Browsing by Title

Sort by: Order: Results:

Now showing items 1947-1966 of 24304
  • Kärenlampi, Rauni (Helsingin yliopisto, 2007)
    Campylobacter jejuni and C. coli are the most frequent causes of bacterial gastroenteritis in Finland. Most Campylobacter infections are sporadic and the sources of infection remain unidentified. Risk factors for Campylobacter infections include eating undercooked meat, especially chicken, drinking unpasteurized milk or contaminated water, having contact with pets and foreign travel. The asaccharolytic nature and inertness in traditional biochemical tests makes the identification of Campylobacter spp. difficult. We studied the phylogeny of 12 Campylobacter spp. based on partial 593-bp groEL gene sequences. In general, lower interspecies sequence similarities were observed for groEL (range from 65% to 94%) than for the 16S rRNA gene (range from 90% to 99%). However, the intraspecies groEL sequence similarities were high (range from 95% to 100%) making groEL gene sequencing and the PCR-RFLP method developed in our study valuable for the identification of Campylobacter species. The minimum growth temperature of around 30°C makes multiplication of Campylobacter in foods highly unlikely. However, the survival in cool and humid conditions, such as chicken meat stored refrigerated, has been shown to be good. The survival of C. jejuni was investigated on iceberg lettuce, cantaloupe, cucumber, carrot and strawberries. Survival on strawberries was significantly lower than on the other produce, as was survival at 21°C compared to 7°C. Survival on the other produce was comparable with earlier reports in water and milk, but not as good as that observed on chicken meat. The association of Penner HS serotypes and pulsed-field gel electrophoresis (PFGE) SmaI/KpnI genotypes of 208 human and 30 chicken caecal C. jejuni isolates was studied during the seasonal peak in 1999 in Finland. Of the strains from humans, 46% had overlapping sero/genotypes with those from chicken. During the seasonal peak in 2003, C. jejuni and C. coli isolates from human fecal samples showed 5.7% and 61% PFGE (KpnI) profile overlap with cattle fecal and poultry retail meat isolates, respectively, demonstrating the importance of genotypes circulating in chicken as compared to those isolated from cattle in human infections. However, in 1999, human cases were also isolated prior to the slaughter of the chicken flock colonized by the same Campylobacter sero/genotype, suggesting that common environmental sources may exist for both human infections and chicken flock contamination. Multilocus sequence typing analysis of 361 Finnish C. jejuni and C. coli isolates from human patients, cattle, chicken and turkey samples, provided new information on the potential association of some clonal groups of this diverse organism with source of isolation as well as demographic characteristics.
  • Sorsa, Johanna (Helsingin yliopisto, 2007)
    Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.
  • Kisko, Kaisa (Helsingin yliopisto, 2008)
    Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.
  • Munsterhjelm, Edward (Helsingin yliopisto, 2006)
    Aim: To characterize the inhibition of platelet function by paracetamol in vivo and in vitro, and to evaluate the possible interaction of paracetamol and diclofenac or valdecoxib in vivo. To assess the analgesic effect of the drugs in an experimental pain model. Methods: Healthy volunteers received increasing doses of intravenous paracetamol (15, 22.5 and 30 mg/kg), or the combination of paracetamol 1 g and diclofenac 1.1 mg/kg or valdecoxib 40 mg (as the pro-drug parecoxib). Inhibition of platelet function was assessed with photometric aggregometry, the platelet function analyzer (PFA-100), and release of thromboxane B2. Analgesia was assessed with the cold pressor test. The inhibition coefficient of platelet aggregation by paracetamol was determined as well as the nature of interaction between paracetamol and diclofenac by an isobolographic analysis in vitro. Results: Paracetamol inhibited platelet aggregation and TxB2-release dose-dependently in volunteers and concentration-dependently in vitro. The inhibition coefficient was 15.2 mg/L (95% CI 11.8 - 18.6). Paracetamol augmented the platelet inhibition by diclofenac in vivo, and the isobole showed that this interaction is synergistic. Paracetamol showed no interaction with valdecoxib. PFA-100 appeared insensitive in detecting platelet dysfunction by paracetamol, and the cold-pressor test showed no analgesia. Conclusions: Paracetamol inhibits platelet function in vivo and shows synergism when combined with diclofenac. This effect may increase the risk of bleeding in surgical patients with an impaired haemostatic system. The combination of paracetamol and valdecoxib may be useful in patients with low risk for thromboembolism. The PFA-100 seems unsuitable for detection of platelet dysfunction and the cold-pressor test seems unsuitable for detection of analgesia by paracetamol.
