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.

Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.

Periodontitis and peri-implantitis biomarkers in human oral fluids and the null-allele mouse model

Tissue destruction associated with the periodontal disease progression is caused by a cascade of host and microbial factors and proteolytic enzymes. Aberrant laminin-332 (Ln-332), human beta defensin (hBD), and matrix metalloproteinase (MMP) functions have been found in oral inflammatory diseases. The null-allele mouse model appears as the next step in oral disease research. The MMP-8 knock-out mouse model allowed us to clarify the involvement of MMP-8 in vivo in oral and related inflammatory diseases where MMP-8 is suggested to play a key role in tissue destruction. The cleaved Ln-332 γ2-chain species has been implicated in the apical migration of sulcular epithelial cells during the formation of periodontal pockets. We demonstrated that increased Ln-332 fragment levels in gingival crevicular fluid (GCF) are strongly associated with the severity of inflammation in periodontitis. Porphyromonas gingivalis trypsin-like proteinase can cleave an intact Ln-332 γ2-chain into smaller fragments and eventually promote the formation of periodontal pockets. hBDs are components of an innate mucosal defense against pathogenic microbes. Our results suggest that P. gingivalis trypsin-like proteinase can degrade hBD and thus reduce the innate immune response. Elevated levels and the increased activity of MMPs have been detected in several pathological tissue-destructive conditions where MMPs are shown to cleave extracellular matrix (ECM) and basement membrane (BM) molecules and to facilitate tissue destruction. Elevated levels of MMP-8 have been reported in many inflammatory diseases. In periodontitis, MMP-8 levels in gingival crevicular fluid (GCF) and in peri-implant sulcular fluid (PISF) are elevated at sites of active inflammation, and the increased levels of MMP-8 are mainly responsible for collagenase activity, which leads to tissue destruction. MMP-25, expressed by neutrophils, is involved in inflammatory diseases and in ECM turnover. MMP-26 can degrade ECM components and serve as an activator of other MMP enzymes. We further confirmed that increased levels and activation of MMP-8, -25, and -26 in GCF, PISF, and inflamed gingival tissue are associated with the severity of periodontal/peri-implant inflammation. We evaluated the role of MMP-8 in P. gingivalis-induced periodontitis by comparing MMP-8 knock-out (MMP8-/-) and wild-type mice. Surprisingly, MMP-8 significantly attenuated P. gingivalis-induced site-specific alveolar bone loss. We also evaluated systemic changes in serum immunoglobulin and lipoprotein profiles among these mouse groups. P. gingivalis infection increased HDL/VLDL particle size in the MMP-8-/- mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total LPS and IgG antibody levels were enhanced in both mice groups. P. gingivalis-induced periodontitis, especially in MMP-8-/- mice, is associated with severe alveolar bone loss and with systemic inflammatory and lipoprotein changes that are likely to be involved in early atherosclerosis.

Studies on Matrix Metalloproteinase (MMP-2, -9 and -14) and β2 Integrin Targeting as Potential Anticancer Therapeutics

Proteolytic enzymes, such as matrix metalloproteinases (MMP), are associated to the progression of several cancers. They degrade extracellular components, which helps tumors to expand and cancer cells to escape from the primary site. Of all MMPs, gelatinases (MMP-2 and -9) and membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), in particular, are often associated to more aggressive types of head and neck carcinomas as well as to a poorer outcome in patient survival. Although therapies during the last decades have advanced, the mortality of the disease is still rather high and adjuvant therapies are searched for continuously. MMP-9 and MT1-MMP are also involved in neo-angiogenesis, which is necessary for tumor expansion. For this reason, we have identified synthetic peptides-targeting gelatinases and MT1-MMP, and have also evaluated their anticancer effects in vitro and in vivo. Antigelatinolytic peptides effectively inhibited tongue-carcinoma cell invasion and reduced the growth of xenografted tumors. In tumor samples of mice that were treated with antigelatinolytic peptides, the micro-vessel density was significantly reduced. We also identified a novel MT1-MMP targeting peptide and demonstrated that it exerted anticancer effects against several malignant cell lines in vitro. The effects of MT1-MMP inhibition on tongue-squamous cell carcinomas were evaluated by using xenograft tumors, which it effectively inhibited. Tranexamic acid was also demonstrated to inhibit tongue-squamous cell carcinoma invasion, most probably due to its ability to prevent the plasmin-mediated activation of proMMP-9. Leukocyte β2 integrins are another interesting option when evaluating targets for the therapeutic intervention of inflammatory conditions or malignancies of hematopoietic origin, since β2 integrins are expressed mainly by leukocytes. We identified a novel technique for screening small-molecule libraries against β2 integrins, and by using this technique we identified a novel αMβ2 integrin-binding chemical (IMB-10). IMB-10 significantly enhances leukocyte adhesion and inhibits their motility. We also demonstrated that IMB-10 can be used to inhibit inflammation and lymphoma growth in vivo. Interestingly, IMB-10 also reduced leukocyte tumor infiltration and inhibited tumor invasion.

