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  • Sadeghi, Mohammadreza (2014)
    Human polyomaviruses (HPyVs) are known to cause benign initial infection at an early age. They have a high prevalence in the population with frequent incidences of reactivation, and pathologic consequences in those who are elderly or immunosuppressed. Thus far 12 HPyV species have been known. The first two HPyVs, JC virus (JCPyV) and BK virus (BKPyV) were described in 1971. They are associated with specific diseases, progressive multifocal leukoencephalopathy (PML) and polyomavirus-associated nephropathy (PVAN), respectively. Since 2007, 10 additional HPyVs have been identified by molecular genetic techniques. Study of the role of these new viruses in human disease is a new challenge in the HPyV area. The Karolinska Institute (KIPyV) and Washington University (WUPyV) viruses were discovered in respiratory secretions of patients with unidentified causes of pneumonia. Other examples of newly found HPyVs are the Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma (MCC), and trichodysplasia spinulosa-associated polyomavirus (TSPyV) in Trichodysplasia spinulosa (TS), both skin diseases in immunocompromised patients. The potential pathogenicity of the remaining HPyVs awaits assessment. Seroprevalence studies indicate that HPyVs infect 30 to 90% of the general population and are transmitted apparently independently of one another. Thus far, although their modes of transmission have yet to be resolved, HPyVs are frequently detectable at different body sites and in bodily fluids of healthy immunocompetent individuals, including the skin, hair follicles, saliva, urine, feces, and respiratory secretions, and they can also be found in the environment. To investigate their tropism, persistence site, reactivation, transmission route, and contribution to disease, we have developed for KI, WU, MC, and TS polyomaviruses comprehensive diagnostic methods. We studied the occurrence of their DNAs and antibodies to these viruses from birth to death. In our study, using recombinant fusion protein antigens, IgG antibodies were detectable for KIPyV in 55% and for WUPyV in 69%. Rapidly increasing and high IgG seroprevalences showed that KIPyV and WUPyV are acquired early in childhood and supported the notion that these polyomaviruses are widespread. Our results not only suggested the significance of protein conformation in immunoreactivity of VP1, the major capsid protein, but also pointed to the antigenic importance of the minor proteins VP2 and VP3. Among aging individuals, by employment of recombinant virus-like particles (VLPs)as antigens in ELISA, MCPyV and TSPyV IgG seroprevalences were 59.6% and 67.3%. Among 462 pregnant women, MCPyV IgG seroprevalence was 46% and in constitutionally healthy individuals, TSPyV IgG seroprevalence among children was 39% and among adults 70%. In addition, our DNA PCR studies of respiratory specimens indicated exposure to KIPyV and WUPyV, as well as to MCPyV. We observed MCPyV and TSPyV DNAs particularly often in tonsillitis or hypertrophic tonsillar tissues, unlike for KIPyV or WUPyV. MCPyV and TSPyV DNA in the tonsillar biopsies suggested lifelong persistence in lymphoid tissue or mucosa. MCPyV DNA occurred in tonsils more frequently in adults than in children. By contrast, WUPyV DNA was found preferentially in children. MCPyV occurred also in nasal swabs and NPAs, at a frequency similar to that of KIPyV and WUPyV. The tonsil may be an initial site of WUPyV infection and a site of MCPyV persistence. On the other hand, TSPyV PCR positivity of tonsillar samples of individuals with long-term immunity provided evidence of TSPyV persistence in tonsils and suggests lymphoid tissue as a latency site also for this emerging human pathogen. Our results indicated that MCPyV DNA, unlike TSPyV DNA, occurs in low copy numbers in serum in a notable proportion of aging individuals. Whether the enhanced viral replication in our elderly participants is a reflection of waning immune surveillance and is correlated with increased MCC risk deserves further exploration. Furthermore, to investigate the frequency of fetal infections by these new viruses, we sought the KIPyV, WUPyV, and MCPyV DNAs by PCR, from 535 fetal autopsy samples (heart, liver, placenta) from intrauterine fetal deaths (IUFDs), miscarriages, or induced abortions. Examining by PCR 535 fetal autopsy samples and the corresponding pregnant women by serology, we obtained data to rule out vertical transmission of the new polyomaviruses KI, WU, and MC. Our data suggest that none of the three often cause miscarriages or IUFDs, nor are they transmitted to fetuses. By means of new molecular methods several emerging polyomaviruses have been discovered. Although it is still too early to reach a conclusion on this point, it seems apparent that these novel viruses follow the pattern established for the JC and BK polyomaviruses: a mild initial infection at an early age, high prevalence in the general population, lymphoid tissue as a latency site, and pathologic consequences among the immunosuppressed and/or the elderly.