  • Jalanka, Jonna (Helsingin yliopisto, 2014)
    The human gastrointestinal microbiota is a complex ecosystem harbouring trillions of bacteria from hundreds of species. Co-evolution has resulted in a mutually beneficial relationship in which gut bacteria make essential contributions to human health, moreover alterations in the microbiota composition and function have been associated to several disease states. In order to identify such changes in patients, it is essential to characterize the microbiota of healthy subjects. In this thesis the intestinal microbiota of healthy adults were thoroughly investigated and shown to display high subject-specificity and stability over time. Moreover, significant correlations between the microbiota and mild intestinal symptoms were identified, including the association of abdominal pain and bloating with the reduction of health-associated bifidobacteria. The intestinal microbiota of healthy subjects was benchmarked against two conditions that were hypothesized to perturb the microbial composition; bowel cleansing, a normal procedure prior colonoscopy and irritable bowel syndrome (IBS), a common disorder estimated to affect 10% of Finnish population. Adequate bowel cleansing by lavage has been shown to be safe for patients, but its long-term effects on the intestinal microbiota, and especially the potential alterations arising from different dosing regimes have not been addressed previously. We showed that there is a short but drastic reduction of the intestinal microbiota after lavage, however the microbiota recovers back to its original form within two weeks after the treatment. Additionally, we found that the recovery rate is dependent on the dosing of the purgative agent, suggesting that two smaller volumes of the purgative should be preferred over a single larger dose. There is growing evidence of the involvement of the intestinal microbiota in the pathophysiology of different IBS subtypes. However, the microbial component in post-infectious IBS patients has previously remained uncharacterised. In parallel to the characterization of intestinal microbiota of PI-IBS patients, the aim of this work was to address the associations between the intestinal microbiota and patient's clinical characteristics. We identified a bacterial signature of 27 taxa and the abundances of implicated bacteria correlated with clinical markers as well as expression levels of several host gene pathways, all suggesting an impaired gut epithelial barrier function in IBS. Observation of these specific associations between the host and intestinal microbiota may provide novel insights into the origin and mechanistic background of intestinal symptoms in IBS as well as enables novel stratification of the IBS patient material with a different aetiology. In summary, this thesis characterised the intestinal microbiota of healthy individuals and showed how perturbations such as IBS and bowel cleansing disrupt the composition, and detected associations between the microbiota and health parameters of the host. The results have clinical relevance by providing novel and a much needed tool for segregating the IBS patient material objectively as well as providing grounds for choosing the split-dosing regime of the purgative agent as an optimal bowel cleansing method. Furthermore, associations between the microbiota and health markers of the host were detected that will give grounds for future research on the aetiology of IBS.