Pharmacogenetic Variation at CYP2D6, CYP2C9, and CYP2C19: Population Genetic and Forensic Aspects

Pharmacogenetics deals with genetically determined variation in drug response. In this context, three phase I drug-metabolizing enzymes, CYP2D6, CYP2C9, and CYP2C19, have a central role, affecting the metabolism of about 20-30% of clinically used drugs. Since genes coding for these enzymes in human populations exhibit high genetic polymorphism, they are of major pharmacogenetic importance. The aims of this study were to develop new genotyping methods for CYP2D6, CYP2C9, and CYP2C19 that would cover the most important genetic variants altering the enzyme activity, and, for the first time, to describe the distribution of genetic variation at these loci on global and microgeographic scales. In addition, pharmacogenetics was applied to a postmortem forensic setting to elucidate the role of genetic variation in drug intoxications, focusing mainly on cases related to tricyclic antidepressants, which are commonly involved in fatal drug poisonings in Finland. Genetic variability data were obtained by genotyping new population samples by the methods developed based on PCR and multiplex single-nucleotide primer extension reaction, as well as by collecting data from the literature. Data consisted of 138, 129, and 146 population samples for CYP2D6, CYP2C9, and CYP2C19, respectively. In addition, over 200 postmortem forensic cases were examined with respect to drug and metabolite concentrations and genotypic variation at CYP2D6 and CYP2C19. The distribution of genetic variation within and among human populations was analyzed by descriptive statistics and variance analysis and by correlating the genetic and geographic distances using Mantel tests and spatial autocorrelation. The correlation between phenotypic and genotypic variation in drug metabolism observed in postmortem cases was also analyzed statistically. The genotyping methods developed proved to be informative, technically feasible, and cost-effective. Detailed molecular analysis of CYP2D6 genetic variation in a global survey of human populations revealed that the pattern of variation was similar to those of neutral genomic markers. Most of the CYP2D6 diversity was observed within populations, and the spatial pattern of variation was best described as clinal. On the other hand, genetic variants of CYP2D6, CYP2C9, and CYP2C19 associated with altered enzymatic activity could reach extremely high frequencies in certain geographic regions. Pharmacogenetic variation may also be significantly affected by population-specific demographic histories, as seen within the Finnish population. When pharmacogenetics was applied to a postmortem forensic setting, a correlation between amitriptyline metabolic ratios and genetic variation at CYP2D6 and CYP2C19 was observed in the sample material, even in the presence of confounding factors typical for these cases. In addition, a case of doxepin-related fatal poisoning was shown to be associated with a genetic defect at CYP2D6. Each of the genes studied showed a distinct variation pattern in human populations and high frequencies of altered activity variants, which may reflect the neutral evolution and/or selective pressures caused by dietary or environmental exposure. The results are relevant also from the clinical point of view since the genetic variation at CYP2D6, CYP2C9, and CYP2C19 already has a range of clinical applications, e.g. in cancer treatment and oral anticoagulation therapy. This study revealed that pharmacogenetics may also contribute valuable information to the medicolegal investigation of sudden, unexpected deaths.

