The Mechanisms of Hedgehog Signal Transduction

Studies in both vertebrates and invertebrates have identified proteins of the Hedgehog (Hh) family of secreted signaling molecules as key organizers of tissue patterning. Initially discovered in Drosophila in 1992, Hh family members have been discovered in animals with body plans as diverse as those of mammals, insects and echinoderms. In humans three related Hh genes have been identified: Sonic, Indian and Desert hedgehog (Shh, Ihh and Dhh). Transduction of the Hh signal to the cytoplasm utilizes an unusual mechanism involving consecutive repressive interactions between Hh and its receptor components, Patched (Ptc) and Smoothened (Smo). Several cytoplasmic proteins involved in Hh signal transduction are known in Drosophila, but mammalian homologs are known only for the Cubitus interruptus (Ci) transcription factor (GLI(1-3)) and for the Ci/GLI-associated protein, Suppressor of Fused (Su(fu)). In this study I analyzed the mechanisms of how the Hh receptor Ptc regulates the signal transducer Smo, and how Smo relays the Shh signal from the cell surface to the cytoplasm ultimately leading to the activation of GLI transcription factors. In Drosophila, the kinesin-like protein Costal2 (Cos2) is required for suppression of Hh target gene expression in the absence of ligand, and loss of Cos2 causes embryonic lethality. Cos2 acts by bridging Smo to the Ci. Another protein, Su(Fu) exerts a weak suppressive influence on Ci activity and loss of Su(Fu) causes subtle changes in Drosophila wing pattern. This study revealed that domains in Smo that are critical for Cos2 binding in Drosophila are dispensable for mammalian Smo function. Furthermore, by analyzing the function of Su(Fu) and the closest mouse homologs of Cos2 by protein overexpression and RNA interference I found that inhibition of the Hh response pathway in the absence of ligand does not require Cos2 activity, but instead critically depends on the activity of Su(Fu). These results indicate that a major change in the mechanism of action of a conserved signaling pathway occurred during evolution, probably through phenotypic drift made possible by the existence in some species of two parallel pathways acting between the Hh receptor and the Ci/GLI transcription factors. In a second approach to unravel Hh signaling we cloned > 90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase-activity deficient mutants. Using this kinome resource as a screening tool, two kinases, MAP3K10 and DYRK2 were found to regulate Shh signaling. DYRK2 directly phosphorylated and induced the proteasome dependent degradation of the key Hh-pathway regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2.

Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

The influence of HIV infection to the periodontium; a clinical, microbiological, and enzymological study

The main targets of human immunodeficiency virus (HIV) are CD4 receptors of CD4+ lymphocytes and many other cells such as monocytes/macrophages, megakaryocytes, peripheral blood dendritic cells, follicular dendritic cells (DC), epidermal Langerhans cells, and astrocytes. Infection and killing of CD4+ lymphocytes or false reaction of the body to HIV infection and the spontaneous apoptosis of CD4+ lymphocytes decrease CD4+ lymphocyte counts leading to immunosuppression, further disease progression, and appearance of opportunistic infections and malignancies. Oral manifestations are considered to be among the first signs of HIV infection. Enhanced degradation of extracellular matrix and basement membrane components in oral diseases including periodontitis is caused by Zn-dependent enzymes called matrix metalloproteinases (MMPs). The levels and degrees of activation of MMP-1, -2, -3, -7, -8, -9, -25, -26, tissue inhibitors of MMPs (TIMP)-1 and -2, and myeloperoxidase (MPO) and collagenolytic/gelatinolytic activities, and also Ig A, -G, and -M, total protein, and albumin levels in a two-year follow-up were studied from salivary samples. The expression of MMP-7, -8, -9, -25, and -26 immunoreactivities in gingival tissue specimens were studied. Healthy HIV-negative subjects served as controls. All studied clinical periodontal parameters and microbiological evaluation of the periodontopathogens showed that periodontal health of the HIV-positive patients was moderately decreased in comparison to the healthy controls. The levels of Candida in the periodontal pockets and salivary MPO increased with the severity of HIV infection. Immunoreactivities and levels of MMPs and TIMPs, and MMP activities (collagenase, gelatinase) were enhanced in the HIV-positive patient salivary samples relative to the healthy controls regardless of the phase of HIV infection. However, these parameters did not reflect periodontal status in a similar way as in the generally healthy periodontitis patients. Salivary total protein, albumin, IgA, -G, and -M levels were significantly higher in all phases of HIV infection compared to the controls, and salivary total protein, IgG and IgM levels remained higher after two years follow-up, partly correlating with the disease progression and which may reflect the leakage of serum components into the mouth and thus a decreased mucosal barrier. Salivary analyses of MMPs and TIMPs with immunohistochemical analyses showed that HIV infection could predispose to periodontal destruction when compared with healthy controls or the body s defence reactions associated with HIV infection may have been reflected or mediated by MMPs.