  • Hänninen, Kaisa (Helsingin yliopisto, 2008)
    Increasing attention has been focused on methods that deliver pharmacologically active compounds (e.g. drugs, peptides and proteins) in a controlled fashion, so that constant, sustained, site-specific or pulsatile action can be attained. Ion-exchange resins have been widely studied in medical and pharmaceutical applications, including controlled drug delivery, leading to commercialisation of some resin based formulations. Ion-exchangers provide an efficient means to adjust and control drug delivery, as the electrostatic interactions enable precise control of the ion-exchange process and, thus, a more uniform and accurate control of drug release compared to systems that are based only on physical interactions. Unlike the resins, only few studies have been reported on ion-exchange fibers in drug delivery. However, the ion-exchange fibers have many advantageous properties compared to the conventional ion-exchange resins, such as more efficient compound loading into and release from the ion-exchanger, easier incorporation of drug-sized compounds, enhanced control of the ion-exchange process, better mechanical, chemical and thermal stability, and good formulation properties, which make the fibers attractive materials for controlled drug delivery systems. In this study, the factors affecting the nature and strength of the binding/loading of drug-sized model compounds into the ion-exchange fibers was evaluated comprehensively and, moreover, the controllability of subsequent drug release/delivery from the fibers was assessed by modifying the conditions of external solutions. Also the feasibility of ion-exchange fibers for simultaneous delivery of two drugs in combination was studied by dual loading. Donnan theory and theoretical modelling were applied to gain mechanistic understanding on these factors. The experimental results imply that incorporation of model compounds into the ion-exchange fibers was attained mainly as a result of ionic bonding, with additional contribution of non-specific interactions. Increasing the ion-exchange capacity of the fiber or decreasing the valence of loaded compounds increased the molar loading, while more efficient release of the compounds was observed consistently at conditions where the valence or concentration of the extracting counter-ion was increased. Donnan theory was capable of fully interpreting the ion-exchange equilibria and the theoretical modelling supported precisely the experimental observations. The physico-chemical characteristics (lipophilicity, hydrogen bonding ability) of the model compounds and the framework of the fibrous ion-exchanger influenced the affinity of the drugs towards the fibers and may, thus, affect both drug loading and release. It was concluded that precisely controlled drug delivery may be tailored for each compound, in particularly, by choosing a suitable ion-exchange fiber and optimizing the delivery system to take into account the external conditions, also when delivering two drugs simultaneously.
  • Hakala, Terhi (Helsingin yliopisto, 2007)
    White-rot fungi are wood degrading organisms that are able to decompose all wood polymers; lignin, cellulose and hemicellulose. Especially the selective white-rot fungi that decompose preferentially wood lignin are promising for biopulping applications. In biopulping the pretreatment of wood chips with white-rot fungi enhances the subsequent pulping step and substantially reduces the refining energy consumption in mechanical pulping. Because it is not possible to carry out biopulping in industrial scale as a closed process it has been necessary to search for new selective strains of white-rot fungi which naturally occur in Finland and cause selective white-rot of Finnish wood raw-material. In a screening of 300 fungal strains a rare polypore, Physisporinus rivulosus strain T241i isolated from a forest burn research site, was found to be a selective lignin degrader and promising for the use in biopulping. Since selective lignin degradation is apparently essential for biopulping, knowledge on lignin-modifying enzymes and the regulation of their production by a biopulping fungus is needed. White-rot fungal enzymes that participate in lignin degradation are laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP) and hydrogen peroxide forming enzymes. In this study, P. rivulosus was observed to produce MnP, laccase and oxalic acid during growth on wood chips. In liquid cultures manganese and veratryl alcohol increased the production of acidic MnP isoforms detected also in wood chip cultures. Laccase production by P. rivulosus was low unless the cultures were supplemented with sawdust and charred wood, the components of natural growth environment of the fungus. In white-rot fungi the lignin-modifying enzymes are typically present as multiple isoforms. In this study, two MnP encoding genes, mnpA and mnpB, were cloned and characterized from P. rivulosus T241i. Analysis of the N-terminal amino acid sequences of two purified MnPs and putative amino acid sequence of the two cloned mnp genes suggested that P. rivulosus possesses at least four mnp genes. The genes mnpA and mnpB markedly differ from each other by the gene length, sequence and intron-exon structure. In addition, their expression is differentially affected by the addition of manganese and veratryl alcohol. P. rivulosus produced laccase as at least two isoforms. The results of this study revealed that the production of MnP and laccase was differentially regulated in P. rivulosus, which ensures the efficient lignin degradation under a variety of environmental conditions.
