Responses of Scots pine to nickel and copper exposure and herbivory

The goal of this thesis was to examine the ecophysiological responses of Scots pine (Pinus sylvestris L.), with an emphasis on the oxidative enzyme peroxidase and plant phenolics to environmental stresses like elevated levels of nickel (Ni) and copper (Cu), and herbivory. The effects of Ni and Cu were studied in a gradient survey at a sulphur dioxide contaminated site in the Kola Peninsula, and with experiments in which seedlings were exposed to Ni mist or to Ni and Cu amended into the soil. In addition, experimental Ni exposure was combined with disturbance of the natural lichen cover of the forest ground layer. Pine sawfly attack was simulated in the early season defoliation experiment, in which mature Scots pine were defoliated (100 %) during two successive years in a dry, nutrient-poor Scots pine stand. In addition, the effect of previous defoliation on the growth of sawfly (Diprion pini L.) larvae was studied. Apoplastic peroxidase activity was elevated in the needles of pine in a Ni- , Cu- and SO2- polluted environment, which indicated an increased oxidative stress. Increased foliar peroxidase activity due to Ni contamination was shown in the experiment, in which Ni was added as mist. No such response was found in peroxidase acitivity of the roots exposed to elevated Ni and/or Cu in the soil. Elevated Ni in the soil increased the concentration of foliar condensed tannins, which are able to bind heavy metals in the cells. Addition of low levels of Ni in the soil appeared to benefit pine seedlings, which was seen as promoted shoot growth and better condition of the roots. Wet Ni deposition of 2000 mg m-2 reduced growth and survival of pine seedlings, whereas deposition levels 200 mg m-2 or 20 mg m-2 caused no effects in a 2-y lasting experiment. The lichen mat on the forest floor did not act as an effective buffer against the adverse impacts of heavy metals on pine seedlings. However, some evidence was found indicating that soil microbes profited from the lichen mat. Artificial defoliation increased peroxidase activity in the Scots pine needles. In addition, defoliation decreased nitrogen, diamine putrescine and glucose concentrations in the needles and increased the concentrations of several phenolic compounds, starch and sucrose. Previous artificial defoliation led to poor growth of sawfly larvae reared on the pines, suggesting delayed induced resistance in Scots pine. However, there was no consistent relationship between inducibility (proportional increase in a compound following defoliation) and adverse effects on the growth of pine sawfly larvae. The observed inducible responses in needle phenolics due to previous defoliation thus appear to represent non-specific responses against sawflies.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.

Oscillatory brain activity in memory disorders

Neuronal oscillations are thought to underlie interactions between distinct brain regions required for normal memory functioning. This study aimed at elucidating the neuronal basis of memory abnormalities in neurodegenerative disorders. Magnetoencephalography (MEG) was used to measure oscillatory brain signals in patients with Alzheimer s disease (AD), a neurodegenerative disease causing progressive cognitive decline, and mild cognitive impairment (MCI), a disorder characterized by mild but clinically significant complaints of memory loss without apparent impairment in other cognitive domains. Furthermore, to help interpret our AD/MCI results and to develop more powerful oscillatory MEG paradigms for clinical memory studies, oscillatory neuronal activity underlying declarative memory, the function which is afflicted first in both AD and MCI, was investigated in a group of healthy subjects. An increased temporal-lobe contribution coinciding with parieto-occipital deficits in oscillatory activity was observed in AD patients: sources in the 6 12.5 Hz range were significantly stronger in the parieto-occipital and significantly weaker in the right temporal region in AD patients, as compared to MCI patients and healthy elderly subjects. Further, the auditory steady-state response, thought to represent both evoked and induced activity, was enhanced in AD patients, as compared to controls, possibly reflecting decreased inhibition in auditory processing and deficits in adaptation to repetitive stimulation with low relevance. Finally, the methodological study revealed that successful declarative encoding and retrieval is associated with increases in occipital gamma and right hemisphere theta power in healthy unmedicated subjects. This result suggests that investigation of neuronal oscillations during cognitive performance could potentially be used to investigate declarative memory deficits in AD patients. Taken together, the present results provide an insight on the role of brain oscillatory activity in memory function and memory disorders.