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Women, environmental changes and forestry-related development : Gender-affected roles of rural people in land degradation and environmental rehabilitation in a dry region of Sudan

The purpose of the present study was to increase understanding of the interaction of rural people and, specifically, women with the environment in a dry area in Sudan. The study that included both nomadic pastoralists and farmers aimed at answering two main research questions, namely: What kinds of roles have the local people, and the women in particular, had in land degradation in the study area and what kinds of issues would a gender-sensitive, forestry-related environmental rehabilitation intervention need to consider there? The study adopted the definition of land degradation as proposed by the United Nations Convention to Combat Desertification (UNCCD), which describes land degradation as reduction or loss the biological or economic productivity and complexity of land in arid, semi-arid and dry sub-humid areas. The Convention perceives desertification as land degradation. The dry study area in Sudan, South of the Sahara, has been the subject of land degradation or desertification discussions since the 1970s, and other studies have been also conducted to assess the degradation in the area. Nevertheless, the exact occurrence, scale and local significance of land degradation in the area is still unclear. This study explored how the rural population whose livelihood depended on the area, perceived environmental changes occurring there and compared their conceptions with other sources of information of the area such as research reports. The main fieldwork methods included interviews with open-ended questions and observation of people and the environment. The theoretical framework conceptualised the rural population as land users whose choices of environmental activities are affected by multiple factors in the social and biophysical contexts in which they live. It was emphasised that these factors have their own specific characteristics in different contexts, simultaneously recognising that there are also factors that generally affect environmental practices in various areas such as the land users' environmental literacy (conceptions of the environment), gender and livelihood needs. The people studied described that environmental changes, such as reduced vegetation cover and cropland production, had complicated the maintenance of their livelihoods in the study area. Some degraded sites were also identified through observations during the fieldwork. Whether a large-scale reduction of cropland productivity had occurred in the farmers' croplands remained, however, unclear. The study found that the environmental impact of the rural women's activities varied and was normally limited. The women's most significant environmental impact resulted from their cutting of trees, which was likely to contribute, at least in some places, to land degradation, affecting the environment together with climate and livestock. However, when a wider perspective is taken, it becomes questionable whether the women have really played roles in land degradation, since gender, poverty and the need to maintain livelihood had caused them to conduct environmentally harmful activities. The women have had, however, no power to change the causes of their activities. The findings further suggested that an inadequate availability of food was the most critical problem in the study area. Therefore, an environmental programme in the area was suggested to include technical measures to increase the productivity of croplands, opportunities for income generation and readiness to co-operate with other programmes to improve the local people's abilities to maintain their livelihoods. In order to protect the environment and alleviate the women's work burden, the introduction of fuel-saving stoves was also suggested. Furthermore, it was suggested that increased planting of trees on homesteads would be supported by an easy availability of tree seedlings. Planting trees on common property land was, however, perceived as extremely demanding in the study area, due to scarcity of such land. In addition, it became apparent that the local land users, and women in particular, needed to allocate their labour to maintain the immediate livelihood of their families and were not motivated to allocate their labour solely for environmental rehabilitation. Nonetheless, from the point of view of the existing social structures, women's active participation in a community-based environmental programme would be rather natural, particularly among the farmer women who had already formed a women's group and participated in communal decision making. Forming of a women group or groups was suggested to further support both the farmer women's and pastoral women's active participation within an environmental programme and their general empowerment. An Environmental programme would need to acknowledge that improving rural people's well-being and maintaining their livelihood in the study area requires development and co-operation with various sectors in Sudan.