  • Laurén, Juha (Helsingin yliopisto, 2007)
    Some leucine-rich repeat (LRR) -containing membrane proteins are known regulators of neuronal growth and synapse formation. In this work I characterize two gene families encoding neuronal LRR membrane proteins, namely the LRRTM (leucine-rich repeat, transmembrane neuronal) and NGR (Nogo-66 receptor) families. I studied LRRTM and NGR family member's mRNA tissue distribution by RT-PCR and by in situ hybridization. Subcellular localization of LRRTM1 protein was studied in neurons and in non-neuronal cells. I discovered that LRRTM and NGR family mRNAs are predominantly expressed in the nervous system, and that each gene possesses a specific expression pattern. I also established that LRRTM and NGR family mRNAs are expressed by neurons, and not by glial cells. Within neurons, LRRTM1 protein is not transported to the plasma membrane; rather it localizes to endoplasmic reticulum. Nogo-A (RTN4), MAG, and OMgp are myelin-associated proteins that bind to NgR1 to limit axonal regeneration after central nervous system injury. To better understand the functions of NgR2 and NgR3, and to explore the possible redundancy in the signaling of myelin inhibitors of neurite growth, I mapped the interactions between NgR family and the known and candidate NgR1 ligands. I identified high-affinity interactions between RTN2-66, RTN3-66 and NgR1. I also demonstrate that Rtn3 mRNA is expressed in the same glial cell population of mouse spinal cord white matter as Nogo-A mRNA, and thus it could have a role in myelin inhibition of axonal growth. To understand how NgR1 interacts with multiple structurally divergent ligands, I aimed first to map in more detail the nature of Nogo-A:NgR1 interactions, and then to systematically map the binding sites of multiple myelin ligands in NgR1 by using a library of NgR1 expression constructs encoding proteins with one or multiple surface residues mutated to alanine. My analysis of the Nogo-A:NgR1 -interactions revealed a novel interaction site between the proteins, suggesting a trivalent Nogo-A:NgR1-interaction. Our analysis also defined a central binding region on the concave side of NgR1's LRR domain that is required for the binding of all known ligands, and a surrounding region critical for binding MAG and OMgp. To better understand the biological role of LRRTMs, I generated Lrrtm1 and Lrrtm3 knock out mice. I show here that reporter genes expressed from the targeted loci can be used for maping the neuronal connections of Lrrtm1 and Lrrtm3 expressing neurons in finer detail. With regard to LRRTM1's role in humans, we found a strong association between a 70 kb-spanning haplotype in the proposed promoter region of LRRTM1 gene and two possibly related phenotypes: left-handedness and schizophrenia. Interestingly, the responsible haplotype was linked to phenotypic variability only when paternally inherited. In summary, I identified two families of neuronal receptor-like proteins, and mapped their expression and certain protein-protein interactions. The identification of a central binding region in NgR1 shared by multiple ligands may facilitate the design and development of small molecule therapeutics blocking binding of all NgR1 ligands. Additionally, the genetic association data suggests that allelic variation upstream of LRRTM1 may play a role in the development of left-right brain asymmetry in humans. Lrrtm1 and Lrrtm3 knock out mice developed as a part of this study will likely be useful for schizophrenia and Alzheimer s disease research.
  • Zhang, Zhen (Helsingin yliopisto, 2014)
    Clostridium botulinum is an infamous pathogen that produces botulinum neurotoxin, the causative agent of potentially fatal, neuroparalytic disease in humans and animals called botulism. Despite decades of research, much remains unknown regarding neurotoxin gene location, toxigenesis, and physiology of C. botulinum. First, it is unclear whether mobile genetic elements can mediate transmission of type E neurotoxin gene. Second, the regulatory circuits of botulinum neurotoxin synthesis remain largely unknown. Third, the physiological mechanism of C. botulinum cold stress tolerance is of great importance in cold chain food processing and preservation, but remains poorly understood. The aim of this study was to investigate the location of the type E botulinum neurotoxin gene, identify the potential regulators of neurotoxin synthesis in Group I C. botulinum type A1 strain ATCC 3502, and to characterize the regulon of the two-component signal transduction system (TCS) CBO0366/CBO0365 and its role in cold stress tolerance in C. botulinum ATCC 3502. A group of 36 C. botulinum type E strains was examined by pulsed-field gel electrophoresis and Southern hybridization with probes specific for type E neurotoxin gene (botE) and orfX1, a member of type E neurotoxin gene cluster. The results suggest that three C. botulinum strains encode botE and orfX1 in large plasmids of about 146 kb in size. In C. botulinum ATCC 3502, TCS CBO0787/CBO0786 was found to negatively regulate botulinum neurotoxin expression. Inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ntnh-botA and ha operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ntnh-botA and ha promoters, demonstrating direct transcriptional repression of the ntnh-botA and ha operons by CBO0786. CodY, a global regulator conserved in low-G+C Gram-positive bacteria, was shown to positively regulate the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased neurotoxin gene expression levels and reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to probes possessing the neurotoxin gene promoters, but strongly interacted only with a 30-bp region in the promoter of ntnh-botA operon, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the ntnh-botA promoter, suggesting CodY-dependent neurotoxin regulation is associated with nutritional status. A total of about 150 genes regulated by TCS CBO0366/CBO0365, were identified by DNA microarray. Inactivation of CBO0365 regulon genes encoding acetone-butanol-ethanol fermentation pathway, phosphate transport and arsenical resistance did not affect the growth of C. botulinum ATCC 3502 at 37 °C, but significantly impacted growth after a temperature downshift to 17 °C. These results suggest TCS CBO0366/CBO0365-regulated acetone-butanol-ethanol fermentation, phosphate transport and arsenical resistance play an important role in cold tolerance in C. botulinum ATCC 3502.