Complement system and alcoholic liver disease

Alcoholic liver disease (ALD) is a well recognized and growing health problem worldwide. ALD advances from fatty liver to inflammation, necrosis, fibrosis and cirrhosis. There is accumulating evidence that the innate immune system is involved in alcoholic liver injury. Within the innate and acquired immune systems, the complement system participates in inflammatory reactions and in the elimination of invading foreign, as well as endogenous apoptotic or injured cells. The present study aimed at evaluating the role of the complement system in the development of alcoholic liver injury. First, in order to study the effects of chronic ethanol intake on the complement system, the deposition of complement components in liver and the expression of liver genes associated with complement in animals with alcohol-induced liver injury were examined. It was demonstrated that chronic alcohol exposure leads to hepatic deposition of the complement components C1, C3, C8 and C9 in the livers of rats. Liver gene expression analysis showed that ethanol up-regulated the expression of transcripts for complement factors B, C1qA, C2, C3 and clusterin. In contrast, ethanol down-regulated the expression of the complement regulators factor H, C4bp and factor D and the terminal complement components C6, C8α and C9. Secondly, the role of the terminal complement pathway in the development of ALD was evaluated by using rats genetically deficient in the complement component C6 (C6-/-). It was found that chronic ethanol feeding induced more liver pathology (steatosis and inflammatory changes) in C6-/- rats than in wild type rats. The hepatic triacylglyceride content and plasma alanine aminotransferase activity increased in C6-/- rats, supporting the histopathological findings and elevation of the plasma pro-/anti-inflammatory TNF-/IL-10 ratio was also more marked in C6-/- rats. Third, the role of the alternative pathway in the development of alcoholic liver steatosis was characterized by using C3-/- mice. In C3-/- mice ethanol feeding tended to reduce steatosis and had no further effect on liver triacylglyceride, liver/body weight ratio nor on liver malondialdehyde level and serum alanine aminotransferase activity. In C3-/- mice alcohol-induced liver steatosis was reduced also after an acute alcohol challenge. In both wild type and C3-/- mice ethanol markedly reduced serum cholesterol and ApoA-I levels, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid binding proteins and fatty acid -oxidation enzymes. In contrast, exclusively in C3-/- mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated the expression of transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. In conclusion, this study has demonstrated that the complement system is involved in the development of alcohol-induced liver injury. Chronic alcohol exposure causes local complement activation and induction of mRNA expression of classical and alternative pathway components in the liver. In contrast expression of the terminal pathway components and soluble regulators were decreased. A deficient terminal complement pathway predisposes to alcoholic liver damage and promotes a pro-inflammatory cytokine response. Complement component C3 contributes to the development of alcohol-induced fatty liver and its consequences by affecting regulatory and specific transcription factors of lipid homeostasis.

Oncolytic adenoviruses for treatment of ovarian cancer

Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.

Fibroblast nemosis in malignant progression of epithelial cells

Paracrine regulation between the components of the tumour microenvironment cancer cells, activated fibroblasts, immune and endothelial cells is under intense investigation. The signals between the different cell types are mediated by soluble factors, such as growth factors, proinflammatory cytokines and proteolytic enzymes. Nemosis is an experimental in vitro model of fibroblast activation, leading to increased production of such mediators. Nemotic activation of fibroblasts occurs as they are forced to cluster thereby forming a multicellular spheroid. The aim of the present studies was to elucidate the mechanisms underlying the nemotic response of cancer-associated fibroblasts (CAF) and the role of nemosis in paracrine regulation between activated fibroblasts and benign and malignant epithelial cells. The results presented in this thesis demonstrate that the nemotic response of CAFs and normal fibroblasts differs, and inter-individual variations exist between fibroblast populations. In co-culture experiments, fibroblasts increased colony formation of squamous cell carcinoma (SCC) cells, and CAFs further augmented this, highlighting the tumour-evolving properties of CAFs. Furthermore, fibroblast monolayers in those co-cultures started to cluster spontaneously. This kind of spontaneous nemosis response might take place also in vivo, although more direct evidence of this still needs to be obtained. The HaCaT skin carcinoma progression model was used to study the effects of benign and malignant keratinocytes on fibroblast nemosis. Benign HaCaT cells inhibited fibroblast nemosis, observed as inhibition of cyclooxygenase 2 (COX-2) induction in nemotic spheroids. In contrast, malignant HaCaTs further augmented the nemotic response by increasing expression of COX-2 and the growth factors hepatocyte growth factor / scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF), as well as causing a myofibroblastic differentiation of nemotic fibroblasts into fibroblasts resembling CAFs. On the other side of this reciprocal signalling, factors secreted into conditioned medium by the nemotic fibroblasts promoted proliferation and motility of the HaCaT cell lines. Notably, the nemotic fibroblast medium increased the expression of p63, a transcription factor linked to carcinogenesis, also in the highly metastatic HaCaT cells. These results emphasize the paracrine role of factors secreted by activated fibroblasts in driving tumour progression. We also investigated the epithelial-mesenchymal transition (EMT) of the HaCaT clones in response to transforming growth factor β (TGF-β), which is a well-characterized inducer of EMT. TGF-β caused growth arrest and loss of epithelial cell junctions in the HaCaT derivatives, but mesenchymal markers were not induced, suggesting a partial, but not complete EMT response. Inflammation induced by COX-2 has been proposed to be a key mechanism in EMT of benign cells. Corroborating this notion, COX-2 was induced only in benign, not in malignant HaCaT derivatives. Furthermore, in cells in which TGF-β caused COX-2 induction, migration was clearly augmented. The concept of treating cancer is changing from targeting solely the cancer cells to targeting the whole microenvironment. The results of this work emphasise the role of activated fibroblasts in cancer progression and that CAFs should also be taken into consideration in the treatment of cancer. The results from these studies suggests that nemosis could be used as a diagnostic tool to distinguish in vitro activated fibroblasts from tumour stroma and also in studying the paracrine signalling that is mediated to other cell types via soluble factors.

Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Expression Analysis of Host-Induced Genes and Proteins of Potato Pathogen Pectobacterium atrosepticum

Pectobacterium atrosepticum on Gram-negatiivinen bakteeri, joka aiheuttaa perunan tyvi- ja märkämätää. P. atrosepticum bakteerin optimilämpötila on melko alhainen ja se on yleinen lauhkeilla alueilla. Tyvimätä leviää pääasiassa siemenperunan välityksellä ja siksi se on ongelma erityisesti siemenperunan tuotannossa. P. atrosepticum kannan SCRI1043 genomi on julkaistu ja sitä tutkitaan malliorganismina märkä- ja tyvimädän taudinaiheuttamisen ymmärtämiseksi. Tämä opportunistinen taudinaiheuttaja voi elää isäntäkasvissa kuukausia piilevänä, aiheuttamatta näkyviä oireita. Suotuisissa olosuhteissa bakteerit alkavat jakautua ja tuottaa kasvin kudoksia hajottavia entsyymejä. Mädäntyvä kasvimassa tarjoaa ravinteita bakteerien kasvuun ja mahdollistaa isäntäkasvin asuttamisen. Soluseiniä hajottavien entsyymien merkitys taudinaiheuttamisessa on hyvin tunnettu, mutta oireettomasta jaksosta ja taudin alkuvaiheista tiedätään vain vähän. Bakteerin genomi sisältää monia toksiineja, adhesiineja, hemolysiineja ja muita proteiineja, joilla saattaa olla merkitys taudinaiheuttamisessa. Tässä työssä käytettiin proteomiikkaa ja mikrosiruanalysiä P. atrosepticum bakteerin erittyvien proteiinien ja geeniekspression tutkimiseen. Proteiinit, jotka eritetään ulos bakteerista, toimivat todennäköisesti taudinaiheuttamisessa, koska ne ovat suorassa kontaktissa isäntäkasvin kanssa. Analyysit suoritettiin olosuhteissa, jotka muistuttavat kasvin soluvälitilaa: matala pH, vähän ravinteita ja matala lämpötila. Isäntäkasvin läsnäolon vaikutusta proteiinien tuottoon ja geeniekspressioon tutkittiin lisäämällä perunauutetta kasvatusalustaan. Tutkimuksessa tunnistettiin P. atrosepticum bakteerin monia jo tunnettuja ja mahdollisesti taudinaiheuttamiseen liittyviä proteiineja. Perunauute lisäsi hiljattain tunnistetun, proteiinien eritysreittiä (tyyppi VI sekreetio, T6SS) koodaavien geenien ilmentymistä. Lisäksi bakteerin havaittiin erittävän useita T6SS:n liittyviä proteiineja kasvualustaan, johon oli lisätty perunauutetta. T6SS:n merkitys bakteereille on vielä epäselvä ja sen vaikutuksesta taudinaiheuttamiseen on julkaistu ristiriitaisia tuloksia. Märkä- ja tyvimädän ymmärtäminen molekulaarisella tasolla luo pohjan tautien kontrollointiin tähtäävään soveltavaan tutkimukseen. Tämä tutkimus lisää tietoa kasvi-patogeeni- interaktiosta ja sitä voidaan tulevaisuudessa käyttää hyväksi esimerkiksi diagnostiikassa, resistenttien perunalajikkeiden jalostuksessa tai viljely- ja varastointiolosuhteiden parantamisessa.