Sweet potato chlorotic stunt virus: Studies on viral synergism and suppression of RNA silencing

The studies presented in this thesis aimed to a better understanding of the molecular biology of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus, Closteroviridae) and its role in the development of synergistic viral diseases. The emphasis was on the severe sweet potato virus disease (SPVD) that results from a synergistic interaction of SPCSV and Sweet potato feathery mottle virus (SPFMV, Potyvirus, Potyviridae). SPVD is the most important disease affecting sweetpotato. It is manifested as a significant increase in symptom severity and SPFMV titres. This is accompanied by a dramatic sweetpotato yield reduction. SPCSV titres remain little affected in the diseased plants. Viral synergistic interactions have been associated with the suppression of an adaptive general defence mechanism discovered in plants and known as RNA silencing. In the studies of this thesis two novel proteins (RNase3 and p22) identified in the genome of a Ugandan SPCSV isolate were shown to be involved in suppression of RNA silencing. RNase3 displayed a dsRNA-specific endonuclease activity that enhanced the RNA-silencing suppression activity of p22. Comparative analyses of criniviral genomes revealed variability in the gene content at the 3´end of the genomic RNA1. Molecular analyses of different isolates of SPCSV indicated a marked intraspecific heterogeneity in this region where the p22 and RNase3 genes are located. Isolates of the East African strain of SPCSV from Tanzania and Peru and an isolate from Israel were missing a 767-nt fragment that included the p22 gene. However, regardless of the absence of p22, all SPCSV isolates acted synergistically with SPFMV in co-infected sweetpotato, enhanced SPFMV titres and caused SPVD. These results showed that p22 is dispensable for development of SPVD. The role of RNase3 in SPVD was then studied by generating transgenic plants expressing the RNase3 protein. These plants had increased titres of SPFMV (ca. 600-fold higher in comparison with nontransgenic plants) 2-3 weeks after graft inoculation and displayed the characteristic SPVD symptoms. RNA silencing suppression (RSS) activity of RNase3 was detected in agroinfiltrated leaves of Nicotiana bethamiana. In vitro studies showed that RNase3 was able to cleave small interferring RNAs (siRNA) to products of ~14-nt. The data thus identified RNase3 as a suppressor of RNA silencing able to cleave siRNAs. RNase3 expression alone was sufficient for breaking down resistance to SPFMV in sweetpotato and for the development of SPVD. Similar RNase III-like genes exist in animal viruses which points out a novel and possibly more general mechanism of RSS by viruses. A reproducible method of sweetpotato transformation was used to target RNA silencing against the SPCSV polymerase region (RdRp) with an intron-spliced hairpin construct. Hence, engineered resistance to SPCSV was obtained. Ten out of 20 transgenic events challenged with SPCSV alone showed significantly reduced virus titres. This was however not sufficient to prevent SPVD upon coinfection with SPFMV. Immunity to SPCSV seems to be required to control SPVD and targeting of different SPCSV regions need to be assessed in further studies. Based on the identified key role of RNase3 in SPVD the possibility to design constructs that target this gene might prove more efficient in future studies.