  • Kukkaro, Petra (Helsingin yliopisto, 2009)
    Viruses of Archaea are the least studied group of viruses. Fewer than 50 archaeal viruses have been reported which constitutes less than one percent of all the isolated prokaryotic viruses. Only about one third of the isolated archaeal viruses infect halophiles. The diversity of haloviruses, virus ecology in highly saline environments and the interactions of haloviruses with their hosts have been little studied. The exiguous knowledge available on halophilic systems is not only due to inadequate sampling but also reflects the extra challenge highly saline systems set on biochemical studies. In this study six new haloviruses were isolated and characterized. Viruses included four archaeal viruses and two bacteriophages. All of the other isolates exhibited head-tail morphology, except SH1 which was the first tailless icosahedral virus isolated from a high salt environment. Production and purification procedures were set up for all of these viruses and they were subjected to stability determinations. Archaeal virus SH1 was studied in more detail. Biochemical studies revealed an internal membrane underneath the protein capsid and a linear dsDNA genome. The overall structure of SH1 resembles phages PRD1, PM2 and Bam35 as well as an archaeal virus STIV. SH1 possesses about 15 structural proteins that form complexes under non-reducing conditions. Quantitative dissociation provided information about the positions of these proteins in the virion. The life cycle of SH1 was also studied. This lytic virus infects Haloarcula hispanica. Adsorption to the host cells is fairly inefficient and the life cycle rather long. Finally, virus responses in a variety of ionic conditions were studied. It was discovered that all of the studied viruses from low salt, marine and high salt environments tolerated larger range of salinities than their bacterial or archaeal hosts. The adsorption efficiency was not determined by the natural environment of a virus. Even though viruses with the slowest binding kinetics were among the haloviruses, fast binders were observed in viruses from all environments. When the salinity was altered, the virus adsorption responses were diverse. Four different behavioral patterns were observed: virus binding increased or decreased in increasing salinity, adsorption maximum was at a particular salt concentration or the salinity did not affect the binding. The way the virus binding was affected did not correlate with the environment, virus morphology or the organism the virus infects.