  • Illman, Sara (Helsingin yliopisto, 2007)
    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.
  • Alakurtti, Kirsi (Helsingin yliopisto, 2006)
    Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by age of onset at 6-15 years, stimulus-sensitive myoclonus, tonic-clonic epileptic seizures and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common mutation underlying EPM1 is a dodecamer repeat expansion in the promoter region of CSTB. In addition, nine other mutations have been identified. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed inhibitor of cathepsins, but its physiological function is unknown. The purpose of this study was to investigate CSTB gene expression and CSTB protein function in normal and pathological conditions. The basal CSTB promoter was mapped and characterized using different promoter-luciferase gene constructs. The binding activity of transcription factors to one ARE half, five Sp1 and four AP1 sites in the CSTB promoter was demonstrated. The CSTB promoter activity was clearly decreased using a CSTB promoter with "premutation" repeat expansions and in individuals with alike expansions. The expression of CSTB mRNA and protein was markedly reduced in patient cells. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes, and in differentiated cells merely to the cytoplasm. This suggests that the subcellular distribution of CSTB is dependent on the differentation status of the cells. The proteins representing patient missense mutations failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. Several alternatively spliced CSTB isoforms were identified. Of these CSTB2 was widely expressed with very low levels whereas the other alternatively spliced forms seemed to have limited tissue expression. In patients CSTB2 expression was reduced similarly to that of CSTB. The physiological relevance of CSTB alternative splicing remains unknown. The mouse Cstb transcript was shown to be present in all embryonic stages and adult tissues examined. The expression was highest at embryonic day 7 and in thymus, as well as in postnatal brain in the cortex, caudate putamen, thalamus, hippocampus, and in the Purkinje cell layer of the cerebellum. Our data implies that CSTB expression is tightly temporally and spatially regulated. The data presented in my thesis lay the basis for further understanding of the role of CSTB in health and disease.
  • Jussila, Minna M. (Helsingin yliopisto, 2006)
    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
  • Arighi, Elena (Helsingin yliopisto, 2005)
  • Lindqvist, Nanna (Elintarviketurvallisuusvirasto Evira, 2008)
    Salmonellosis is one of the most significant zoonoses worldwide and also in Finland. Salmonella Typhimurium definitive phage type (DT) 1 and Salmonella Infantis are considered endemic in Finland. These serovars have frequently caused outbreaks among humans. Salmonella Agona was not frequently encountered in Finland until a small outbreak among cattle farms occurred in 1994-1995. Bacterial typing methods are used for outbreak investigations and for surveillance, where the data can be used for risk assessment calculations in addition to the future prevention of outbreaks.In particular the identification of factors that contribute to the persistence and spread of infection in endemic situations, estimations of the effect of animal reservoirs on human cases, and the identification of other risk factors for human infections are among the important reasons for typing. Salmonella Infantis became more common in cattle in the 1980s, after it established itself in the broiler chicken production in Finland in 1971. In the 1990s, S. Infantis became the predominant serovar among cattle in Finland. In 1995, a feedborne outbreak of S. Infantis in cattle occurred. By XbaI-PFGE methodology we were able to identify the feedstuff-related genotype, which contained a 60 kb plasmid. Farms infected with the feedstuff-related genotype were also identified. The stability of the feedstuff-related genotype was followed on selected farms. The plasmid was stable on the farms during the follow-up period. The feedstuff-related genotype did not persist in the cattle population. Moreover, there was a general decline in bovine salmonella infections from 1997 onwards. The genetic diversity of the S. Infantis isolates taken from Finnish cattle was also assessed. The S. Infantis infection in cattle is highly clonal as 99 per cent of the isolates had XbaI-PFGE profiles clonally related to each other. The major genotype was predominant