Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Modern diagnosis of Epstein-Barr virus infections and post-transplant lymphoproliferative disease

Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.

Improving oncolytic adenoviral therapies for gastrointestinal cancers and tumor initiating cells

Although the treatment of most cancers has improved steadily, only few metastatic solid tumors can be cured. Despite responses, refractory clones often emerge and the disease becomes refractory to available treatment modalities. Furthermore, resistance factors are shared between different treatment regimens and therefore loss of response typically occurs rapidly, and there is a tendency for cross-resistance between agents. Therefore, new agents with novel mechanisms of action and lacking cross-resistance to currently available approaches are needed. Modified oncolytic adenoviruses, featuring cancer-celective cell lysis and spread, constitute an interesting drug platform towards the goals of tumor specificity and the implementation of potent multimodal treatment regimens. In this work, we demonstrate the applicability of capsid-modified, transcriptionally targeted oncolytic adenoviruses in targeting gastric, pancreatic and breast cancer. A variety of capsid modified adenoviruses were tested for transductional specificity first in gastric and pancreatic cancer cells and patient tissues and then in mice. Then, oncolytic viruses featuring the same capsid modifications were tested to confirm that successful transductional targeting translates into enhanced oncolytic potential. Capsid modified oncolytic viruses also prolonged the survival of tumor bearing orthotopic models of gastric and pancreatic cancer. Taken together, oncolytic adenoviral gene therapy could be a potent drug for gastric and pancreatic cancer, and its specificity, potency and safety can be modulated by means of capsid modification. We also characterized a new intraperitoneal virus delivery method in benefit for the persistence of gene delivery to intraperitoneal gastric and pancreatic cancer tumors. With a silica implant a steady and sustained virus release to the vicinity of the tumor improved the survival of the orthotopic tumor bearing mice. Furthermore, silica gel-based virus delivery lowered the toxicity mediating proimflammatory cytokine response and production of total and anti-adenovirus neutralizing antibodies (NAbs). On the other hand, silica shielded the virus against pre-excisting NAbs, resulting in a more favourable biodistribution in the preimmunized mice. The silica implant might therefore be of interest in treating intraperitoneally disseminated disease. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in cancer relapse and the formation of metastasis. Therefore, we examined if transcriptionally modified oncolytic adenoviruses are able to kill these cells. Complete eradication of CD44+CD24-/low putative breast cancer stem cells was seen in vitro, and significant antitumor activity was detected in CD44+CD24-/low –derived tumor bearing mice. Thus, genetically engineered oncolytic adenoviruses have potential in destroying cancer initiating cells, which may have relevance for the elimination of cancer stem cells in humans.

VEGF-C and angiopoietins in lymphangiogenesis

Angiogenesis and lymphangiogenesis occur during development as the result of tightly coordinated signalling programs to generate two hierarchically organised vascular systems. All tissues and organs are dependent on a functional blood vasculature for oxygen and nutrients, whereas the lymphatic vasculature functions to collect excess tissue fluid, passing it through lymph nodes for immune surveillance, and returning it to the blood circulation. Effectors that control developmental angiogenesis and lymphangiogenesis are also involved in pathological settings, and therefore potential targets for therapy. Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) growth factors, signalling through endothelial VEGFR and Tie receptors, have been established as key regulators of angiogenic and lymphangiogenic processes in development and disease. In this study, we aimed to obtain a clearer understanding of the vascular effects of stimulation by VEGF-C, Ang1 and Ang2, all known to be involved in lymphangiogenesis. In cell culture models, we found that both intrinsic and microenvironmental regulatory mechanisms are involved in the regulation of endothelial cell phenotypes, and distinct responses to VEGF signalling are induced by specific receptor pathways in different endothelial cell types. Surprisingly, we also found that Ang1 induces sprouting lymphangiogenesis in vivo by a VEGFR-3 dependent mechanism, establishing Ang1 as a novel lymphangiogenic factor. Using inducible transgenic mouse models, we found that VEGF-C-induced lymphatic hyperplasia persisted independently of the growth factor, indicating that short pro-lymphangiogenic therapy could lead to lasting improvements in tissue oedema. While VEGF-C had blood vessel effects in embryos, no angiogenic side effects were observed in adult tissues. Furthermore, inducible transgenic expression of Ang2 during embryonic development confirmed Ang2 as an important regulator of lymphatic remodelling and mural cell contacts. The unexpected similarity of the lymphatic maturation defects caused by excess Ang2 to those observed in Ang2 deficient mice demonstrated that correct doses of Ang2 are crucial for the control of lymphatic development. Unlike Ang1, Ang2 did not induce lymphatic sprouting. Although Ang1 has been shown to be able to substitute for Ang2 during developmental lymphangiogenesis, their lymphatic effects are not identical. These findings further our understanding of the basic mechanisms of angiogenesis and lymphangiogenesis, important for the future development of targeted therapies for vascular diseases such as cancer, inflammation, lymphoedema and ischemia. VEGF-C and Ang1 especially emerged as promising candidates for pro-lymphangiogenic therapy.