Monitoring of Fungal Growth and Degradation of Wood

Valko- ja ruskolahosienet tunnetaan luonnossa tehokkaimpina puun ja karikkeen lignoselluloosan lahottajina. Valkolahosienet pystyvät hajottamaan kaikkia puun osia: ligniiniä, selluloosaa ja hemiselluloosaa. Selektiivisesti ligniiniä hajottavat sienet lahottavat puusta suhteessa enemmän vaikeasti hajoavaa ligniiniä kuin selluloosaa tai hemiselluloosaa, jolloin jäljelle jää valkoista ja miltei puhdasta selluloosaa. Bioteknisissä sovelluksissa juuri selektiviiviset valkolahottajat ovat kiinnostavia. Niiden avulla voidaan puuhaketta esikäsitellä esimerkiksi paperinvalmistuksessa haitallisen ligniinin poistamiseksi. Ruskolahosienet ovat huomattavia puun, puutavaran ja puisten rakenteiden lahottajia, kuten tässä työssä käytetty Gloeophyllum trabeum (saunasieni ) ja Poria (Postia) placenta (istukkakääpä). Ruskolahosienet hajottavat puusta hemiselluloosan lisäksi selluloosaa, jolloin jää jäljelle ruskea ja jauhomaiseksi mureneva ligniini. Ruskolahosienet muovaavat ligniiniä jonkin verran. Kahden ruskolahosienen G. trabeumin ja P. placentan lisäksi tutkittiin valkolahosieniä, joista Ceriporiopsis subvermispora (karstakääpä) ja harvinainen Physisporinus rivulosus -sieni (talikääpä) hajottavat ligniiniä erittäin selektiivisesti. Phanerochaete chrysosporium on kaikkialla paljon tutkittu sieni, ja Phlebia radiata valkolahosientä (rusorypykkä) on tutkittu paljon mikrobiologian osastolla. Lisäksi tutkittiin Phlebia tremellosa -sienten (hytyrypykkä) ligninolyyttisten entsyymien tuottoa ja 14C-leimatun synteettisen ligniinin (DHP) hajotusta. P. radiata ja P. tremellosa -sienten on todettu aiemmin hajottavan ligniiniä selektiivisesti. Työssä selvitettiin miten sienten kasvua voi mitata, miten vertailukelpoisia eri mittaamismenetelmillä saadut tulokset ovat ja ilmenevätkö sienten aktiivisimmat kasvuvaiheet samaan aikaan eri menetelmillä mitattuna. Tärkeimmät tulokset olivat seuraavat havainnot: (i) P. radiata ja P. tremellosa -sienikannat tuottivat ligniini- ja mangaaniperoksidaasientsyymejä (LiP ja MnP) sekä lakkaasia, ja sienistä puhdistettiin 2-3 LiP- ja P. radiatasta yksi MnP-entsyymi; (ii) P. tremellosa -sienet hajottivat leimattua synteettistä ligniiniä (DHP) yhtä hyvin kuin paljon tutkitut P. chrysosporium ja P. radiata -sienet; (iii) puu, sienen luonnollinen kasvualusta, lisäsi valkolaho- ja ruskolahosienten demetoksylaatiota [O14CH3]-leimatusta ligniinin malliyhdisteestä 14CO2:ksi ilman puuta olleeseen alustaan verrattuna; (iv) demetoksylaatio (14CO2:n tuotto) oli normaalissa ilma-atmosfäärissä useimmiten parempi happeen verrattuna; (v) hapessa paras 14CO2:n tuotto saatiin puupalakasvatuksissa, joihin oli lisätty ravinnetyppeä tai typen lisäksi glukoosia sekä valkolaho- että ruskolahosienillä; (vi) ilmassa 14CO2:n tuotto oli puulla voimakkainta valkolahosienillä ilman lisäravinteita, kun taas G. trabeum -sienellä se oli yhtä hyvä eri alustoissa; (vii) biomassan muodostuminen rihmastojen ergosterolipitoisuuksista mitattuna oli ruskolahosienillä parempi kuin valkolahosienillä; (viii) ja biomassojen huippupitoisuudet olivat 6:lla sienellä eri suuruisia ja niiden maksimimäärien ajankohdat vaihtelivat viiden viikon kasvatusten kuluessa. Mikrobiologian osastolla Viikissä eristetty ja paljon tutkittu P. radiata -valkolahosieni oli mukana kaikissa tehdyissä kokeissa. Sienen LiP-aktiivisuus ja 14CO2:n tuotto 14C-rengas-leimatusta synteettisestä ligniinistä (DHP) korreloivat erittäin hyvin. Biomassan muodostuminen ergosterolilla määritettynä tuki hyvin entsyymiaktiivisuusmittauksilla ja isotooppikasvatuksilla saatuja tuloksia.