Post-industrial Intervention : An Activity-Theoretical Expedition Tracing the Proximal Development of Forms of Conducting Interventions

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Boundaries in Action : An Activity-theoretical Study of Development, Learning, and Change in Health Care Organization for Patients with Multiple and Chronic Illnesses

This study examines boundaries in health care organizations. Boundaries are sometimes considered things to be avoided in everyday living. This study suggests that boundaries can be important temporally and spatially emerging locations of development, learning, and change in inter-organizational activity. Boundaries can act as mediators of cultural and social formations and practices. The data of the study was gathered in an intervention project during the years 2000-2002 in Helsinki in which the care of 26 patients with multiple and chronic illnesses was improved. The project used the Change Laboratory method that represents a research assisted method for developing work. The research questions of the study are: (1) What are the boundary dynamics of development, learning, and change in health care for patients with multiple and chronic illnesses? (2) How do individual patients experience boundaries in their health care? (3) How are the boundaries of health care constructed and reconstructed in social interaction? (4) What are the dynamics of boundary crossing in the experimentation with the new tools and new practice? The methodology of the study, the ethnography of the multi-organizational field of activity, draws on cultural-historical activity theory and anthropological methods. The ethnographic fieldwork involves multiple research techniques and a collaborative strategy for raising research data. The data of this study consists of observations, interviews, transcribed intervention sessions, and patients' health documents. According to the findings, the care of patients with multiple and chronic illnesses emerges as fragmented by divisions of a patient and professionals, specialties of medicine and levels of health care organization. These boundaries have a historical origin in the Finnish health care system. As an implication of these boundaries, patients frequently experience uncertainty and neglect in their care. However, the boundaries of a single patient were transformed in the Change Laboratory discussions among patients, professionals and researchers. In these discussions, the questioning of the prevailing boundaries was triggered by the observation of gaps in inter-organizational care. Transformation of the prevailing boundaries was achieved in implementation of the collaborative care agreement tool and the practice of negotiated care. However, the new tool and practice did not expand into general use during the project. The study identifies two complementary models for the development of health care organization in Finland. The 'care package model', which is based on productivity and process models adopted from engineering and the 'model of negotiated care', which is based on co-configuration and the public good.

Evaluation of driving ability of the disabled persons in the context of the psychological activity theory

In the future the number of the disabled drivers requiring a special evaluation of their driving ability will increase due to the ageing population, as well as the progress of adaptive technology. This places pressure on the development of the driving evaluation system. Despite quite intensive research there is still no consensus concerning what is the factual situation in a driver evaluation (methodology), which measures should be included in an evaluation (methods), and how an evaluation has to be carried out (practise). In order to find answers to these questions we carried out empirical studies, and simultaneously elaborated upon a conceptual model for driving and a driving evaluation. The findings of empirical studies can be condensed into the following points: 1) A driving ability defined by the on-road driving test is associated with different laboratory measures depending on the study groups. Faults in the laboratory tests predicted faults in the on-road driving test in the novice group, whereas slowness in the laboratory predicted driving faults in the experienced drivers group. 2) The Parkinson study clearly showed that even an experienced clinician cannot reliably accomplish an evaluation of a disabled person’s driving ability without collaboration with other specialists. 3) The main finding of the stroke study was that the use of a multidisciplinary team as a source of information harmonises the specialists’ evaluations. 4) The patient studies demonstrated that the disabled persons themselves, as well as their spouses, are as a rule not reliable evaluators. 5) From the safety point of view, perceptible operations with the control devices are not crucial, but correct mental actions which the driver carries out with the help of the control devices are of greatest importance. 6) Personality factors including higher-order needs and motives, attitudes and a degree of self-awareness, particularly a sense of illness, are decisive when evaluating a disabled person’s driving ability. Personality is also the main source of resources concerning compensations for lower-order physical deficiencies and restrictions. From work with the conceptual model we drew the following methodological conclusions: First, the driver has to be considered as a holistic subject of the activity, as a multilevel hierarchically organised system of an organism, a temperament, an individuality, and a personality where the personality is the leading subsystem from the standpoint of safety. Second, driving as a human form of a sociopractical activity, is also a hierarchically organised dynamic system. Third, in an evaluation of driving ability it is a question of matching these two hierarchically organised structures: a subject of an activity and a proper activity. Fourth, an evaluation has to be person centred but not disease-, function- or method centred. On the basis of our study a multidisciplinary team (practitioner, driving school teacher, psychologist, occupational therapist) is recommended for use in demanding driver evaluations. Primary in a driver’s evaluations is a coherent conceptual model while concrete methods of evaluations may vary. However, the on-road test must always be performed if possible.