Novel Insights into the Molecular Genetic Background of Colorectal Tumors

Many of the genes predisposing to highly penetrant colorectal cancer (CRC) syndromes, including hereditary non-polyposis colorectal cancer (MLH1, MSH2, MSH6, PMS2), familial adenomatous polyposis (APC), Peutz-Jeghers syndrome (LKB1), juvenile polyposis (SMAD4, BMPR1A), MYH-associated polyposis (MYH), and Cowden syndrome (PTEN) have already been discovered. Identification of these genes has allowed a more precise classification of the hereditary CRC syndromes and provided a means for predictive genetic testing and surveillance. Some of the genes are also involved in sporadic cancer forms, and therefore the investigation of the rare CRC syndromes has been a breakthrough for general cancer research. Despite the accumulating knowledge on hereditary cancer syndromes, a significant number of familial CRCs remain molecularly unexplained after genetic testing, reflecting the possibility of other predisposing genes or existence of novel syndromes. Moreover, genetic variants conferring low-penetrance risk are still largely unknown. In this study, we examined the role of some new high- and low-penetrance alleles on CRC predisposition. We identified disease causing MYH mutations in a subset (9%) of patients with APC and AXIN2 mutation negative adenomatous polyposis. Due to differences in the pattern of inheritance and clinical manifestation, screening for mutations in MYH is beneficial in view of genetic counselling and surveillance. A novel functionally deficient MYH founder mutation A459D was identified in the Finnish population, and this finding had immediate clinical implications for genetic counselling of at risk families. Many patients with hamartomatous polyposis remain without molecular diagnosis due to atypical phenotypes. We therefore sought to classify 49 patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analyses of PTEN, LKB1, BMPR1A, SMAD4, ENG, BRAF, MYH, and BHD along with revision of polyp histology. Mutations were identified in 11/49 (22%) of the patients. In 6 cases the molecular diagnosis was re-classified guiding surveillance and decisions for prophylactic surgery. Re-evaluation of polyp histology with subsequent more accurate selection of candidate gene analyses is beneficial and can be recommended for patients with unexplained polyposis. Furthermore, germline mutations in ENG underlying juvenile polyposis were described for the first time, characterizing a possible novel genetically defined form of hereditary CRC. Association analyses on two putative low-penetrance alleles, NOD2 3020insC and MDM2 SNP309 were performed in a population-based series of 1042 Finnish CRC patients and in cancer-free controls. In contrast to previous results, NOD2 3020insC did not associate with CRC or age at disease onset in the Finnish population. These data suggest that NOD2 3020insC alone might not be sufficient for CRC predisposition. MDM2 SNP309 was as common in the CRC cohort as in the healthy controls. Interesting trends, however, were observed, which after correction for multiple testing did not reach statistical significance. SNP309 was more common in female CRC patients and a trend towards an earlier age at disease onset was observed in women with SNP309. Subsequent studies have supported this observation and SNP309 could affect gender- or hormone-related tumorigenesis. Finally, a large-scale unbiased effort was designed to characterize the complete mutatome of CRC with microsatellite instability (MSI). Using an approach combining expression microarray and genome database searches, we were able to identify putative MSI target genes. Further characterization of one of the genes suggested that it might play a role also in microsatellite stable CRC and Peutz-Jeghers syndrome pathogenesis.