both at the starting year of our analysis in 1985 and as the infection seemed to fade out in 2003. There is a trend towards less genetic diversity of the S. Infantis infection among domestic isolates from humans and poultry. The ribo/IS200-types and the most common XbaI-PFGE profiles determined among the analysed cattle isolates could also be detected among domestic isolates from poultry and humans. In our analysis of successive isolates from the same cattle herds, we frequently detected minor changes in banding patterns during long-lasting infections in individual herds. The sampling and testing of several isolates from a herd in outbreak investigations is therefore advisable. In 1997, another small outbreak of Salmonella Agona among cattle farms occurred. The PFGE genotype of that outbreak was possibly related to that of the first outbreak, in 1994-1995. In 1999, a large outbreak of S. Agona of domestic origin involving more than 50 human cases occurred. Despite epidemiological investigations carried out by the local authorities, the source of the outbreak remained unknown. Based on our typing data, this outbreak was unrelated to the cattle farm outbreaks, though it did occur in the same region of Finland. The outbreak profile for the 1999 outbreak could not be found in any of the older isolates. Salmonella Typhimurium DT1 has become the most common S. Typhimurium phage type among cattle farms in Finland, and has been detected annually since 1980. The infection is highly homogenous. The most common XbaI-PFGE profile can be seen in isolates from humans, domestic animals, hedgehogs and wild birds, so molecular subtyping by XbaI-PFGE alone is not discriminatory enough in analysing our endemic infection. A combination of XbaI-, BlnI-, and SpeI-PFGE can be applied in the analysis of outbreaks. Two clusters were formed by the combination of the XbaI-, BlnI-, and SpeI-PFGE profiles, IS200-profiles and possession of the serovar-specific virulence plasmid. The major cluster, typical of our endemic infection, had no virulence plasmid.
  • Kaukinen, Anne (Helsingin yliopisto, 2010)
    Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessive disease, enriched in the Finnish population. NPHS1 is caused by a mutation in the NPHS1 gene. This gene encodes for nephrin, which is a major structural component of the slit diaphragm connecting podocyte foot processes in the glomerular capillary wall. In NPHS1, the genetic defect in nephrin leads to heavy proteinuria already in the newborn period. Finnish NPHS1 patients are nephrectomized at infancy, and after a short period of dialysis the patients receive a kidney transplant, which is the only curative therapy for the disease. In this thesis, we examined the cellular and molecular mechanisms leading to the progression of glomerulosclerosis and tubulointerstitial fibrosis in NPHS1 kidneys. Progressive mesangial expansion in NPHS1 kidneys is caused by mesangial cell hyperplasia and the accumulation of extracellular matrix proteins. Expansion of the extracellular matrix was caused by the normal mesangial cell component, collagen IV. However, no significant changes in mesangial cell phenotype or extracellular matrix component composition were observed. Endotheliosis was the main ultrastructural lesion observed in the endothelium of NPHS1 glomeruli. The abundant expression of vascular endothelial growth factor and its transcription factor hypoxia inducible factor-1 alpha were in accordance with the preserved structure of the endothelium in NPHS1 kidneys. Hypoperfusion of peritubular capillaries and tubulointerstitial hypoxia were evident in NPHS1 kidneys, indicating that these may play an important role in the rapid progression of fibrosis in the kidneys of NPHS1 patients. Upregulation of Angiotensin II was obvious, emphasizing its role in the pathophysiology of NPHS1. Excessive oxidative stress was evident in NPHS1 kidneys, manifested as an increase expression of p22phox, superoxide production, lipid oxide peroxidation and reduced antioxidant activity. In conclusion, our data indicate that mesangial cell proliferation and the accumulation of extracellular matrix accumulation are associated with the obliteration of glomerular capillaries, causing the reduction of circulation in peritubular capillaries. The injury and rarefaction of peritubular capillaries result in impairment of oxygen and nutrient delivery to the tubuli and interstitial cells, which correlates with the fibrosis, tubular atrophy and oxidative stress observed in NPHS1 kidneys.
