Below-ground processes in meadow soil under elevated ozone and carbon dioxide : - Greenhouse gas fluxes, N cycling and microbial communities

This thesis focuses on how elevated CO2 and/or O3 affect the below-ground processes in semi-natural vegetation, with an emphasis on greenhouse gases, N cycling and microbial communities. Meadow mesocosms mimicking lowland hay meadows in Jokioinen, SW Finland, were enclosed in open-top chambers and exposed to ambient and elevated levels of O3 (40-50 ppb) and/or CO2 (+100 ppm) for three consecutive growing season, while chamberless plots were used as chamber controls. Chemical and microbiological analyses as well as laboratory incubations of the mesocosm soils under different treatments were used to study the effects of O3 and/or CO2. Artificially constructed mesocosms were also compared with natural meadows with regards to GHG fluxes and soil characteristics. In addition to research conducted at the ecosystem level (i.e. the mesocosm study), soil microbial communities were also examined in a pot experiment with monocultures of individual species. By comparing mesocosms with similar natural plant assemblage, it was possible to demonstrate that artificial mesocosms simulated natural habitats, even though some differences were found in the CH4 oxidation rate, soil mineral N, and total C and N concentrations in the soil. After three growing seasons of fumigations, the fluxes of N2O, CH4, and CO2 were decreased in the NF+O3 treatment, and the soil NH4+-N and mineral N concentrations were lower in the NF+O3 treatment than in the NF control treatment. The mesocosm soil microbial communities were affected negatively by the NF+O3 treatment, as the total, bacterial, actinobacterial, and fungal PLFA biomasses as well as the fungal:bacterial biomass ratio decreased under elevated O3. In the pot survey, O3 decreased the total, bacterial, actinobacterial, and mycorrhizal PLFA biomasses in the bulk soil and affected the microbial community structure in the rhizosphere of L. pratensis, whereas the bulk soil and rhizosphere of the other monoculture, A. capillaris, remained unaffected by O3. Elevated CO2 caused only minor and insignificant changes in the GHG fluxes, N cycling, and the microbial community structure. In the present study, the below-ground processes were modified after three years of moderate O3 enhancement. A tentative conclusion is that a decrease in N availability may have feedback effects on plant growth and competition and affect the N cycling of the whole meadow ecosystem. Ecosystem level changes occur slowly, and multiplication of the responses might be expected in the long run.

Structural and Functional Studies on Trimeric Autotransporters

Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.

Innate immunity and its control in the pathogenesis of inflammatory rheumatic diseases

The pathogenesis of inflammatory rheumatic diseases, including rheumatoid arthritis (RA) and spondyloarthropathies (SpAs) such as reactive arthritis (ReA), is incompletely understood. ReA is a sterile joint inflammation, which may follow a distal infection caused by Gram-negative bacteria that have lipopolysaccharide (LPS) in their outer membrane. The functions of innate immunity that may affect the pathogenesis, prognosis and treatment of these diseases were studied in this thesis. When compared with healthy controls, whole blood monocytes of healthy subjects with previous ReA showed enhanced capacity to produce TNF, an essential proinflammatory cytokine, in response to adherent conditions (mimicking vascular endothelium made adherent by inflammatory signals) and non-specific protein kinase C stimulation. Also, blood neutrophils of these subjects showed high levels of CD11b, an important adhesion molecule, in response to adherence or LPS. Thus, high responsiveness of monocytes and neutrophils when encountering inflammatory stimuli may play a role in the pathogenesis of ReA. The results also suggested that the known risk allele for SpAs, HLA-B27, may be an additive contributor to the observed differences. The promoter polymorphisms TNF 308A and CD14 (gene for an LPS receptor component) 159T were found not to increase the risk of acute arthritis. However, all female patients who developed chronic SpA had 159T and none of them had 308A, possibly reflecting an interplay between hormonal and inflammatory signals in the development of chronic SpA. Among subjects with early RA, those having the polymorphic TLR4 +896G allele (causing the Asp299Gly change in TLR4, another component of LPS receptor) required a combination of disease-modifying antirheumatic drugs to achieve remission. It is known that rapid treatment response is essential in order to maintain the patients work ability. Hence, +896G might be a candidate marker for identifying the patients who need combination treatment. The production of vascular endothelial growth factor (VEGF), which strongly promotes vascular permeability and angiogenesis that takes place e.g. early in rheumatic joints, was induced by LPS and inhibited by interferon (IFN)-alpha in peripheral blood mononuclear cells. These long-living cells might provide a source of VEGF when stimulated by LPS and migrating to inflamed joints, and the effect of IFN-alpha may contribute to the clinical efficacy of this cytokine in inhibiting joint inflammation.

Identification of molecules relevant for the invasiveness of fibrosarcomas and melanomas