Target Cell Topography and Cytoskeletal Reorganization in Natural Killer Cell Activity

The immune system has to recognize and destroy abnormal or infected cells to maintain homeostasis. Natural killer (NK) cells directly recognize and kill transformed or virus-infected cells without prior sensitization. We have studied both virus-infected and tumor cells in order to identify the target structures involved in triggering NK activity. Mouse/human cell hybrids containing various human chromosomes were used as targets. The human chromosome responsible for activating NK cell killing was identified to chromosome number 6. The results suggest that activated NK cells recognize ligands that are encoded on human chromosome 6. We showed that the ligand on the target cell side was intercellular adhesion molecule 2 (ICAM-2). There was no difference in the level of expression of ICAM-2, however, but a drastic difference was seen in the distribution of the molecule: ICAM-2 was evenly distributed on the surface of the NK-resistant cells, but almost totally redistributed to the tip of uropods, bud-like extensions, which were absent from the parental cells. Interestingly, the gene coding for cytoskeletal linker protein ezrin has been localized to human chromosome 6, and there was a colocalization of ezrin and ICAM-2 in the uropods. Furthermore, the transfected human ezrin into NK cell-resistant cells induced uropod formation, ICAM-2 and ezrin redistribution to newly formed uropods, and sensitized target cells to NK cell killing. These data reveal a novel form of NK cell recognition: target structures are already present on normal cells; they become detectable only after abnormal redistribution into hot spots on the target cell membrane. NK cells are central players in the defence against virus infections. They inhibit the spread of infection, allowing time for specific immune responses to develop. The virus-proteins that directly activate human NK cell killing are largely unknown. We studied the sensitivity of virus-specific early proteins of Semliki Forest virus (SFV) to NK killing. The viral non-structural proteins (nsP1-4) translated early in the virus cycle were transfected in NK-resistant cells. Viral early gene nsP1 alone efficiently sensitized target cells to NK activity, and the tight membrane association of nsP1 seems to be critical in the triggering of NK killing. NsP1 protein colocalized with (redistributed) ezrin in filopodia-like structures to which the NK cells were bound. The results suggest that also in viral infections NK cells react to rapid changes in membrane topography. Based on the results of this thesis, a new model of target cell recognition of NK cells can be suggested: reorganization of the cytoskeleton induces alterations in cell surface topography, and this new pattern of surface molecules is recognized as "altered-self".

Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

Antizyme Inhibitor 2 : A Novel Regulator of Cellular Polyamine Homeostasis

Polyamines are organic polycations that participate in various physiological functions, including cell proliferation, differentiation and apoptosis. Cellular polyamines originate from endogenous biosynthesis and exogenous sources. Their subcellular pool is under strict control, achieved by regulating their uptake and metabolism. Polyamine-induced proteins called antizymes (AZ) act as key regulators of intracellular polyamine concentration. They regulate both the transport of polyamines and the activity and degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. AZs themselves are negatively regulated by antizyme inhibitor (AZIN). AZIN functions as a positive regulator of cellular polyamine homeostasis, which by binding to AZs reactivates ODC and induces the uptake of polyamines. In various pathological conditions, including cancer, polyamine levels are misregulated. Polyamine homeostasis has therefore become an attractive target for therapeutic interventions and it is thus crucial to characterize the molecular basis underlying the homeostatic regulation. A novel human AZIN-resembling protein was previously identified in our group. The purpose of this study was to elucidate the function and distribution of this protein, termed as an antizyme inhibitor 2 (AZIN2). According to my results, AZIN2 functions as a novel regulator of polyamine homeostasis. It shows no enzymatic activity, but instead it binds AZs and negates their activity, which subsequently leads to reactivation of ODC and inhibition of its degradation. Expression of AZIN2 is restricted to terminally differentiated cells, such as mast cells (MC) and neurosecretory cells. In these actively secreting cell types, AZIN2 localizes to subcellular vesicles or granules where its function is important for the vesicle-mediated secretion. In MCs, AZIN2 localizes to the serotonin-containing subset of MC granules, and its expression is coupled to MC activation. The functional role of polyamines as potential mediators of MC activity was also investigated, and it was observed that the secretion of serotonin is selectively dependent on activation of ODC. In neurosecretory cells, AZIN2-positive vesicles localize mainly to the trans-Golgi network (TGN). Depletion of AZIN2 or cellular polyamines causes selective fragmentation of the TGN and retards secretion of proteins. Since addition of exogenous polyamines reverses these effects, the data indicate that AZIN2 and its downstream effectors, polyamines, are functionally implicated in the regulation of secretory vesicle transport. My studies therefore reveal a novel function for polyamines as modulators of both constitutive and regulated secretion. Based on the results, I propose that the role of AZIN2 is to act as a local in situ activator of polyamine biosynthesis.

