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  • Hoppu, Pekka (Helsingin yliopisto, 2008)
    The number of drug substances in formulation development in the pharmaceutical industry is increasing. Some of these are amorphous drugs and have glass transition below ambient temperature, and thus they are usually difficult to formulate and handle. One reason for this is the reduced viscosity, related to the stickiness of the drug, that makes them complicated to handle in unit operations. Thus, the aim in this thesis was to develop a new processing method for a sticky amorphous model material. Furthermore, model materials were characterised before and after formulation, using several characterisation methods, to understand more precisely the prerequisites for physical stability of amorphous state against crystallisation. The model materials used were monoclinic paracetamol and citric acid anhydrate. Amorphous materials were prepared by melt quenching or by ethanol evaporation methods. The melt blends were found to have slightly higher viscosity than the ethanol evaporated materials. However, melt produced materials crystallised more easily upon consecutive shearing than ethanol evaporated materials. The only material that did not crystallise during shearing was a 50/50 (w/w, %) blend regardless of the preparation method and it was physically stable at least two years in dry conditions. Shearing at varying temperatures was established to measure the physical stability of amorphous materials in processing and storage conditions. The actual physical stability of the blends was better than the pure amorphous materials at ambient temperature. Molecular mobility was not related to the physical stability of the amorphous blends, observed as crystallisation. Molecular mobility of the 50/50 blend derived from a spectral linewidth as a function of temperature using solid state NMR correlated better with the molecular mobility derived from a rheometer than that of differential scanning calorimetry data. Based on the results obtained, the effect of molecular interactions, thermodynamic driving force and miscibility of the blends are discussed as the key factors to stabilise the blends. The stickiness was found to be affected glass transition and viscosity. Ultrasound extrusion and cutting were successfully tested to increase the processability of sticky material. Furthermore, it was found to be possible to process the physically stable 50/50 blend in a supercooled liquid state instead of a glassy state. The method was not found to accelerate the crystallisation. This may open up new possibilities to process amorphous materials that are otherwise impossible to manufacture into solid dosage forms.
  • Niemi, Jarkko (Helsingin yliopisto, 2007)
    Aerosol particles can cause detrimental environmental and health effects. The particles and their precursor gases are emitted from various anthropogenic and natural sources. It is important to know the origin and properties of aerosols to efficiently reduce their harmful effects. The diameter of aerosol particles (Dp) varies between ~0.001 and ~100 μm. Fine particles (PM2.5: Dp < 2.5 μm) are especially interesting because they are the most harmful and can be transported over long distances. The aim of this thesis is to study the impact on air quality by pollution episodes of long-range transported aerosols affecting the composition of the boundary-layer atmosphere in remote and relatively unpolluted regions of the world. The sources and physicochemical properties of aerosols were investigated in detail, based on various measurements (1) in southern Finland during selected long-range transport (LRT) pollution episodes and unpolluted periods and (2) over the Atlantic Ocean between Europe and Antarctica during a voyage. Furthermore, the frequency of LRT pollution episodes of fine particles in southern Finland was investigated over a period of 8 years, using long-term air quality monitoring data. In southern Finland, the annual mean PM2.5 mass concentrations were low but LRT caused high peaks of daily mean concentrations every year. At an urban background site in Helsinki, the updated WHO guideline value (24-h PM2.5 mean 25 μg/m3) was exceeded during 1-7 LRT episodes each year during 1999-2006. The daily mean concentrations varied between 25 and 49 μg/m3 during the episodes, which was 3-6 times higher than the mean concentration in the long term. The in-depth studies of selected LRT episodes in southern Finland revealed that biomass burning in agricultural fields and wildfires, occurring mainly in Eastern Europe, deteriorated air quality on a continental scale. The strongest LRT episodes of fine particles resulted from open biomass-burning fires but the emissions from other anthropogenic sources in Eastern Europe also caused significant LRT episodes. Particle mass and number concentrations increased strongly in the accumulation mode (Dp ~ 0.09-1 μm) during the LRT episodes. However, the concentrations of smaller particles (Dp < 0.09 μm) remained low or even decreased due to the uptake of vapours and molecular clusters by LRT particles. The chemical analysis of individual particles showed that the proportions