Role of heat treatment in the processing and quality of oat flakes

This thesis reports on investigations into the influence of heat treatment on the manufacturing of oat flakes. Sources of variation in the oat flake quality are reviewed, including the whole chain from the farm to the consumer. The most important quality parameters of oat flakes are the absence of lipid hydrolysing enzymes, specific weight, thickness, breakage (fines), water absorption. Flavour, colour and pasting properties are also important, but were not included in the experimental part of this study. Of particular interest was the role of heat processing. The first possible heat treatment may occur already during grain drying, which in Finland generally happens at the farm. At the mill, oats are often kilned to stabilise the product by inactivating lipid hydrolysing enzymes. Almost invariably steaming is used during flaking, to soften the groats and reduce flake breakage. This thesis presents the use of a material science approach to investigating a complex system, typical of food processes. A combination of fundamental and empirical rheological measurements was used together with a laboratory scale process to simulate industrial processing. The results were verified by means of industrial trials. Industrially produced flakes at three thickness levels (nominally 0.75, 0.85 and 0.90 mm) were produced from kilned and unkilned oat groats, and the flake strength was measured at different moisture contents. Kilning was not found to significantly affect the force required to puncture a flake with a 2mm cylindrical probe, which was taken as a measure of flake strength. To further investigate how heat processing contributes to flake quality, dynamic mechanical analysis was used to characterise the effect of heat on the mechanical properties of oats. A marked stiffening of the groat, of up to about 50% increase in storage modulus, was observed during first heating at around 36 to 57°C. This was also observed in tablets prepared from ground groats and extracted oat starch. This stiffening was thus attributed to increased adhesion between starch granules. Groats were steamed in a laboratory steamer and were tempered in an oven at 80 110°C for 30 90 min. The maximum force required to compress the steamed groats to 50% strain increased from 50.7 N to 57.5 N as the tempering temperature was increased from 80 to 110°C. Tempering conditions also affected water absorption. A significantly higher moisture content was observed for kilned (18.9%) compared to unkilned (17.1%) groats, but otherwise had no effect on groat height, maximum force or final force after a 5 s relaxation time. Flakes were produced from the tempered groats using a laboratory flaking machine, using a roll gap of 0.4 mm. Apart from specific weight, flake properties were not influenced by kilning. Tempering conditions however had significant effects on the specific weight, thickness and water absorption of the flakes, as well as on the amount of fine material (<2 mm) produced during flaking. Flake strength correlated significantly with groat strength and flake thickness. Trial flaking at a commercial mill confirmed that groat temperature after tempering influenced water absorption. Variation in flake strength was observed , but at the groat temperatures required to inactivate lipase, it was rather small. Cold flaking of groats resulted in soft, floury flakes. The results presented in this thesis suggest that heating increased the adhesion between starch granules. This resulted in an increase in the stiffness and brittleness of the groat. Brittle fracture, rather than plastic flow, during flaking could result in flaws and cracks in the flake. These would be expected to increase water absorption. This was indeed observed as tempering temperature increased. Industrial trials, conducted with different groat temperatures, confirmed the main findings of the laboratory experiments. The approach used in the present study allowed the systematic study of the effect of interacting process parameters on product quality. There have been few scientific studies of oat processing, and these results can be used to understand the complex effects of process variables on flake quality. They also offer an insight into what happens as the oat groat is deformed into a flake.