The decline of northern malaria and population dynamics of Plamodium vivax

Europe was declared malaria free in 1975. The disappearance of malaria has traditionally been attributed to numerous deliberate actions like vector control, the screening of houses, more efficient medication etc. Malaria, however, disappeared from many countries like Finland before any counter measures had even started. The aim of this thesis is to study the population ecology of P. vivax and its interaction with the human host and the vector. By finding the factors that attributed to the extinction of vivax malaria it might be possible to improve the modern strategy against P. vivax. The parasite was studied with data from Finland, which provides the longest time series (1749-2008) of malaria statistics in the world. The malaria vectors, Anopheles messeae and A. beklemishevi are still common species in the country. The eradication of vivax malaria is difficult because the parasite has a dormant stage that can cause a relapse long after a primary infection. It was now shown that P. vivax is able to detect the presence of a potential vector. A dormant stage is triggered even from a bite of an uninfected Anopheles mosquito. This optimizes the chances for the Plasmodium to reach a mosquito vector for sexual reproduction. The longevity of the dormant stage could be shown to be at least nine years. The parasite spends several years in its human host and the behaviour of the human carrier had a profound impact on the decline of the disease in Finland. Malaria spring epidemics could be explained by a previous warm summer. Neither annual nor summer mean temperature had any impact on the long term malaria trend. Malaria disappeared slowly from Finland without mosquito control. The sociological change from extended families to nuclear families led to decreased household size. The decreased household size correlated strongly with the decline of malaria. That led to an increased isolation of the subpopulations of P. vivax. Their habitat consisted of the bedrooms in which human carriers slept together with the overwintering vectors. The isolation of the parasite ultimately led to the extinction of vivax malaria. Metapopulation models adapted to local conditions should therefore be implemented as a tool for settlement planning and socio-economic development and become an integrated part of the fight against malaria.

Suunnittelunäkemys metsäorganisaatioissa ja sen vaikutus tietojärjestelmälle asetettaviin vaatimuksiin

Department of Forest Resource Management in the University of Helsinki has in years 2004?2007 carried out so-called SIMO -project to develop a new generation planning system for forest management. Project parties are organisations doing most of Finnish forest planning in government, industry and private owned forests. Aim of this study was to find out the needs and requirements for new forest planning system and to clarify how parties see targets and processes in today's forest planning. Representatives responsible for forest planning in each organisation were interviewed one by one. According to study the stand-based system for managing and treating forests continues in the future. Because of variable data acquisition methods with different accuracy and sources, and development of single tree interpretation, more and more forest data is collected without field work. The benefits of using more specific forest data also calls for use of information units smaller than tree stand. In Finland the traditional way to arrange forest planning computation is divided in two elements. After updating the forest data to present situation every stand unit's growth is simulated with different alternative treatment schedule. After simulation, optimisation selects for every stand one treatment schedule so that the management program satisfies the owner's goals in the best possible way. This arrangement will be maintained in the future system. The parties' requirements to add multi-criteria problem solving, group decision support methods as well as heuristic and spatial optimisation into system make the programming work more challenging. Generally the new system is expected to be adjustable and transparent. Strict documentation and free source code helps to bring these expectations into effect. Variable growing models and treatment schedules with different source information, accuracy, methods and the speed of processing are supposed to work easily in system. Also possibilities to calibrate models regionally and to set local parameters changing in time are required. In future the forest planning system will be integrated in comprehensive data management systems together with geographic, economic and work supervision information. This requires a modular method of implementing the system and the use of a simple data transmission interface between modules and together with other systems. No major differences in parties' view of the systems requirements were noticed in this study. Rather the interviews completed the full picture from slightly different angles. In organisation the forest management is considered quite inflexible and it only draws the strategic lines. It does not yet have a role in operative activity, although the need and benefits of team level forest planning are admitted. Demands and opportunities of variable forest data, new planning goals and development of information technology are known. Party organisations want to keep on track with development. One example is the engagement in extensive SIMO-project which connects the whole field of forest planning in Finland.