of several anthropogenic particle types increased (e.g. tar balls, metal oxides/hydroxides, spherical silicate fly ash particles and various calcium-rich particles) in southern Finland during an LRT episode, when aerosols originated from the polluted regions of Eastern Europe and some open biomass-burning smoke was also brought in by LRT. During unpolluted periods when air masses arrived from the north, the proportions of marine aerosols increased. In unpolluted rural regions of southern Finland, both accumulation mode particles and small-sized (Dp ~ 1-3 μm) coarse mode particles originated mostly from LRT. However, the composition of particles was totally different in these size fractions. In both size fractions, strong internal mixing of chemical components was typical for LRT particles. Thus, the aging of particles has significant impacts on their chemical, hygroscopic and optical properties, which can largely alter the environmental and health effects of LRT aerosols. Over the Atlantic Ocean, the individual particle composition of small-sized (Dp ~ 1-3 μm) coarse mode particles was affected by continental aerosol plumes to distances of at least 100-1000 km from the coast (e.g. pollutants from industrialized Europe, desert dust from the Sahara and biomass-burning aerosols near the Gulf of Guinea). The rate of chloride depletion from sea-salt particles was high near the coasts of Europe and Africa when air masses arrived from polluted continental regions. Thus, the LRT of continental aerosols had significant impacts on the composition of the marine boundary-layer atmosphere and seawater. In conclusion, integration of the results obtained using different measurement techniques captured the large spatial and temporal variability of aerosols as observed at terrestrial and marine sites, and assisted in establishing the causal link between land-bound emissions, LRT and air quality.
  • Salusjärvi, Tuomas (Helsingin yliopisto, 2006)
    The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.
  • Blom, Titta S. (Helsingin yliopisto, 2003)
  • Vainio, Ulla (Helsingin yliopisto, 2007)
    Wood is an important material for the construction and pulping industries. Using x-ray diffraction the microfibril angle of Sitka spruce wood was studied in the first part of this thesis. Sitka spruce (Picea sitchensis [Bong.] Carr.) is native to the west coast of North America, but due to its fast growth rate, it has also been imported to Europe. So far, its nanometre scale properties have not been systematically characterised. In this thesis the microfibril angle of Sitka spruce was shown to depend significantly on the origin of the tree in the first annual rings near the pith. Wood can be further processed to separate lignin from cellulose and hemicelluloses. Solid cellulose can act as a reducer for metal ions and it is also a porous support for nanoparticles. By chemically reducing nickel or copper in the solid cellulose support it is possible to get small nanoparticles on the surfaces of the cellulose fibres. Cellulose supported metal nanoparticles can potentially be used as environmentally friendly catalysts in organic chemistry reactions. In this thesis the size of the nickel and copper containing nanoparticles were studied using anomalous small-angle x-ray scattering and wide-angle x-ray scattering. The anomalous small-angle x-ray scattering experiments showed that the crystallite size of the copper oxide nanoparticles was the same as the size of the nanoparticles, so the nanoparticles were single crystals. The nickel containing nanoparticles were amorphous, but crystallised upon heating. The size of the nanoparticles was observed to be smaller when the reduction of nickel was done in aqueous ammonium hydrate medium compared to reduction made in aqueous solution. Lignin is typically seen as the side-product of wood industries. Lignin is the second most abundant natural polymer on Earth, and it possesses potential to be a useful material for many purposes in addition to being an energy source for the pulp mills. In this thesis, the morphology of several lignins, which were produced by different separation methods from wood, was studied using small-angle and ultra small-angle x-ray scattering. It was shown that the fractal model previously proposed for the lignin structure does not apply to most of the extracted lignin types. The only lignin to which the fractal model could be applied was kraft lignin. In aqueous solutions the average shape of the low molar mass kraft lignin particles was observed to be elongated and flat. The average shape does not necessarily correspond to the shape of the individual particles because of the polydispersity of the fraction and due to selfassociation of the particles. Lignins, and especially lignosulfonate, have many uses as dispersants, binders and emulsion stabilisers. In this thesis work the selfassociation of low molar mass lignosulfonate macromolecules was observed using small-angle x-ray scattering. By taking into account the polydispersity of the studied lignosulfonate fraction, the shape of the lignosulfonate particles was determined to be flat by fitting an oblate ellipsoidal model to the scattering intensity.