  • Hakonen, Anna H (Anna Hakonen, 2008)
    Uusi hermoston rappeumasairaus MIRAS: Suomessa kantajia joka 125. väestöstä Tässä väitöskirjatyössä on kuvattu uusi peittyvästi periytyvä hermoston rappeumasairaus, MIRAS (mitochondrial recessive ataxia syndrome), ja sen geenitausta. Tauti osoittautui tutkimuksessamme Suomen yleisimmäksi perinnölliseksi ataksiasairaudeksi. Tutkimuksessa on tutkittu perinnöllisiä aivosairauksia, joissa yhtenä oireena on ataksia (kävelyn epävarmuus, tasapainovaikeus ja liikkeiden haparointi), sekä lukuisia muita aivojen toimintahäiriöstä johtuvia oireita. Seuloessamme suomalaisilta ataksiapotilailta MIRAS-geenivirhettä, 27 potilasta sai diagnoosin aikaisemmin tuntemattomalle, etenevälle ataksiasairaudelleen. Tutkimuksen tuloksena kyseisen geenivirheen DNA-diagnostiikka on otettu käyttöön suomalaisissa ja eurooppalaisissa laboratorioissa, ja toista sataa potilasta ympäri maailman on saanut diagnoosin. Suomen väestössä joka 125. kantaa MIRAS geenivirhettä, mutta taudin saa vain, jos perii geenivirheen molemmilta vanhemmiltaan. MIRAS on taudinkuvaltaan vaihteleva, mutta vaikea etenevä neurologinen sairaus. Useilla potilailla esiintyvät oireet ovat ataksia, puheen puuromaisuus (dysartria), ääreishermorappeuma (neuropatia), pakkoliikkeet, psykiatriset oireet sekä vaikea epilepsia. Erityisen tärkeää MIRAS-taudin tunnistaminen on siihen liittyvän epilepsian hoitopäätöksessä: valproaatti-lääkitys voi aiheuttaa MIRAS-potilaille vaikean maksavaurion. Väitöskirjatyön tuloksena selvisi, että kaikki suomalaiset, norjalaiset, belgialaiset, englantilaiset, australialaiset ja uusi-seelantilaiset MIRAS potilaat olivat kaukaista sukua samalle, tuhansia vuosia sitten eläneelle eurooppalaiselle esivanhemmalle. Ataksiasairauksien tautimekanismeja selvitimme tutkimalla MIRAS-ataksiaa ja sitä muistuttavaa IOSCA sairautta (infantile onset spinocerebellar ataxia), jonka aiheuttaa peittyvästi periytyvä geenivirhe Twinkle-geenissä. Tutkimuksessa löydettiin myös uusi, Twinkle-geenin geenivirheestä johtuva taudinkuva: vaikea-asteinen, varhaisella iällä alkava aivosairaus, jossa on lisäksi viitteitä maksasairaudesta. Löysimme potilaiden aivoista muutoksia mitokondrioiden eli solun voimalaitosten perimän määrässä. Nämä tulokset antavat arvokasta lisätietoa ataksiasairauksien taustalla olevista muutoksista, joiden ymmärtäminen on välttämätön edellytys hoitomahdollisuuksien tutkimiselle tulevaisuudessa.