Microclimate and gas emissions in dairy buildings : Instrumentation, theory and measurements

The aim of this thesis was to develop measurement techniques and systems for measuring air quality and to provide information about air quality conditions and the amount of gaseous emissions from semi-insulated and uninsulated dairy buildings in Finland and Estonia. Specialization and intensification in livestock farming, such as in dairy production, is usually accompanied by an increase in concentrated environmental emissions. In addition to high moisture, the presence of dust and corrosive gases, and widely varying gas concentrations in dairy buildings, Finland and Estonia experience winter temperatures reaching below -40 ºC and summer temperatures above +30 ºC. The adaptation of new technologies for long-term air quality monitoring and measurement remains relatively uncommon in dairy buildings because the construction and maintenance of accurate monitoring systems for long-term use are too expensive for the average dairy farmer to afford. Though the documentation of accurate air quality measurement systems intended mainly for research purposes have been made in the past, standardised methods and the documentation of affordable systems and simple methods for performing air quality and emissions measurements in dairy buildings are unavailable. In this study, we built three measurement systems: 1) a Stationary system with integrated affordable sensors for on-site measurements, 2) a Wireless system with affordable sensors for off-site measurements, and 3) a Mobile system consisting of expensive and accurate sensors for measuring air quality. In addition to assessing existing methods, we developed simplified methods for measuring ventilation and emission rates in dairy buildings. The three measurement systems were successfully used to measure air quality in uninsulated, semi-insulated, and fully-insulated dairy buildings between the years 2005 and 2007. When carefully calibrated, the affordable sensors in the systems gave reasonably accurate readings. The spatial air quality survey showed high variation in microclimate conditions in the dairy buildings measured. The average indoor air concentration for carbon dioxide was 950 ppm, for ammonia 5 ppm, for methane 48 ppm, for relative humidity 70%, and for inside air velocity 0.2 m/s. The average winter and summer indoor temperatures during the measurement period were -7º C and +24 ºC for the uninsulated, +3 ºC and +20 ºC for the semi-insulated and +10 ºC and +25 ºC for the fully-insulated dairy buildings. The measurement results showed that the uninsulated dairy buildings had lower indoor gas concentrations and emissions compared to fully insulated buildings. Although occasionally exceeded, the ventilation rates and average indoor air quality in the dairy buildings were largely within recommended limits. We assessed the traditional heat balance, moisture balance, carbon dioxide balance and direct airflow methods for estimating ventilation rates. The direct velocity measurement for the estimation of ventilation rate proved to be impractical for naturally ventilated buildings. Two methods were developed for estimating ventilation rates. The first method is applicable in buildings in which the ventilation can be stopped or completely closed. The second method is useful in naturally ventilated buildings with large openings and high ventilation rates where spatial gas concentrations are heterogeneously distributed. The two traditional methods (carbon dioxide and methane balances), and two newly developed methods (theoretical modelling using Fick s law and boundary layer theory, and the recirculation flux-chamber technique) were used to estimate ammonia emissions from the dairy buildings. Using the traditional carbon dioxide balance method, ammonia emissions per cow from the dairy buildings ranged from 7 g day-1 to 35 g day-1, and methane emissions per cow ranged from 96 g day-1 to 348 g day-1. The developed methods proved to be as equally accurate as the traditional methods. Variation between the mean emissions estimated with the traditional and the developed methods was less than 20%. The developed modelling procedure provided sound framework for examining the impact of production systems on ammonia emissions in dairy buildings.