  • Koskinen, Kaisa (Helsingin yliopisto, 2013)
    Microbes are essential for all life on Earth. They are found in all viable habitats from deep sea sediments and bedrock to high up in the atmosphere with a variety that exceeds by far the eukaryotic diversity. Ecosystem services provided by microorganisms, such as degradation of organic material and mediation of biogeochemical cycles are fundamentally important for the whole biosphere and its inhabitants. Microbes also form symbiotic relationships with multicellular organisms, and play important roles in nutrition and disease. Recent developments in molecular techniques, especially the next generation sequencing technologies and microarray applications, have opened new possibilities in studying diverse microbial communities. In this thesis, the aim was to determine the diversity and community structure of environmental samples collected from the northern Baltic Sea water column and anaerobic digestion reactor, and to assess how the prevailing abiotic factors affect the microbial community structure. We applied 16S rRNA and ITS gene amplicon sequencing method with 454 sequencing technology to form a detailed taxonomic description of studied communities. The produced sequence data was further utilised in designing probes for a new padlock probe based ligation detection reaction (LDR) microarray that could be employed for specific and sensitive taxonomic identification of microbial groups in diverse communities. The functionality, specificity and sensitivity of the microarray were assessed using artificial and real environmental samples. Additionally, selected amplicon sequencing data analysis methods were compared in order to discover which algorithms work most reliably. In this subproject, we aimed to clarify how significantly the selected analysis methods, specifically denoising and clustering algorithms, affect the results and how comparable the results derived from different analysis pipelines are. Amplicon sequencing revealed diverse microbial communities in the northern Baltic Sea water column and anaerobic digestion reactor. The pelagic bacterial communities in the northern Baltic Sea were strongly stratified, with aerobic Bacteria such as Pseudomonas and Flavobacterium dominating in the surface layer and Oleispira and sulfate-reducing bacteria in the anoxic deep waters. Based on the sequence data the diversity was assessed one order of magnitude less diverse compared to Atlantic and Pacific ocean bacterial communities. The anaerobic digestion reactor communities were dominated by Bacteria belonging to phyla Bacteroidetes, Firmicutes and Thermotogae and methanogenic Archaea, all essential and typical degraders in anaerobic digestion. The process also supported a diverse fungal community of phyla Ascomycota and Basidiomycota, including several taxa capable of degrading organic material in anaerobic conditions. The LDR microarray technology proved sensitive, specific and semiquantitative method for identifying microbes in diverse communities. The proof of principle tests and experiments with real environmental samples showed that if the probes are designed carefully, the detection is comparable to qPCR and amplicon sequencing. The detection limit was 0.01 fmol/µl/template. Data analysis method comparisons revealed prominent differences in observed operational taxonomic units and relative abundance of identified taxa. The majority of tested methods assessed the species richness too high. Using a functioning denoising method evened out the differences in the number of observed OTUs caused by various clustering algorithms. The ability to filter out the spurious taxa produced by amplification and sequencing, but still retain all the real diversity varied between methods. This study shows both the potential and the challenges in the use of amplicon sequencing and microarray technologies in studying diverse microbial communities. The results indicate that the padlock based LDR microarray can be designed for very accurate and sensitive identification of microbial groups of interest. The data suggest that amplicon sequencing is a powerful tool in identifying microbes and assessing the diversity but distinguishing between spurious and true community members remain a challenge. There is still work to be done in the development and application of data analysis tools.