  • Alha, Lauri (Helsingin yliopisto, 2010)
    The first observations of solar X-rays date back to late 1940 s. In order to observe solar X-rays the instruments have to be lifted above the Earth s atmosphere, since all high energy radiation from the space is almost totally attenuated by it. This is a good thing for all living creatures, but bad for X-ray astronomers. Detectors observing X-ray emission from space must be placed on-board satellites, which makes this particular discipline of astronomy technologically and operationally demanding, as well as very expensive. In this thesis, I have focused on detectors dedicated to observing solar X-rays in the energy range 1-20 keV. The purpose of these detectors was to measure solar X-rays simultaneously with another X-ray spectrometer measuring fluorescence X-ray emission from the Moon surface. The X-ray fluorescence emission is induced by the primary solar X-rays. If the elemental abundances on the Moon were to be determined with fluorescence analysis methods, the shape and intensity of the simultaneous solar X-ray spectrum must be known. The aim of this thesis is to describe the characterization and operation of our X-ray instruments on-board two Moon missions, SMART-1 and Chandrayaan-1. Also the independent solar science performance of these two almost similar X-ray spectrometers is described. These detectors have the following two features in common. Firstly, the primary detection element is made of a single crystal silicon diode. Secondly, the field of view is circular and very large. The data obtained from these detectors are spectra with a 16 second time resolution. Before launching an instrument into space, its performance must be characterized by ground calibrations. The basic operation of these detectors and their ground calibrations are described in detail. Two C-flares are analyzed as examples for introducing the spectral fitting process. The first flare analysis shows the fit of a single spectrum of the C1-flare obtained during the peak phase. The other analysis example shows how to derive the time evolution of fluxes, emission measures (EM) and temperatures through the whole single C4 flare with the time resolution of 16 s. The preparatory data analysis procedures are also introduced in detail. These are required in spectral fittings of the data. A new solar monitor design equipped with a concentrator optics and a moderate size of field of view is also introduced.
  • Jakava-Viljanen, Miia (Helsingin yliopisto, 2007)
    Lactobacilli, common members of porcine intestinal microbiota, have been considered to be an important group of bacteria in maintaining the stability of gastrointestinal tract (GIT), preventing intestinal infections and supporting intestinal health. Because several species of lactobacilli have GRAS (generally regarded as safe) status and some of them have an ability to interact with intestinal epithelial cells, their possible applications as mucosal vaccine vector and/or probiotics have aroused interest. Selection criteria for lactobacilli to be used as vaccine vector or probiotic include the abilities to adhere to the intestinal epithelium cells and colonize the lumen of the GIT. Bacterial adhesins are often found in hair-like appendages called pili or fimbriae that extend outward from bacterial surface. Alternatively, they can be directly associated with the microbial cell surface. Surface layer proteins (Slps) of lactobacilli have been shown to confer tissue adherence. In this study, S-layer carrying lactobacilli from the intestine and faeces of pigs were isolated and their ability to adhere was studied. Besides the putative binding properties of Slps, a very large number of Slp subunits present in an S-layer make the use of the S-layer structure a very interesting alternative to surface display antigens. Therefore, the aim was to characterize the S-layer proteins. Two new surface layer proteins with potential to be tested as antigen carriers were characterized, and three slp genes were isolated, sequenced, and studied for their expression. To identify the S-layer carrying lactobacilli strains of porcine origin, a polyphasic taxonomic approach was applied. These results indicated that strains from Finland and the related L. sobrius strains, originating from elsewhere, constitute a single species, L. amylovorus, and that the name L. sobrius should be considered as a later synonym of L. amylovorus. The F18 fimbriae carrying Escherichia coli strains cause post-weaning diarrhoea and edema disease in pigs. The adhesin FedF of E. coli F18 fimbriae was characterized. The work aims at developing lactobacilli as a live mucosal vaccine vector for pigs against diseases caused by F18+ E. coli. Oral immunization of weaned piglets with adhesins is known to induce a protective mucosal immune response. Naked FedF appeared to be very unstable but we could produce it as a fusion protein with maltose binding protein (MBP). Specific adhesion to isolated porcine intestinal epithelial cells was demonstrated with MBP-FedF fusions as well as the ability of anti-MBP-FedF antibodies to prevent binding of E. coli F18 to porcine epithelial cells.
