of the Health Behaviour among the Finnish Adult Population surveys. Among Finnish men smoking initiation declined from earlier to later cohorts, whereas among women it increased in successive birth cohorts born before 1956. The lasting differences between birth cohorts as regards ever daily smoking reflected well the impact of measures to reduce smoking in Finland in 1976. Smoking initiation in the birth cohorts (born in 1961 or later) which were in critical age as regards the risk of smoking initiation when the TCA came into force was less common than could be expected according to the trends seen in the earlier birth cohorts. Marked socioeconomic differences were found in smoking in the different birth cohorts. Smoking was more prevalent in the lower socioeconomic groups than in the higher ones, and the differences were larger in the later birth cohorts compared to the earlier ones. The differences between the birth cohorts in ever daily smoking were compatible with the hypothetical impact of the TCA in almost all socioeconomic groups, except farmers. Among men the 1976 TCA appears to have had the greatest impact on white-collar employees. Among women the effect of the act was highly significant in all socioeconomic groups. However, female smoking prevalence continues to show wide socioeconomic disparities. Daily smoking decreased among employees after the 1995 TCAA, supporting the hypothesis of the lowering impact of the amendment on daily smoking due to increased smoking cessation. No parallel change in daily smoking was found in the population without direct expose to ETS legislation (farmers, students, housewives, pensioners or unemployed). Exposure to ETS decreased markedly among non-smokers at work after the 1995 TCAA. The 1976 TCA and the 1995 TCAA were useful in controlling smoking initiation and cessation, but their impact was not equal across the population groups. The results of this study strongly suggested that tobacco control policies markedly contribute to the decrease in smoking and in exposure to environmental tobacco smoke.

Latent TGF-β binding proteins -3 and -4 : transcriptional control and extracellular matrix targeting

Extracellular matrix (ECM) is a complex network of various proteins and proteoglycans which provides tissues with structural strength and resilience. By harvesting signaling molecules like growth factors ECM has the capacity to control cellular functions including proliferation, differentiation and cell survival. Latent transforming growth factor β (TGF-β) binding proteins (LTBPs) associate fibrillar structures of the ECM and mediate the efficient secretion and ECM deposition of latent TGF-β. The current work was conducted to determine the regulatory regions of LTBP-3 and -4 genes to gain insight into their tissue-specific expression which also has impact on TGF-β biology. Furthermore, the current research aimed at defining the ECM targeting of the N-terminal variants of LTBP-4 (LTBP-4S and -4L), which is required to understand their functions in tissues and to gain insight into conditions in which TGF-β is activated. To characterize the regulatory regions of LTBP-3 and -4 genes in silico and functional promoter analysis techniques were employed. It was found that the expression of LTBP-4S and -4L are under control of two independent promoters. This finding was in accordance with the observed expression patterns of LTBP-4S and -4L in human tissues. All promoter regions characterized in this study were TATAless, GC-rich and highly conserved between human and mouse species. Putative binding sites for Sp1 and GATA family of transcription factors were recognized in all of these regulatory regions. It is possible that these transcription factors control the basal expression of LTBP-3 and -4 genes. Smad binding element was found within the LTBP-3 and -4S promoter regions, but it was not present in LTBP-4L promoter. Although this element important for TGF-β signaling was present in LTBP-4S promoter, TGF-β did not induce its transcriptional activity. LTBP-3 promoter activity and mRNA expression instead were stimulated by TGF-β1 in osteosarcoma cells. It was found that the stimulatory effect of TGF-β was mediated by Smad and Erk MAPK signaling pathways. The current work explored the ECM targeting of LTBP-4S and identified binding partners of this protein. It was found that the N-terminal end of LTBP-4S possesses fibronectin (FN) binding sites which are critical for its ECM targeting. FN deficient fibroblasts incorporated LTBP-4S into their ECM only after addition of exogenous FN. Furthermore, LTBP-4S was found to have heparin binding regions, of which the C-terminal binding site mediated fibroblast adhesion. Soluble heparin prevented the ECM association of LTBP-4S in fibroblast cultures. In the current work it was observed that there are significant differences in the secretion, processing and ECM targeting of LTBP-4S and -4L. Interestingly, it was observed that most of the secreted LTBP-4L was associated with latent TGF-β1, whereas LTBP-4S was mainly secreted as a free form from CHO cells. This thesis provides information on transcriptional regulation of LTBP-3 and -4 genes, which is required for the deeper understanding of their tissue-specific functions. Further, the current work elucidates the structural variability of LTBPs, which appears to have impact on secretion and ECM targeting of TGF-β. These findings may advance understanding the abnormal activation of TGF-β which is associated with connective tissue disorders and cancer.