Protein cross-linking with oxidative enzymes and transglutaminase : Effects in meat protein systems

The effects of tyrosinase, laccase and transglutaminase (TG) were studied in different meat protein systems. The study was focused on the effects of the enzymes on the gel formation properties of myofibrils, and on the textural and water-holding properties of the heated meat systems. The cross-linking efficiency of a novel Trichoderma reesei tyrosinase was compared to that of the commercial Agaricus bisporus tyrosinase. Trichoderma tyrosinase was found to be superior compared to the Agaricus enzyme in its protein cross-linking efficiency and in the incorporation of a small molecule into a complex proteinaceous substrate. Tyrosinase, laccase and TG all polymerised myofibrillar proteins, but laccase was also found to cause protein fragmentation. A positive connection between covalent cross-link and gel formation was observed with tyrosinase and TG. Laccase was able to increase the gel formation only slightly. With an excessive laccase dosage the gel formation declined due to protein fragmentation. Tyrosinase, laccase and TG had different effects on the texture and water-holding of the heated chicken breast meat homogenates. Tyrosinase improved the firmness of the homogenate gels free of phosphate and with a low amount of meat. TG improved the firmness of all studied homogenates. Laccase weakened the gel firmness of the low-meat, low-salt and low-salt/phosphate homogenates and maintained the firmness on the control level in the homogenate free of phosphate. Tyrosinase was the only enzyme capable of reducing the weight loss in the homogenates containing a low amount of meat and a low amount of NaCl. TG was the only enzyme that could positively affect the firmness of the homogenate gel containing both low NaCl and phosphate amounts. In pilot scale the test products were made of coarsely ground chicken breast fillet with a moderate amount of salt. Increasining the amount of meat, salt and TG contents favoured the development of firmness of the test products. The evaporation loss decreased slightly along with increasing TG and NaCl amounts in the experimental conditions used, indicating a positive interaction between these two factors. In this work it was shown that tyrosinase, laccase and TG affected the same myofibrillar proteins, i.e. myosin and troponin T. However, these enzymes had distinguishable effects on the gel formation of a myofibril system as well as on the textural and water-holding properties of the finely ground meat homogenates, reflecting distinctions at least in the reaction mechanisms and target amino acid availability in the protein substrates for these enzymes.

Molecular phylogeny of Archaea in boreal forest soil, freshwater and temperate estuarine sediment

Suurin osa luonnossa havaitsemistamme mikrobeista on sellaisia, joita emme edelleenkään osaa kasvattaa laboratorio-oloissa, vaikka tietomme mikrobien monimuotoisuudesta paranevat koko ajan. Luonnontilaisen mikrobieliöstön kokoonpano eri ympäristöissä on paljolti epäselvä ja ymmärrämme vielä hyvin puutteellisesti mikrobien ekologiaa ja niiden rooleja eliöyhteisöissä. Nykyaikaiset molekulaariset tutkimusmenetelmät auttavat selvittämään mikrobien monimuotoisuutta kokonaisvaltaisesti ja nopeasti. Ympäristöstä kemiallisesti puhdistetut ribosomaalista RNA:ta koodaavat geenit edustavat periaatteessa kaikkia eliöyhteisön geneettisesti toisistaan poikkeavia eliöitä. Niistä voidaan valikoida halutut genomit jatkotutkimuksia varten. Uusien menetelmien käyttö on tuonut esiin sen merkittävän seikan, että "tavanomaisten" elinympäristöjen eliöyhteisöihin kuuluu suuri joukko entuudestaan tuntemattomia arkkieliöitä. Aiemmin kuviteltiin, että arkkieliöt asuttavat vain sellaisia "epätavallisia" tai "äärimmäisiä" elinympäristöjä, joita luonnehtii joku seuraavista ominaisuuksista: hyvin korkea lämpötila, korkea suolapitoisuus, korkea happamuus tai emäksisyys, hapettomuus. Tutkijat ovat viimeisen noin kymmenen vuoden aikana osoittaneet, että arkkieliöt asuttavat hyvin monenlaisia kylmän ja lauhkean vyöhykkeen ympäristöjä, yhtä hyvin maaperää kuin suolaisen ja makean veden pohjaa tai pintakerroksia. Nämä löydöt ovat avanneet uuden alun arkkieliöiden tutkimukselle, erityisesti sen selvittämiselle, mitkä ovat niiden fysiologiset ja ekologiset roolit monimuotoisissa mikrobiyhteisöissä. Tämä väitöskirja kuvaa entuudestaan tuntemattomien arkkieliöiden löytymistä havumetsävyöhykkeen metsämaasta. Arkkieliöitä löytyi myös lauhkean vyöhykkeen vuorovesialueelta, murtoveden huuhtelemasta pohjasta. Nämä löydöt ovat perustavalaatuisia vuorovesialueen eliöyhteisöjen ymmärtämiseksi. Suomalaisen metsäjärven vedestä määritettiin molempien arkkieliöiden pääryhmien - tieteellisiltä nimiltään Crenarchaeota ja Euryarchaeota - edustajia. Euryarchaeota-ryhmän edustajia voitiin havainnoida myös fluoresenssi-mikroskopoinnilla. Löydöt viittaavat siihen, että arkkieliöillä on oma biogeokemiallinen roolinsa makeanveden ravintoketjujen hiilen käytössä. Tässä työssä määritetyt uudet arkkieliöiden genomien nukleotidisekvenssit on toimitettu ARB-tietokantaan, jonka kasvava vertailuaineisto edelleen parantaa uusien arkkieliösekvenssien analyysiä ja auttaa hybridisaatiokoetinten ja polymeraasiketjureaktioalukkeiden suunnittelussa ja arvioinnissa. Tässä väitöskirjassa esitellyt tulokset yhdessä lukuisien vesi-, maaperä- ja muiden ympäristöjen arkkieliöitä käsittelevien julkaisujen kanssa osoittavat, että arkkieliöt asuttavat monia erilaisia elinympäristöjä ja että ne ovat ekologisesti paljon menestyneempiä, kuin tieteenalalla on kuviteltu. Voimme olettaa, että heti kun joitain näistä eliöistä onnistutaan kasvattamaan ja ylläpitämään laboratorio-oloissa, niiden joukosta löydetään aivan uusia, entuudestaan tuntemattomia fysiologisia fenotyyppejä, jotka avaavat mielenkiintoisia näkymiä aineenvaihdunnan ja perinnöllisten ominaisuuksien tutkimukselle.