  • Montesano, Marcos (Helsingin yliopisto, 2002)
  • Tamminen, Manu (Helsingin yliopisto, 2011)
    Fish farming introduces nutrients, microbes and a wide variety of chemicals such as heavy metals, antifoulants and antibiotics to the surrounding environment. Introduction of antibiotics has been linked with the increased incidence of antibiotic resistant pathogenic bacteria in the farm vicinities. In this thesis molecular methods such as quantitative PCR and DNA sequencing were applied to analyze bacterial communities in sediments from fish farms and pristine locations. Altogether four farms and four pristine sites were sampled in the Baltic Sea. Two farm and two pristine locations were sampled over a surveillance period of four years. Furthermore, a new methodology was developed as a part of the study that permits amplifying single microbial genomes and capturing them according to any genetic traits, including antibiotic resistance genes. The study revealed that several resistance genes for tetracycline were found at the sediment underneath the aquaculture farms. The copy number of these genes remained elevated even at a farm that had not used any antibiotics since year 2000, six years before this study started. Similarly, an increase in the amount of mercury resistance gene merA was observed at the aquaculture sediment. The persistence of the resistance genes in absence of any selection pressure from antibiotics or heavy metals suggests that the genes may be introduced to the sediment by the farming process. This is also supported by the diversity pattern of the merA gene between farm and pristine sediments. The bacterial community-level changes in response to fish farming were very complex and no single phylogenetic groups were found that would be typical to fish farm sediments. However, the community structures had some correlation with the exposure to fish farming. Our studies suggest that the established approaches to deal with antibiotic resistance at the aquaculture, such as antibiotic cycling, are fundamentally flawed because they cannot prevent the introduction of the resistance genes and resistant bacteria to the farm area by the farming process. Further studies are required to study the entire fish farming process to identify the sources of the resistance genes and the resistant bacteria. The results also suggest that in order to prevent major microbiological changes in the surrounding aquatic environment, the farms should not be founded in shallow water where currents do not transport sedimenting matter from the farms. Finally, the technique to amplify and select microbial genomes will potentially have a considerable impact in microbial ecology and genomics.
  • Mbanzibwa, Deusdedith R (Helsingin yliopisto, 2011)
    Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.
  • Laiho, Päivi (Helsingin yliopisto, 2005)
  • Ollikainen, Miina (Helsingin yliopisto, 2007)
    Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common known clearly hereditary cause of colorectal and endometrial cancer (CRC and EC). Dominantly inherited mutations in one of the known mismatch repair (MMR) genes predispose to HNPCC. Defective MMR leads to an accumulation of mutations especially in repeat tracts, presenting microsatellite instability. HNPCC is clinically a very heterogeneous disease. The age at onset varies and the target tissue may vary. In addition, families that fulfill the diagnostic criteria for HNPCC but fail to show any predisposing mutation in MMR genes exist. Our aim was to evaluate the genetic background of familial CRC and EC. We performed comprehensive molecular and DNA copy number analyses of CRCs fulfilling the diagnostic criteria for HNPCC. We studied the role of five pathways (MMR, Wnt, p53, CIN, PI3K/AKT) and divided the tumors into two groups, one with MMR gene germline mutations and the other without. We observed that MMR proficient familial CRC consist of two molecularly distinct groups that differ from MMR deficient tumors. Group A shows paucity of common molecular and chromosomal alterations characteristic of colorectal carcinogenesis. Group B shows molecular features similar to classical microsatellite stable tumors with gross chromosomal alterations. Our finding of a unique tumor profile in group A suggests the involvement of novel predisposing genes and pathways in colorectal cancer cohorts not linked to MMR gene defects. We investigated the genetic background of familial ECs. Among 22 families with clustering of EC, two (9%) were due to MMR gene germline mutations. The remaining familial site-specific ECs are largely comparable with HNPCC associated ECs, the main difference between these groups being MMR proficiency vs. deficiency. We studied the role of PI3K/AKT pathway in familial ECs as well and observed that PIK3CA amplifications are characteristic of familial site-specific EC without MMR gene germline mutations. Most of the high-level amplifications occurred in tumors with stable microsatellites, suggesting that these tumors are more likely associated with chromosomal rather than microsatellite instability and MMR defect. The existence of site-specific endometrial carcinoma as a separate entity remains equivocal until predisposing genes are identified. It is possible that no single highly penetrant gene for this proposed syndrome exists, it may, for example be due to a combination of multiple low penetrance genes. Despite advances in deciphering the molecular genetic background of HNPCC, it is poorly understood why certain organs are more susceptible than others to cancer development. We found that important determinants of the HNPCC tumor spectrum are, in addition to different predisposing germline mutations, organ specific target genes and different instability profiles, loss of heterozygosity at MLH1 locus, and MLH1 promoter methylation. This study provided more precise molecular classification of families with CRC and EC. Our observations on familial CRC and EC are likely to have broader significance that extends to sporadic CRC and EC as well.