Distribution and adhesion-promoting activity of alpha4 and alpha5 chain laminins in human endothelia and carcinomas

Basement membranes are specialized sheets of extracellular matrix found in contact with epithelia, endothelia, and certain isolated cells. They support tissue architecture and regulate cell behaviour. Laminins are among the main constituents of basement membranes. Due to differences between laminin isoforms, laminins confer structural and functional diversity to basement membranes. The first aim of this study was to gain insights into the potential functions of the then least characterized laminins, alpha4 chain laminins, by evaluating their distribution in human tissues. We thus created a monoclonal antibody specific for laminin alpha4 chain. By immunohistochemistry, alpha4 chain laminins were primarily localized to basement membranes of blood vessel endothelia, skeletal, heart, and smooth muscle cells, nerves, and adipocytes. In addition, alpha4 chain laminins were found in the region of certain epithelial basement membranes in the epidermis, salivary gland, pancreas, esophagus, stomach, intestine, and kidney. Because of the consistent presence of alpha4 chain laminins in endothelial basement membranes of blood vessels, we evaluated the potential roles of endothelial laminins in blood vessels, lymphatic vessels, and carcinomas. Human endothelial cells produced alpha4 and alpha5 chain laminins. In quantitative and morphological adhesion assays, human endothelial cells barely adhered to alpha4 chain-containing laminin-411. The weak interaction of endothelial cells with laminin-411 appeared to be mediated by alpha6beta1 integrin. The alpha5 chain-containing laminin-511 promoted endothelial cell adhesion better than laminin-411, but it did not promote the formation of cell-extracellular matrix adhesion complexes. The adhesion of endothelial cells to laminin-511 appeared to be mediated by Lutheran glycoprotein together with beta1 and alphavbeta3 integrins. The results suggest that these laminins may induce a migratory phenotype in endothelial cells. In lymphatic capillaries, endothelial basement membranes showed immunoreactivity for laminin alpha4, beta1, beta2, and gamma1 chains, type IV and XVIII collagens, and nidogen-1. Considering the assumed inability of alpha4 chain laminins to polymerize and to promote basement membrane assembly, the findings may in part explain the incomplete basement membrane formation in these vessels. Lymphatic capillaries of ovarian carcinomas showed immunoreactivity also for laminin alpha5 chain and its receptor Lutheran glycoprotein, emphasizing a difference between normal and ovarian carcinoma lymphatic capillaries. In renal cell carcinomas, immunoreactivity for laminin alpha4 chain was found in stroma and basement membranes of blood vessels. In most tumours, immunoreactivity for laminin alpha4 chain was also observed in the basement membrane region of tumour cell islets. Renal carcinoma cells produced alpha4 chain laminins. Laminin-411 did not promote adhesion of renal carcinoma cells, but inhibited their adhesion to fibronectin. Renal carcinoma cells migrated more on laminin-411 than on fibronectin. The results suggest that alpha4 chain laminins have a counteradhesive function, and may thus have a role in detachment and invasion of renal carcinoma cells.