  • Seppälä, Ulla (Helsingin yliopisto, 2001)
  • Savelainen, Matti (Helsingin yliopisto, 2014)
    We constrain cosmological models where the primordial perturbations have an adiabatic and a (possi- bly correlated) cold dark matter (CDI), neutrino density (NDI) or neutrino velocity (NVI) isocurvature component. We use both a phenomenological approach, where the power spectra of primordial per- turbations are parametrized with two amplitudes at two different scales, and a slow-roll two-field inflation approach where inflation slow-roll parameters are used as primary parameters, determining the spectral indices and the ratio of tensor perturbations to scalar perturbations. We use WMAP 7- year and 9-year data combined with other CMB data and Planck 2013 CMB temperature anisotropy data. Bayesian methods indicate no preference for any of the isocurvature modes: the CMB data set tight upper bounds on any non-adiabatic contribution to the observed CMB temperature variance. We show that allowing for a primordial tensor contribution has a negligible effect on the determi- nation of the non-adiabatic contribution and vice versa, as long as the tensor spectral index obeys the first inflationary consistency relation. On large scales, the WMAP CMB data seem to constrain isocurvature tighter than the Planck data. This is due to the lack of power at low multipoles, l ∼ 2...40, in the Planck data compared to the prediction of the best-fitting adiabatic ΛCDM model. Hence the Planck data prefer a power-reducing mechanism, which the mixed adiabatic and isocurva- ture models with negative correlation or full anticorrelation can offer. With WMAP 9-year data we find that in the NDI and NVI cases larger isocurvature fractions are allowed than in the correspond- ing models with CDI. For uncorrelated perturbations, the upper limit to the primordial NDI (NVI) fraction is 24% (20%) at k = 0.002Mpc−1 and 28% (16%) at k = 0.01Mpc−1. For maximally correlated (anticorrelated) perturbations, the upper limit to the NDI fraction is 3.0% (0.9%). The non-adiabatic contribution to the CMB temperature variance can be 10% ( 13%) for the NDI (NVI) modes. For Planck data the non-adiabatic contribution to the temperature variance can be up to 7%, 9%, 5% in the CDI, NDI, NVI models. The Planck data constrain the primordial CDI fraction in specific curvaton and axion scenarios to 0.25% and 3.9%, respectively. All bounds above are at 95% CL. With the WMAP data, relaxing the pure adiabaticity assumption leads to large shifts of the preferred values of standard cosmological parameters and broadening of their posterior probability distributions. In contrast, as the Planck data determines the acoustic peak structure precisely up to the sixth acoustic peak, allowing for a mixture of the primordial adiabatic and an isocurvature mode does not significantly affect the determination of standard cosmological parameters.
  • Hanif, Mubashir (Helsingin yliopisto, 2004)
    Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.
  • Strengell, Satu (Helsingin yliopisto, 2014)
    Functional lung CT using synchrotron radiation was applied for studying regional ventilation distribution in vivo in animal lungs. This quantitative method employs the K-edge subtraction (KES) technique, which takes advantage of the sharp rise of the absorption coefficient at the binding energy of K-shell electron of a contrast agent. In KES technique two images are taken simultaneously at energies bracketing the K-edge energy of stable xenon (Xe) gas. This technique produces absorption CT images for structural evaluation and Xe density images and at the same time it allows the study of the regional ventilation heterogeneity, calculated based on wash-in series of Xe inhalation images. In this work the analysis of KES CT images was developed to show the regional differences in the airway reactions produced by the direct constrictor agonist, methacholine (MCh), and the indirect constrictor agonist, ovalbumin (OVA), in healthy and asthmatic rabbits. Moreover these constrictors were tested under the influence of cigarette smoke. The effect in airways of different administration routes of MCh, inhaled or intravenous (i.v.), was studied using low radiation dose Xe density CT images. The radiation dose in synchrotron radiation imaging of rabbit lungs in vivo, with and without Xe, was calculated and measured using several methods and compared to the image quality. Results showed that i.v. MCh caused bronchoconstriction primarily in central airways in healthy and asthmatic animals, whereas i.v. OVA only in asthmatic animals. Inhaled MCh, in both groups, affected more the periphery. On the other hand cigarette smoke inhibited the bronchoconstriction in both healthy and asthmatic animals. Theoretical and measured values of synchrotron radiation dose were consistent in this setup. Dose dependency of the image quality, defined by signal to noise ratio, was evaluated, and a threshold for detectable signal contrast was found. The optimal imaging setup was estimated using these results. These findings show the feasibility of the KES CT method for functional lung imaging of airway reactions by providing information on the regional ventilation distributions. By optimizing the image acquisition and reconstruction algorithms with existing methods, KES CT lung imaging could be adaptable for human studies in the near future.
  • Polianskyte, Zydrune (Helsingin yliopisto, 2009)
    Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases.
  • Saariaho, Anna-Helena (Helsingin yliopisto, 2006)
    Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.