  • Govindan, Ashokkumar (2014)
    Ubiquitin-mediated proteolysis regulates many basic cellular processes in plant development. Especially, in Arabidopsis thaliana over 1400 genes encode components of the ubiquitin/26S proteasome pathway. Approximately, 90% of the genes encode subunits of the E3 ubiquitin ligases, which confer substrate specificity. However, till now few E3 target proteins were known. Hence, identification and characterization of E3-substrate interaction is essential for understanding the role of ubiquitinylation in various plant developmental pathways. The main objectives of my Master’s thesis were derived from the Flower- specific Ubiquitin Proteasome System (FUPS) research project on the identification of proteins related to FUPS by the candidate and genomic approaches. The candidate approach involved molecular cloning of candidate ubiquitin E3 ligase gene RFI2 and its interaction substrates SLK1 and SLK2 (SLKs). The genomic approach includes genotypic and phenotypic characterization of the Salk T-DNA mutant lines corresponding to the selected FUPS E3 components. The cloning of candidate genes RFI2 and SLKs were done by Gateway cloning technology in order to generate overexpression and inducible expression gene constructs. For functional characterization of candidate proteins in vivo, these constructs were transformed into plants by floral dip Agrobacterium mediated transformation. The yeast two hybrid (Y2H) system was employed to study protein-protein interactions. Genotypic characterization of the selected T-DNA mutant lines was carried out by genetic screening through kanamycin selection. Molecular cloning of candidate genes RFI2 and SLKs into various expression vectors was accomplished. The RFI2 expression constructs were successfully transformed into Arabidopsis plants. The overexpression construct of SLK2 and the RFI2+SLK2 double construct were successfully transformed into root callus. However, the Y2H interaction assay was inconclusive about RFI2-SLK protein interaction, but RFI2 was shown to form homodimers. The transformed plants and the tagged protein constructs of RFI2 and SLK2 will be utilized for protein purification, biochemical assay and in vitro ubiquitination assay to study protein interaction, stability and modification. The genetic screening of T-DNA lines resulted in identification of 10 homozygous lines. In phenotypic analysis of these homozygous lines, early-stage growth and developmental phenotypes such as germination, juvenile leaf emergence, rosette size, colour and root growth were observed. The phenotypic analysis is crucial in identification of the informative phenotype changes in the E3 T-DNA mutant lines. This approach has been successfully employed in understanding of genetic and molecular basis for many biological traits in plants.