  • Sarén, Matti-Paavo (Helsingin yliopisto, 2006)
    In recent years there has been growing interest in selecting suitable wood raw material to increase end product quality and to increase the efficiency of industrial processes. Genetic background and growing conditions are known to affect properties of growing trees, but only a few parameters reflecting wood quality, such as volume and density can be measured on an industrial scale. Therefore research on cellular level structures of trees grown in different conditions is needed to increase understanding of the growth process of trees leading to desired wood properties. In this work the cellular and cell wall structures of wood were studied. Parameters, such as the mean microfibril angle (MFA), the spiral grain angles, the fibre length, the tracheid cell wall thickness and the cross-sectional shape of the tracheid, were determined as a function of distance from the pith towards the bark and mutual dependencies of these parameters were discussed. Samples from fast-grown trees, which belong to a same clone, grown in fertile soil and also from fertilised trees were measured. It was found that in fast-grown trees the mean MFA decreased more gradually from the pith to the bark than in reference stems. In fast-grown samples cells were shorter, more thin-walled and their cross-sections were rounder than in slower-grown reference trees. Increased growth rate was found to cause an increase in spiral grain variation both within and between annual rings. Furthermore, methods for determination of the mean MFA using x-ray diffraction were evaluated. Several experimental arrangements including the synchrotron radiation based microdiffraction were compared. For evaluation of the data analysis procedures a general form for diffraction conditions in terms of angles describing the fibre orientation and the shape of the cell was derived. The effects of these parameters on the obtained microfibril angles were discussed. The use of symmetrical transmission geometry and tangentially cut samples gave the most reliable MFA values.
  • Rantakari, Anssi (Helsingin yliopisto, 2005)
  • Tsitko, Irina (Helsingin yliopisto, 2007)
    Species of the genera Rhodococcus, Gordonia and Mycobacterium are known as degraders of recalcitrant pollutants. These bacteria are good survivors in harsh environments. Due to such properties these organisms are able to occupy a wide range of environmental niches. The members of these taxa have been suggested as tools for biotechnical applications such as bioremediation and biosynthesis. At the same time several of the species are known as opportunistic human pathogens. Therefore, the detailed characterization of any isolate that has potential for biotechnological applications is very important. This thesis deals with several corynebacterial strains originating from different polluted environments: soil, water-damaged indoor walls, and drinking water distribution systems. A polyphasic taxonomic approach was applied for characterization of the isolates. We found that the strains degrading monoaromatic compounds belonged to Rhodococcus opacus, a species that has not been associated with any health problem. The taxonomic position of strain B293, used for many years in degradation research under different names, was clarified. We assigned it to the species Gordonia polyisoprenivorans. This species is classified under European Biohazard grouping 1, meaning that it is not considered a health hazard for humans. However, there are reports of catheter-associated bacteraemia caused by G. polyisoprenivorans. Our results suggested that the ability of the organism to grow on phthalate esters, used as softeners in medical plastics, may be associated with the colonization of catheters and other devices. In this thesis Mycobacterium lentiflavum, a new emerging opportunistic human pathogen, was isolated from biofilms growing in public drinking water distribution systems. Our report on isolation of M. lentiflavum from water supplies is the second report on this species from drinking water systems, which may thus constitute a reservoir of M. lentiflavum. Automated riboprinting was evaluated for its applicability in rapidly identifying environmental mycobacteria. The technique was found useful in the characterization of several species of rapidly and slowly growing environmental mycobacteria. The second aspect of this thesis refers to characterization of the degradation and tolerance power of several R. opacus, M. murale and G. polyisoprenivorans strains. R. opacus GM-14 utilizes a wide range of aromatic substrates, including benzene, 15 different halobenzenes, 18 phenols and 7 benzoates. This study revealed the high tolerance of R. opacus strains toward toxic hydrophobic compounds. R. opacus GM-14 grew in mineral medium to which benzene or monochlorobenzene was added in amounts of 13 or 3 g l-1, respectively. R. opacus GM-29 utilized toluene and benzene for growth. Strain GM-29 grew in mineral medium with 7 g l-1 of liquid toluene or benzene as the sole carbon source, corresponding to aqueous concentrations of 470 and 650 mg l-1, respectively. Most organic solvents, such as toluene and benzene, due to their high level of hydrophobicity, pass through the bacterial membrane, causing its disintegration. In this thesis the mechanisms of adaptation of rhodococci to toxic hydrophobic compounds were investigated. The rhodococcal strains increased the level of saturation of their cellular fatty acids in response to challenge with phenol, chlorophenol, benzene, chlorobenzene or toluene. The results indicated that increase in the saturation level of cellular fatty acids, particularly that in tuberculostearic acid, is part of the adaptation mechanism of strains GM-14 and GM-29 to the presence of toxic hydrophobic compounds.