Cancer is becoming the leading cause of deaths in the world. As 90% of all deaths from cancer are caused by metastasis, discovery of the mechanisms behind cancer cell invasion and metastasis is of utmost importance. Only new effective therapies targeting cancer progression can reduce cancer mortality rates. The aim of this study was to identify molecules that are relevant for tumor cell invasion and spreading in fibrosarcomas and melanomas, and to analyze their potential for cancer biomarkers or therapeutic targets. First, the gene expression changes of normal cells and transformed cells showing high invasiveness, S-adenosylmethionine decarboxylase (AdoMetDC)-transfected murine fibroblasts and human melanoma cells, were studied by microarray analyses. The function of the identified candidate molecules were then studied in detail in these cell lines. Finally, the physiological relevance of the identified changes was studied by immunohistochemical analyses of human sarcoma and melanoma specimens or by a mouse xenograft model. In fibrosarcoma cells, the most remarkable change detected was a dramatic up-regulation of the actin-sequestering molecule thymosin beta 4 (TB4), which was shown to be important for the transformed phenotype of the AdoMetDC-transfected cells (Amdc-s and -as). A sponge toxin latrunculin A, inhibiting the binding of TB4 to actin, was found to selectively inhibit the migration and invasion of these cells. Further, Amdc-s-induced mouse tumors and human high-grade sarcomas were found to show intense TB4 immunostaining. In addition to TB4, integrin subunits alfa 6 and beta 7 (ItgA6 and ItgB7) were found to be up-regulated in Amdc-s and -as cells. ItgA6 was shown to dimerize mainly with ItgB1 in Amdc-s. Inhibition of ItgA6 or ItgB1 function with neutralizing antibodies fully blocked the invasiveness of Amdc-s cells, and importantly also human HT-1080 fibrosarcoma cells, in three-dimensional (3D)-Matrigel mimicking tumor extracellular matrix (ECM). By immunohistochemical analyses, strong staining for ITGA6 was detected in human high-grade fibrosarcomas and other sarcomas, especially at the invasion fronts of the tumors. In the studied melanoma cell lines, the expression levels of the adhesion-related ECM proteins tenascin-C (TN-C), fibronectin (FN), and transforming growth factor beta-induced (TGFBI) were found to be highly up-regulated. By immunohistochemistry, intense TN-C and FN staining was detected in invasive and metastatic melanoma tumors, showing co-localization (together with procollagen-I) in tubular meshworks and channels around the invading melanoma cells. In vitro, TN-C and FN were further found to directly stimulate the migration of melanoma cells in 3D-collagen-I matrix. The third candidate protein, TGFBI, was found to be an anti-adhesive molecule for melanoma cells, and knockdown of its expression in metastatic melanoma cells (TGFBI-KD cells) led to dramatically impaired tumor growth in immunocompromized mice. Interestingly, the control tumors showed intense TGFBI immunostaining in the invasion fronts, showing partial co-localization with the fibrillar FN staining, whereas the small TGFBI-KD cell-induced tumors displayed amorphous, non-fibrillar FN staining. These data suggest an important role for TGFBI in FN fibrillogenesis and melanoma progression. In conclusion, we have identified several invasion-related molecules, which show potential for cancer diagnostic or prognostic markers, or therapeutic targets. Based on our previous and present fibrosarcoma studies, we propose the possibility of using ITGA6 antagonists (affecting tumor cell adhesion) in combination with TB4 inhibitors (affecting tumor cell migration) and cathepsin L inhibitors (affecting the degradation of basement membrane and ECM proteins) for the treatment of fibrosarcomas and other tumors overexpressing these molecules. With melanoma cells, in turn, we point to the importance of three secreted ECM proteins, TN-C, FN, and TGFBI, in melanoma progression. Of these, especially the potential of TN-C as a prognostic melanoma biomarker and TGFBI as a promising therapeutic target molecule are clearly worth additional studies.

Jätteenkeräyksen ja -siirron yhteiskunnalliset kustannukset Punavuoressa – Tarkastelussa seka-, bio-, kartonki- ja paperijäte

Tämän pro gradu -tutkielman tarkoituksena on määrittää jätteenkeräyksen ja -siirron yhteiskunnalliset kustannukset valitulla tutkimusalueella Helsingin Punavuoressa. Jätteenkeräyksen ja -siirron kustannukset vastaavat suuruudeltaan merkittävää osaa jätehuollon kokonaiskustannuksista, minkä vuoksi kustannusten tutkimiselle ja tarkastelulle löytyy kysyntää. Lisäksi keräyksen ja siirron kustannukset saattavat vaihdella suuresti johtuen erilaisista kaupunkirakenteista,keräysmenetelmistä ja teknologioista, joten tapaustarkastelun avulla pystytään selvittämään yksityiskohtaisesti alueen jätteenkeräyksen ja -siirron kustannukset. Tutkimusalue Helsingin Punavuoressa on yksi Suomen tiheimmin asutuista alueista, missä jätteidenkeräystä hankaloittaa kapeat kadut, useat sisäpihoille sijoitetut jätehuoneet ja vilkas liikenne. Erityispiirteidensä vuoksi jätteenkeräys- ja siirto aiheuttaa tutkimusalueella yksityisten kustannusten lisäksi myös useita ulkoisvaikutuksia muun muassa ilmansaasteiden ja viihtyvyyshaittojen muodossa. Tässä työssä lasketaan jätteenkeräyksen ja -siirron yhteiskunnalliset kustannukset neljän eri jätelajin osalta huomioimalla sekä yksityiset kustannustekijät että ulkoiskustannuksina syntyvien päästöjen kustannukset. Työn aineistona on käytetty erilaisia kustannuslaskelmien kirjallisuuslähteitä, asiantuntija-arvioita ja tutkimusalueella tehtyjä kellotusmittauksia. Alueen kellotusmittauksiin perustuvalla aikaperusteisella laskentatavalla jätteenkeräyksen ja -siirron jätetonnikohtaisiksi keskimääräisiksi kustannuksiksi saatiin 73 €/t. Kustannuksissa havaittiin kuitenkin suuria jätelajikohtaisia eroja, jolloin keräyksen ja siirron kustannukset heittelivät 49–125 €/t välillä. Suuret jätelajikohtaiset kustannuserot ovat selitettävissä pitkälti jätteiden koostumuksella, koska kevyiden ja paljon tilaa vievien jätelajien jätetonnikohtaiset kustannukset olivat suurimpia. Teoriataustan ja lähdeaineiston perusteella saadut tulokset myös osoittavat, että jätteenkeräyksen ja siirron kustannuksista huomioitujen ulkoiskustannusten osuus on häviävän pieni verrattuna yksityisten kustannusten tasoon.