Probiotic activity of Lactobacillus delbrueckii subsp. bulgaricus in the oral cavity

Yogurt consumption has been related to longevity of some populations living on the Balkans. Yogurt starter L. delbrueckii subsp. bulgaricus and Str. thermophilus have been recognized as probiotics with verified beneficial health effects. The oral cavity emerges as a arget for probiotic applications. Probiotics have demonstrated promising results in controlling dental diseases and oral yeast infections. However, L. bulgaricus despite its broad availability in dairy products has not been evaluated for probiotic activity in the mouth. These series of studies investigated in vitro properties of L. bulgaricus to outline its potential as an oral probiotic. Prerequisite probiotic properties in the mouth are resistance to oral defense mechanisms, adherence to saliva-coated surfaces, and inhibition of oral pathogens. L. bulgaricus strains showed a strain-dependent inhibition of oral streptococci and Aggregatibacter actinomycetemcomitans, whereas none of the dairy starter strains could affect growth of Porphyromonas gingivalis and Fusobacterium nucleatum. Adhesion is a factor contributing to colonization of the species at the target site. Radiolabeled L. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated surfaces. The effects of lysozyme on adhesion and adhesion of Streptococcus sanguinis after lactobacilli pretreatment were also assessed. Adhesion of L. bulgaricus remained lower in comparison to L. rhamnosus GG. One L. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment significantly increased Lactobacillus adhesion. Low gelatinolytic activity was observed for all strains and no conversion of proMMP-9 to its active form was induced by L. bulgaricus. Safety assessment ruled out deleterious effects of L. bulgaricus on extracellular matrix structures. Cytokine response of oral epithelial cells was assessed by measuring IL-8 and TNF-α in cell culture supernatants. The effect of P. gingivalis on cytokine secretion after lactobacilli pretreatment was also assessed. A strain- and time-dependent induction of IL-8 was observed with live bacteria inducing the highest levels of cytokine secretion. Levels of TNF-α were low and only one of ten L. bulgaricus strains stimulated TNF-α secretion similar to positive control. The addition of P. gingivalis produced immediate reduction of cytokine levels within the first hours of incubation irrespective of lactobacilli strains co-cultured with epithelial cells. According to these studies strains among the L. delbrueckii subsp. bulgaricus species may have beneficial probiotic properties in the mouth. Their potential in prevention and management of common oral infectious diseases needs to be further studied.

Trends and Explanations for Socioeconomic Differences in Physical Activity

Very limited scientific knowledge exists on the trends and explanations of socioeconomic differences in physical activity among adults. There is a paucity of studies examining whether determinants vary across socioeconomic position and different life stages. This study examines a) how socioeconomic differences in leisure-time and commuting physical activity have changed in Finland from 1978 to 2002 and b) the contribution of childhood socioeconomic position, adolescence sports and exercise, adulthood socioeconomic position, working conditions and other adulthood health behaviours to socioeconomic differences in leisure-time physical activity. This study utilised three population-based datasets collected by the National Institute for Health and Welfare (THL, formerly National Institute for Public Health): the Health Behaviour and Health among the Finnish Adult Population Study from 1978 to 2002 (N=96 105), the National FINRISK Study 2002 and its physical activity sub-study (N= 9 179), and the Health 2000 Study (N=8 028). Survey information was collected by self-administered questionnaires, interviews at home, and measurements made at the study site. The response rates varied from 69 to 89 per cent. Several socioeconomic measures were linked from the national population registers. Based on the results, those with low income were physically inactive during leisure-time and while commuting from 1978 to 2002. Manual worker women, however, were more physically active commuters compared to their counterparts. Parental socioeconomic position contributed directly to adulthood educational differences in leisure-time physical inactivity but also indirectly through adulthood socioeconomic position (occupation, household income) and other unhealthy behaviours (mainly smoking). Among those with low education participation in competitive sports in youth and among those with high education exercise in late adolescence contributed to leisure-time physical activity in adulthood. Long exposure to physically strenuous working conditions in men and current job strain in women contributed to occupational class differences in leisure-time physical activity. Socioeconomic differences in physical activity have remained similar for twenty years in Finland. Educational career seems to have a strong contribution to physical activity. To adopt a lifelong physically active life-style, one should participate in a range of different sports and exercise in adolescence and in youth, have a low exposure to physically and mentally strenuous working conditions in later life and have other healthy behaviours in later life.