Interactions between transgenic trees and mycorrhizal and pathogenic fungi

The development of biotechnology techniques in plant breeding and the new commercial applications have raised public and scientific concerns about the safety of genetically modified (GM) crops and trees. To find out the feasibility of these new technologies in the breeding of commercially important Finnish hardwood species and to estimate the ecological risks of the produced transgenic plants, the experiments of this study have been conducted as a part of a larger project focusing on the risk assessment of GM-trees. Transgenic Betula pendula and Populus trees were produced via Agrobacterium mediated transformation. Stilbene synthase (STS) gene from pine (Pinus sylvestris) and chitinase gene from sugar beet (Beta vulgaris) were transferred to (hybrid) aspen and birch, respectively, to improve disease resistance against fungal pathogens. To modify lignin biosynthesis, a 4-coumarate:coenzyme A ligase (4CL) gene fragment in antisense orientation was introduced into two birch clones. In in vitro test, one transgenic aspen line expressing pine STS gene showed increased resistance to decay fungus Phellinus tremulae. In the field, chitinase transgenic birch lines were more susceptible to leaf spot (Pyrenopeziza betulicola) than the non-transgenic control clone while the resistance against birch rust (Melampsoridium betulinum) was improved. No changes in the content or composition of lignin were detected in the 4CL antisense birch lines. In order to evaluate the ecological effects of the produced GM trees on non-target organisms, an in vitro mycorrhiza experiment with Paxillus involutus and a decomposition experiment in the field were performed. The expression of a transgenic chitinase did not disturb the establishment of mycorrhizal symbiosis between birch and P. involutus in vitro. 4CL antisense transformed birch lines showed retarded root growth but were able to form normal ectomycorrhizal associations with the mycorrhizal fungus in vitro. 4CL lines also showed normal litter decomposition. Unexpected growth reductions resulting from the gene transformation were observed in chitinase transgenic and 4CL antisense birch lines. These results indicate that genetic engineering can provide a tool in increasing disease resistance in Finnish tree species. More extensive data with several ectomycorrhizal species is needed to evaluate the consequences of transgene expression on beneficial plant-fungus symbioses. The potential pleiotropic effects of the transgene should also be taken into account when considering the safety of transgenic trees.