Bioavailability aspects of hydrophobic contaminant degradation in soils

This thesis concentrates on bioavailability of organic soil contaminants in the context of bioremediation of soil contaminated with volatile or non-volatile hydrophobic pollutants. Bioavailability and biodegradation was studied from four viewpoints: (i) Improvement of bioavailability and biodegradation of volatile hydrocarbons in contained bioremediation systems at laboratory - and pilot-scale. (ii) Improvement of bioavailability of non-volatile, hydrophobic compounds in such systems. (iii) Biodegradation of a non-volatile hydrophobic compound in soil organic matter in microcosms. (iiii) Bioavailability of nitrogen in an open, full-scale bioremediation system. It was demonstrated that volatility of organic compounds can be controlled by amending the soil with adsorbents. The sorbed hydrocarbons were shown to be available to soil microbiota. As the result, biodegradation of the volatile hydrocarbons was greatly favored at the expense of volatilization. PAH compounds were shown to be mobilized and their bioavailability improved by a hydrophobic, non-toxic additive, vegetable oil. Bioavailability of the PAHs was recorded as an increased toxicity of the soil. In spite of the increased bioavailability, biodegradation of the PAHs decreased. In microcosms simulating boreal forest organic surface soil, PAH-compound (pyrene) was shown to be removed from soil biologically. Therefore hydrophobicity of the substrate does not necessarily mean low availability and biodegradation in organic soil. Finally, in this thesis it was demonstrated that an unsuitable source of nitrogen or its overdose resulted in wasteful spending of this nutrient and even harmful effects on soil microbes. Such events may inhibit rather than promote the bioremediation process in soil.

Prolamin degradation in sourdoughs

This thesis examines protein behaviours that occur during cereal fermentations. The focus is on the prolamin degradation in sourdoughs. The thesis also looks at what happens to the oat globulins during an oat bran acidification process. The cereal prolamins are unique proteins in many respects. The wheat prolamins (glutenins and gliadins) are responsible for the formation of the gluten that provides the viscoelastic properties to wheat doughs whereas the rye prolamins (secalins) are unable to develop gluten-like structures. In addition, many baking technological features, such as flavour, shelf-life and dough properties are affected by the protein degradation that might occur during processing. On the other hand, the prolamins contain protein structures that are harmful to gluten sensitive people. It is thus evident that the degradation of the prolamins in sourdough processes may be approached from various aspects. This thesis describes some of these approaches. Four different cereal fermentations were carried out. Wheat sourdough (WSD) and rye sourdough (RSD) fermentations represented traditional sourdoughs. A germinated-wheat sourdough (GWSD) was a novel sourdough type that was prepared using germinated wheat grains that had high and diverse proteolytic activities. The oat bran fermentation (OBF) represented a fermentation system that lacked functional cereal proteases. The high molecular weight glutenins and rye secalins were degraded during the WSD and RSD fermentations, respectively. It was noteworthy that in WSD only a very limited degradation of the gliadins occurred. The gliadins were, however, hydrolysed very extensively during the GWSD fermentation. No protein degradation was observable in the OBF system. Instead the acidification altered the solubility of the oat globulins and this finally led to their aggregation. This thesis confirms that the endogenous proteases of cereals hydrolyse cereal prolamins in sourdoughs. The thesis also shows that the proteolytic activity of the used cereal raw material determines the extent of proteolysis that occurs in sourdough. This means that bakers may adjust the protein degradation in their sourdoughs by selecting the raw material based on its proteolytic activity. The thesis also demonstrates that by using germinated grains, with high and diverse proteolytic activity in sourdough preparations, the prolamins can be extensively degraded. Whether such highly proteolytic food technology could be used to manufacture new gluten-free cereal-based products for gluten sensitive people remains to be solved.