  • Mouhu, Katriina (Helsingin yliopisto, 2014)
    Strawberries (Fragaria sp.) are found throughout the Northern Hemisphere, growing in a wide variety of climatic conditions. The economically most important species is the garden strawberry (Fragaria x ananassa Duch.). Strawberries are perennial rosette plants with distinct developmental phases regulated by day length during the growing season. During long days (LDs) in spring and summer, strawberries grow actively with axillary buds developing into stolons called runners. During short days (SDs) in autumn, runner formation is replaced by branch crown formation and an inflorescence is initiated in the shoot meristem of a rosette crown. After winter rest, the inflorescence formed in the previous autumn flowers and vegetative growth is again activated by LDs. The wild strawberry (F. vesca L.) has been used as a model plant in strawberry research for several years. The wild strawberry is a seasonally flowering SD plant, but several perpetually flowering strawberry genotypes have been found. These types differ by a single recessive locus, the SEASONAL FLOWERING LOCUS (SFL), but the regulatory gene behind this trait has not been identified. This thesis aimed to identify the genes related to flowering and vegetative development in the wild strawberry. Expressed sequence tag (EST) sequencing of SD F. vesca and a perpetually flowering genotype were combined with data mining in published Fragaria and Rosaceae EST databases using known Arabidopsis thaliana (L.) Heynh. flowering-related genes as a reference. The results revealed that most genes in the Arabidopsis flowering pathways could be identified among strawberry ESTs, indicating putative conservation in flowering pathway genes between these species. Fragaria vesca TERMINAL FLOWER 1 (FvTFL1), a homologue of the Arabidopsis thaliana TFL1, was confirmed to be the SFL, encoding the flowering repressor in wild strawberry. Fragaria vesca SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (FvSOC1), a homologue of Arabidopsis SOC1, represses flowering via FvTFL1 activation in shoot apices. Both FvSOC1 and FvTFL1 expression are under photoperiodic regulation, controlled at least in part via F. vesca FLOWERING LOCUS T 1 (FvFT1), encoding a putative homologue of the Arabidopsis mobile flowering signal FT, and expressed specifically under LDs. It was concluded that FvTFL1 functions as the flowering repressor and FvTFL1 activation under LDs occurs by FvFT1 via FvSOC1 in wild strawberry. FvSOC1 regulates vegetative growth independently of FvTFL1. FvSOC1 enhanced runner formation from the axillary buds, which involves changes in the levels of gibberellin biosynthesis genes. These results were used to construct a model of the yearly growth cycle in wild strawberry.
  • Kiuru-Kuhlefelt, Sonja (Helsingin yliopisto, 2002)
  • Koivunen, Minni (Helsingin yliopisto, 2006)
    For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis.
  • Biedzka-Sarek, Marta (Helsingin yliopisto, 2008)
    In complement activation, Factor H (FH) and C4b-binding protein (C4bp) are the key regulators that prevent the complement cascade from attacking host tissues. Some bacteria may bind and deposit these regulators on their own surfaces and thus provide themselves with an efficient means to avoid complement activation. In consequence, bacteria resist complement-mediated lysis and opsonin-dependent phagocytosis. This study has demonstrated that Y. enterocolitica, similar to many other pathogens, recruits both FH and C4bp to its surface to ensure protection against the complement-mediated killing. YadA and Ail, the most crucial serum resistance factors of Y.enterocolitica, mediate the binding of FH and C4bp. FH - YadA interaction involves multiple higher structural motifs on the YadA stalk and the short consensus repeats (SCRs) of the entire polypeptide chain of FH. The Ail binding site on FH has been located to SCRs 6 and 7. The binding site for FH on Ail, however, remains undetermined. Both YadA- and Ail-bound regulators display full cofactor activity for FI-mediated cleavage of C3b/C4b. FH/C4bp-binding characteristics do, however, differ between YadA and Ail. In addition, Ail captures the regulators only in the absence of blocking lipopolysaccharide O-antigen and outer core, whereas YadA binds FH/C4bp independent of the presence of other surface factors Independent of mode of binding, however, YadA and Ail provide Y. enterocolitica a means to avoid complement-mediated lysis, enhancing chances for the bacteria to survive in the host during various phases of infection.