Glycogen debranching enzyme activity in the muscles of meat producing animals

Muscle glycogen exists in two forms: low molecular weight pro-glycogen and high molecular weight macro-glycogen. The degradation of glycogen to glucose 1 phosphate and free glucose is catalysed by glycogen phosphorylase together with glycogen debranching enzyme (GDE). The process in which glycogen is broken down via anaerobic pathways to lactate, results in the acidification of the muscles and has a great influence on meat quality. Thus, the overall aim of this thesis was to characterise the post mortem action of GDE in muscles of meat production animals (pigs, cattle and chickens). Interest was focused on the differences in GDE activity between fast twitch glycolytic muscles and slow twitch oxidative muscles. The effects of pH, temperature, RN genotype (PRKAG3 gene), and of time post mortem on GDE activity were also investigated. This thesis showed that there are differences in GDE activity between animal species and between different muscles of an animal. It was shown that in pigs and cattle, higher GDE activity and phosphorylase activity exists in the fast twitch glycolytic muscles than in slow twitch oxidative muscles of the same animal. Thus, the high activity of these enzymes enables a faster rate of glycogenolysis in glycolytic M. longissimus dorsi compared to oxidative M. masseter. In chicken muscles, the GDE activity was low compared to pig or cattle muscles. Furthermore, the GDE activity in the glycolytic M. pectoralis superficialis was lower than in more oxidative M. quadriceps femoris despite the high phosphorylase activity in the former. The relative ratios between phosphorylase and GDE activity were higher in fast twitch glycolytic muscles than in slow twitch oxidative muscles of all studied animals. This suggests that the relatively low GDE activity compared to the phosphorylase activity in fast twitch glycolytic muscles may be a protection mechanism in living muscle against a very fast pH decrease. Chilling significantly decreased GDE activity and below 15 C porcine GDE was almost inactive. The effect of pH on GDE activity was only minor at the range normally found in post mortem muscles (pH 7.4 to 5.0). The GDE activity remained level for several hours after slaughter. During the first hours post mortem, GDE activity was similar in RN- carrier pigs and in wild type pigs. However, the GDE activity declined faster in M. longissimus dorsi from wild type pigs than in the RN carrier pigs, the difference between genotypes was significant after 24 h post mortem. Pro-glycogen and macro-glycogen contents were higher, pH decrease was faster and ultimate pH was lower in RN- carrier pigs than in wild type pigs. In the RN- carriers, the prolonged high GDE activity level may enable an extended pH decrease and lower ultimate pH in their muscles. In conclusion, GDE is not the main factor determining the rate or the extent of post mortem glycogenolysis, but under certain conditions, such as in very fast chilling, the inhibition of GDE activity in meat may reduce the rate of pH decrease and result in higher ultimate pH. The rate and extent of pH decrease affects several meat quality traits.

Microbes in the tailoring of barley malt properties

Microbes have a decisive role in the barley-malt-beer chain. A major goal of this thesis was to study the relationships between microbial communities and germinating grains during malting. Furthermore, the study provided a basis for tailoring of malt properties with natural, malt-derived microbes. The malting ecosystem is a dynamic process, exhibiting continous change. The first hours of steeping and kilning were the most important steps in the process with regard to microbiological quality. The microbial communities consisting of various types of bacteria, yeasts and filamentous fungi formed complex biofilms in barley tissues and were well-protected. Inhibition of one microbial population within the complex ecosystem led to an increase of non-suppressed populations, which must be taken into account because a shift in microbial community dynamics may be undesirable. Both bacterial and fungal communities should be monitored simultaneously. Using different molecular approaches we showed that the diversity of microbes in the malting ecosystem was greater than expected. Even some new microbial groups were found in the malting ecosystem. Suppression of Gram-negative bacteria during steeping was advanategous for grain germination and malt brewhouse performance. Fungal communities including both filamentous fungi and yeasts significantly contributed to the production of microbial beta-glucanases and xylanases, and were also involved in proteolysis. Well-characterized lactic acid bacteria (Lactobacillus plantarum VTT E-78076 and Pediococcus pentosaceus VTT E-90390) proved to be an effective way of balancing the microbial communities in malting. Furthermore, they had positive effects on malt characteristics and notably improved wort separation. Previously the significance of yeasts in the malting ecosystem has been largely underestimated. This study showed that yeast community was an important part of the industrial malting ecosystem. Yeasts produced extracellular hydrolytic enzymes with a potentially positive contribution to malt processability. Furthermore, several yeasts showed strong antagonistic activity against field and storage moulds. Addition of a selected yeast culture (Pichia anomala VTT C-04565) into steeping restricted Fusarium growth and hydrophobin production and thus prevented beer gushing. Addition of P. anomala C565 into steeping water tended to retard wort filtration, but the filtration was improved when the yeast culture was combined with L. plantarum E76. The combination of different microbial cultures offers a possibility to use ther different properties, thus making the system more robust. Improved understanding of complex microbial communities and their role in malting enables a more controlled process management and the production of high quality malt with tailored properties

Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

The white-rot fungi Phlebia radiata and Dichomitus squalens in wood-based cultures: expression of laccases, lignin peroxidases, and oxalate decarboxylase

Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.