Degradation of lignin and other 14C-labelled compounds in compost and soil with an emphasis on white-rot fungi

Puu, ruohokasvit ja näistä tehdyt tuotteet kuten mekaanisesta massasta valmistettu sanomalehtipaperi sisältävät ligniiniä, joka hajoaa yleensä hyvin hitaasti luonnossa. Valkolahosienet hajottavat ligniiniä tehokkaimmin, ja koska niiden tuottamat entsyymit hajottavat myös muita vaikeasti hajoavia yhdisteitä, voidaan valkolahosienten avulla mahdollisesti puhdistaa saastuneita maita. Tässä työssä haluttiin selvittää, säilyttävätkö valkolahosienet (Abortiporus biennis, Bjerkandera adusta, Dichomitus squalens, Phanerochaete chrysosporium, Phanerochaete sordida, Phlebia radiata, Pleurotus ostreatus, Trametes hirsuta ja Trametes versicolor) aktiivisuutensa ja kasvavatko ne maassa. Aktiivisuutta mitattiin seuraamalla sienten synteettisen ligniinin (14C-DHP) hajotuskykyä. T. versicolor (silkkivyökääpä) osoittautui tehokkaimmaksi ligniinin hajottajaksi ja sen pentakloorifenolin (PCP) hajotuskykyä tutkittiin erillisessä kokeessa. Entiset tai pitkään käytössä olleet saha-alueet ovat yhä saastuneet puun käsittelyaineista peräisin olevilla kloorifenoleilla. Biohajoavien muovien kehitystyö sekä kompostoinnin yleistyminen jätteiden käsittelymenetelmänä ovat luoneet tarpeen materiaalien biohajoavuuden määrittämiseen. Euroopan standardisoimisjärjestön (CEN) kontrolloidussa kompostitestissä biohajoavuus määritetään materiaalin hajoamisen aikana muodostuvan hiilidioksidin perusteella. Hiilidioksidin tuotto mitataan sekä näytettä sisältävästä kompostista että kompostista ilman näytettä, ja tällöin oletetaan, että kompostin orgaaninen aines molemmissa komposteissa (tausta) tuottaa yhtä paljon hiilidioksidia. Testin puutteeksi saattaa osoittautua kompostissa tai maassa esiintyvä "priming effect". Tällä tarkoitetaan materiaalin lisäämisen jälkeen esiintyvää epänormaalin suurita tai pientä hiilidioksidin muodostusta, minkä seurauksena testin tulosksena saatava biohajoavuus on virheellinen. Ligniinin hajotessa muodostuu enemmän humusta kuin hiilidioksidia, koska ligniini on humuksen tärkein lähtöaine. Näin ollen ligniiniä sisältävät paperituotteet saattavat testin mukaan vaikuttaa biologisesti hajoamattomilta. Valkolahosienet hajottivat 4-23% ligniinistä hiilidioksidiksi ja T. versicolor 29% PCP:sta. Kompostissa ligniini hajosi hiilidioksidiksi 58°C:ssa huomattavasti vähemmän (8%) kuin lämpötiloissa 35°C ja 50°C (23-24%). Kompostin todennäköisesti tärkeimpien ligniinin hajottajien, termofiilisten sienten, tyypillinen optimilämpötila on 45°C, eivätkä ne ole enää aktiivisia 58°C:ssa. Sekä maassa että kompostissa ligniini sitoutui kuitenkin suurimmaksi osaksi humukseen. Valkolahosienet hajottivat sekä humukseen sitoutunutta ligniiniä että PCP:ia, mutta kompostin sekapopulaatio ei tähän pystynyt, ja ligniiniä sitoutui humukseen yhä enemmän kompostoinnin aikana. T. versicolor hajotti PCP:ia tehokkaasti, eikä se tuottanut myrkyllisiä kloorianisoleja, joita jotkut valkolahosienet saattavat muodostaa kloorifenoleista. Priming effect ilmiötä tutkittiin eri ikäisissä ja kypsyydeltään erilaisissa komposteissa. Kompostit erosvat toisistaan myös hajoamattoman jätteen määrän ja mikrobipopulaation suhteen. Negatiivinen priming effect havaittiin kaikissa epästabiileissa komposteissa (ikä enintään 6 kk), ja sen lisäksi yhdessä näistä komposteista positiivinen priming effect kokeen lopussa. Stabiileissa komposteissa (ikä vähintään 6 kk) ilmiötä ei sen sijaan havaittu. Epästabiileissa komposteissa biohajoavuudelle saadut tulokset eivät siis ole luotettavia. Työn tulosten perusteella valkolahosienet, ja erityisesti T. versicolor, ovat lupaavia saastuneen maan puhdistukseen, joskin sienirihmaston mahdollisuudet säilyä aktiivisena maan alkuperäisen mikrobipopulaation kanssa täytyy vielä selvittää. Kompostin sekapopulaatio, joka ei sisällä valkolahosieniä, hajotti ligniiniä yllättävän tehokkaasti termofiilisille sienille sopivissa lämpötiloissa, vaikka ligniini sitoutuikin pääasiallisesti humukseen. Kompostin kypsyys osoittautui tärkeäksi tekijäksi kontrolloidun kompostitestin onnistumisen kannalta. Priming effect ilmiön välttämiseksi on varmistettava, että testissä käytetty komposti on riittävän kypsä. Kompostien mikrobipopulaation koostumusta kompostoinnin eri vaiheissa tulisi tarkemmin selvittää, koska stabiilien ja epästabiilien kompostien ero aiheutui todennäköisesti populaatioiden rakenteessa vallitsevista eroista. Näin myös priming effect ilmiön syyt voitaisiin selittää paremmin.