Median muotoilema : Muotoilun mediajulkisuus suomalaisessa talouslehdistössä

Designed by the Media The Media publicity of Design in the Finnish Economic Press The meaning of design has increased in consumer societies. Design is the subject of debate and the number of media discussions has also increased steadily. Especially the role of industrial design has been emphasised. In this study I examine the media publicity of design in the Finnish economic press from the late 1980s to the beginning of the 2000s. The research question is connected to media representations: How is design represented in the Finnish economic press? In other words, what are the central topics of design in the economic press, and to what issues are the media debates connected? The usually repeated phrase that design discussions take place only on the cultural pages of the daily press or in cultural contexts is being changed. Design is also linked to the consumer culture and consumers everyday practices. The research material has been collected from the Finnish economic press. The qualitative sample consists of articles from Kauppalehti, Taloussanomat and from several economic papers published by the Talentum Corporation. The approach of the research is explorative, descriptive and hermeneutic. This means that the economic press articles are used to explore how design is represented in the media. In addition, the characteristics of design represented in the media are described in detail. The research is based on the interpretive tradition of studying textual materials. Background assumptions are thus grounded in hermeneutics. Erving Goffman s frame analysis is applied in analysing the economic press materials. The frames interpreted from the articles depict the media publicity of design in the Finnish economic press. The research opens up a multidimensional picture of design in the economic press. The analysis resulted in five frames that describe design from various points of view. In the personal frame designers are described in private settings and through their personal experiences. The second frame relates to design work. In the frame of mastery of the profession, the designers work is interpreted widely. Design is considered from the aspects of controlling personal know-how, co-operation and the overall process of design. The third frame is connected to the actual substance of the economic press. In the frame of economy and market, design is linked to international competitiveness, companies competitive advantage and benefit creation for the consumers. The fourth frame is connected to the actors promoting design on a societal level. In the communal frame, the economic press describes design policy, design research and education and other actors that actively develop design in the societal networks. The last frame is linked to the traditions of design and above all to the examination of the cultural transition. In the frame of culture the traditions of design are emphasised. Design is also connected to the industrial culture and furthermore to the themes of the consumer culture. It can be argued that the frames construct media publicity of design from various points of view. The frames describe situations, action and the actors of design. The interpreted media frames make it possible to understand the relation of interpreted design actions and the culture. Thus, media has a crucial role in representing and recreating meanings related to design. The publicity of design is characterised by the five focal themes: personification, professionalisation, commercialisation, communalisation and transition of cultural focus from the traditions of design to the industrial culture and the consumer culture. Based on my interpretation these themes are guided by the mediatisation of design. The design phenomenon is defined more often on the basis of the media representations in the public discourses. The design culture outlined in this research connects socially constructed and structurally organised action. Socially constructed action in design is connected to the experiences, social recreation and collective development of design. Structurally, design is described as professional know-how, as a process and as an economic profit generating action in the society. The events described by the media affect the way in which people experience the world, the meanings they connect to the events around themselves and their life in the world. By affecting experiences, the media indirectly affects human actions. People have become habituated to read media representations on a daily basis, but they are not used to reading and interpreting the various meanings that are incorporated in the media texts.