Biological Control of Oilseed Rape Pests with Entomopathogenic Nematodes

Biological control techniques attract increasing attention as one of the sustainable alternatives to pesticide use in integrated pest management programs. In order to develop sustainable pest management methods for arable crops based on entomopathogenic nematodes (EPN), their efficacy and persistence needed to be investigated, and an economically feasible delivery system had to be developed. In this study, first a survey of entomopathogens was conducted, and a system approach was tested, using the oilseed Brassica (OSB) growing system (OSB, spring wheat, and red clover) as a model. The system approach aimed at determining the potential of Steinernema feltiae (Filipjev) for the control of OSB pests, developing OSB rotation schemes that support EPN persistence, and investigating the impact of the selected biotic and abiotic factors on efficacy and persistence of EPN. This study employed abductive logic (which employs constant interplay between the theory and empirical observation), quantitative methods, and a case study on OSB. Laboratory and field experiments were carried out, and two types of pathogen surveys. A horizontal survey included OSB fields across Estonia, Germany, Poland, Sweden and the UK, while a vertical survey included sampling from two sets of differently managed experimental fields during three years. A new approach was introduced for measuring occurrence, where the prevalence and relative intensity of entomopathogens, biotic agents, and unidentified insect antagonists were determined. The effect of dose, timing, and the application method on S. feltiae in the control of pests in OSB, and the potential of a controlled release delivery system (CRS) were evaluated in the field. Studies on the impact of selected biotic and abiotc factors (Brassica plant, bait insects, developmental stages of Meligethes aeneus Fab., Isaria fumosorosea Wize (Ifr), and organic and synthetic fertilizers) on the efficacy of S. feltiae were conducted in the laboratory. Persistence of S. feltiae in the OSB growing system, and the effect of dose, timing, and the application method, was assessed in the field as part of the efficacy experiments. The impact of selected biotic and abiotic factors on S. feltiae persistence was assessed in laboratory experiments. The pathogen survey showed that the occurrence of entomopathogens is low in the OSB growing system, and that a management system causing less disturbance (ICM) to the soil increases the relative intensity of insect parasitic nematodes and other insect antagonists. A longer study period is required to show any possible impact of ICM on the relative intensity of entomopathogenic fungi, or on the prevalence of entomopathogens. Two different measures of the occurrence yielded different results: the relative intensity revealed the difference between the two different crop management methods, while prevalence did not. The highest efficacy of S. feltiae was achieved by using a low dose and targeting all stages of M. aeneus. When only the larval stage was targeted, the application method and dose had no significant effect. The CRS decreased the pest abundance significantly more than the surface application method. S. feltiae persisted in the OSB fields in Finland for several months, but did not survive the winter. The strain survived for 7 months when it was applied in autumn in Germany, but its populations declined rapidly after winter. The examined biotic and abiotic factors had variable impacts on S. feltiae efficacy and persistence. The two measures, prevalence and relative intensity of entomopathogens, gave valuable information for their use in biocontrol programs. The recommended biocontrol strategy for OSB growing in Finland is inundation and seasonal inoculation of EPN. The impact of some biotic and abiotic factors on S. feltiae efficacy and persistence is significant, and can be used to improve the efficacy of EPN. The CRS is a novel alternative for EPN application, and should also be considered for use on other crops. Keywords: Biological control, inundation, inoculation, conservation, formulation, slow release method, crop rotation, Entomopathogenic nematodes, Steinernema feltiae, oilseed rape pests, Meligethes aeneus, Phyllotreta spp., occurrence, prevalence, intensity, efficacy, persistence, field, Isaria fumosorosea, biotic factors, abiotic factors, interaction, impact, insect stages, integrated crop management, standard (conventional) crop management

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

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.

Characterization of lignin-modifying enzymes of the selective white-rot fungus Physisporinus rivulosus

White-rot fungi are wood degrading organisms that are able to decompose all wood polymers; lignin, cellulose and hemicellulose. Especially the selective white-rot fungi that decompose preferentially wood lignin are promising for biopulping applications. In biopulping the pretreatment of wood chips with white-rot fungi enhances the subsequent pulping step and substantially reduces the refining energy consumption in mechanical pulping. Because it is not possible to carry out biopulping in industrial scale as a closed process it has been necessary to search for new selective strains of white-rot fungi which naturally occur in Finland and cause selective white-rot of Finnish wood raw-material. In a screening of 300 fungal strains a rare polypore, Physisporinus rivulosus strain T241i isolated from a forest burn research site, was found to be a selective lignin degrader and promising for the use in biopulping. Since selective lignin degradation is apparently essential for biopulping, knowledge on lignin-modifying enzymes and the regulation of their production by a biopulping fungus is needed. White-rot fungal enzymes that participate in lignin degradation are laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP) and hydrogen peroxide forming enzymes. In this study, P. rivulosus was observed to produce MnP, laccase and oxalic acid during growth on wood chips. In liquid cultures manganese and veratryl alcohol increased the production of acidic MnP isoforms detected also in wood chip cultures. Laccase production by P. rivulosus was low unless the cultures were supplemented with sawdust and charred wood, the components of natural growth environment of the fungus. In white-rot fungi the lignin-modifying enzymes are typically present as multiple isoforms. In this study, two MnP encoding genes, mnpA and mnpB, were cloned and characterized from P. rivulosus T241i. Analysis of the N-terminal amino acid sequences of two purified MnPs and putative amino acid sequence of the two cloned mnp genes suggested that P. rivulosus possesses at least four mnp genes. The genes mnpA and mnpB markedly differ from each other by the gene length, sequence and intron-exon structure. In addition, their expression is differentially affected by the addition of manganese and veratryl alcohol. P. rivulosus produced laccase as at least two isoforms. The results of this study revealed that the production of MnP and laccase was differentially regulated in P. rivulosus, which ensures the efficient lignin degradation under a variety of environmental conditions.