Lignocellulose degradation and humus modification by the fungus Paecilomyces inflatus

Composting is the biological conversion of solid organic waste into usable end products such as fertilizers, substrates for mushroom production and biogas. Although composts are highly variable in their bulk composition, composting material is generally based on lignocellulose compounds derived from agricultural, forestry, fruit and vegetable processing, household and municipal wastes. Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore lignocellulose degradation is essential for maintaining the global carbon cycle. In compost, the active component involved in the biodegradation and conversion processes is the resident microbial population, among which microfungi play a very important role. In composting pile the warm, humid, and aerobic environment provides the optimal conditions for their development. Microfungi use many carbon sources, including lignocellulosic polymers and can survive in extreme conditions. Typically microfungi are responsible for compost maturation. In order to improve the composting process, more information is needed about the microbial degradation process. Better knowledge on the lignocellulose degradation by microfungi could be used to optimize the composting process. Thus, this thesis focused on lignocellulose and humic compounds degradation by a microfungus Paecilomyces inflatus, which belongs to a flora of common microbial compost, soil and decaying plant remains. It is a very common species in Europe, North America and Asia. The lignocellulose and humic compounds degradation was studied using several methods including measurements of carbon release from 14C-labelled compounds, such as synthetic lignin (dehydrogenative polymer, DHP) and humic acids, as well as by determination of fibre composition using chemical detergents and sulphuric acid. Spectrophotometric enzyme assays were conducted to detect extracellular lignocellulose-degrading hydrolytic and oxidative enzymes. Paecilomyces inflatus secreted clearly extracellular laccase to the culture media. Laccase was involved in the degradation process of lignin and humic acids. In compost P. inflatus mineralised 6-10% of 14C-labelled DHP into carbon dioxide. About 15% of labelled DHP was converted into water-soluble compounds. Also humic acids were partly mineralised and converted into water-soluble material, such as low-molecular mass fulvic acid-like compounds. Although laccase activity in aromatics-rich compost media clearly is connected with the degradation process of lignin and lignin-like compounds, it may preferentially effect the polymerisation and/or detoxification of such aromatic compounds. P. inflatus can degrade lignin and carbohydrates also while growing in straw and in wood. The cellulolytic enzyme system includes endoglucanase and β-glucosidase. In P. inflatus the secretion of these enzymes was stimulated by low-molecular-weight aromatics, such as soil humic acid and veratric acid. When strains of P. inflatus from different ecophysiological origins were compared, indications were found that specific adaptation strategies needed for lignocellulosics degradation may operate in P. inflatus. The degradative features of these microfungi are on relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not established. The results of this study may help to understand, control and better design the process of plant polymer conversion in compost environment, with a special emphasis on the role of ubiquitous microfungi.