Innovatiivisuuden yhteys elintarvikealan pienyrityksen tuotestrategiaan ja laatukäsitykseen

Tutkimuksen tavoitteena oli selvittää pienten elintarvikeyritysten innovatiivisuutta ja sen yhteyttä yrityksen strategisiin toimintoihin. Yritysten erilainen suhtautuminen innovaatioihin ja uusia tuotteita kohtaan pyrittiin esittämään yrityksen eri taustatekijöiden kuten toimialan, resurssien, tuotestrategian ja uusien elintarvikkeiden laatukäsityksen kautta. Teoriaosassa käytiin läpi innovaatioteorioita ja innovaation käsitettä elintarvikealalla. Kirjallisuuden avulla tuotiin esiin yrityksen innovatiivisuuden mittaamiseen käytettyjä menetelmiä. Innovaatioteorian rinnalle nostettiin uusien elintarvikkeiden laatukäsitys ja tuotteen laatu, joiden avulla, kirjallisuuteen pohjautuen, tarkasteltiin elintarviketta tuoteinnovaationa. Empiirinen osa tutkimuksessa kerättiin yrityksille lähetettynä postikyselynä syksyllä 2005. Vastanneita yrityksiä oli 59, jotka olivat suomalaisia elintarvikkeita jalostavia yrityksiä. Kysely muodostui osioista esitiedot, uuden elintarvikkeen ominaisuudet, innovatiivisuus ja tuotestrategia. Yritysten ulkopuoliselle kuluttajaryhmälle (n=97) tehtiin myös kysely liittyen elintarvikkeen laatuun. Yritykset jaettiin kahteen eri ryhmään innovatiivisuuden perusteella ja ryhmien sisältöä tarkasteltiin yritysten elintarviketoimialan, tuotestrategian ja laatukäsityksen näkökulmasta. Ryhmiksi muodostuivat kasvuhaluiset innovoijat (n=24) ja perinteiset kehittäjät (n=25). Uuden elintarvikkeen 22 laatuominaisuudesta kehitettiin uuden elintarvikkeen laatumittaristo hyödyntämällä faktori- ja reliabiliteettianalyysiä. Laatumittaristo muodostui kuudesta eri ulottuvuudesta: ulkonäkö, tunne, käytettävyys, terveydellisyys, hygienia-aistittava laatu ja prosessilaatu. Laatumittarin avulla verrattiin kasvuhaluisten innovoijien ja perinteisten kehittäjien laatukäsitystä. Merkitsevä ero muodostui laadun terveydellisyys-ulottuvuuden arvostuksessa. Tutkimustulosten perusteella elintarvikealan pienyritysten toimialakentällä on strategisesti erilaisia yrityksiä. Kasvuhaluiset innovoijat olivat tuoteorientoituneita, joiden tuotestrategia tähtäsi kasvuun ja tuotteet olivat uutuusasteeltaan korkeita erikoistuotteita. Yritysryhmien innovatiivisuuteen vaikuttavana tekijänä oli yrityksen koko. Kooltaan kasvuhaluiset innovoijat olivat suurimmaksi osaksi mikroyrityksiä (2 - 9 hlöä). Innovatiivisuuden voi katsoa liittyvän yrityksen tiettyyn kasvuhaluiseen vaiheeseen, jolloin myös yritys ajattelee markkinasuuntautuneisemmin. Tuotteiden uudet mahdollisuudet luoda kasvua koetaan tärkeämmiksi kuin kasvuvaiheen ohittaneissa yrityksissä. Tutkimus tukee käsitystä, että innovatiiviset yritykset käyttävät enemmän ulkoista markkinainformaatiota hyväkseen.