  • Gonzalez, Manuel (2012)
    Campylobacter Spp are recognized as a major cause of bacterial food-borne gastroenteritis worldwide, with Campylobacter jejuni and Campylobacter coli being the most common species isolated in human infections (WHO, 2011). The number of registered cases of human campylobacteriosis in Finland has ranged from 3,796 cases in 2001 to 4,231 cases in 2011. The reported incidence in Finland in the last 10 years is higher than the European Union average. In order to compare human, chicken and cattle C. jejuni isolates, the presence or absence of four nonubiquitous genes were determined so that they could be associated with the source of the isolate. First, we tested the presence of dmsA, which encodes a subunit of the putative tripartite anaerobic dimethyl sulfoxide oxidoreductase (DMSO/trimethylamine N-oxide reductase). Second, we detected cj1585c, which encodes another oxidoreductase. Third, the serine protease gene cjj81176-1367/1371 was isolated. Fourth, γ-glutamyl-transpeptidase gene ggt was detected. We ascertained that ggt and dmsA are present more frequently in isolates obtained from humans and chickens, whereas cjj81176-1367/1371 and cj1585c are the most common in bovine isolates. Campylobacter jejuni is able to survive in different environments and in a wide range of temperatures. The study of C. jejuni inactivation in minced chicken meat and dug well water ascertain that the Weibull model could be applied optimally to the data to build a reliable prediction model for the survival of this microorganism as a function of temperature. The longest survival time found for C. jejuni in minced meat chicken was at the storage temperature of -20°C, and that of dug well water was at 4°C. We analyzed the effect of different seasoning as dry marinade combinations on accelerating the reduction of C. jejuni counts on chicken drumsticks and observed a decrease of more than 1 log CFU/g. In addition, our results showed that using some fractions of potato protein in combination with food additives and sodium lactate obtained inactivation levels in excess than 1.66 log CFU/g. The most important C. jejuni counts reductions were always obtained within the first hours after the application of the seasoning combinations onto the chicken meat.
  • Beklen Tanzer, Arzu (Helsingin yliopisto, 2010)
    Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.
  • Savolainen, Laura (Helsingin yliopisto, 2014)
    Tuberculosis (TB) still ranks among the most lethal infectious diseases in the world. The traditional distinction between active tuberculosis and latent tuberculosis infection (LTBI) is changing; at the moment, tuberculosis infection is described as a continuum with different stages of infection. The development of TB prevention, treatment and diagnostics is hampered by the diverse pathogenesis of the disease and the ability of the bacteria to fight host defence mechanisms. Traditional culture methods are slow and staining methods not sufficiently sensitive. Nucleic acid amplification techniques (NAAT) tend to be costly and in some cases lack sensitivity. New methods are needed for rapid and inexpensive detection of active disease and to distinguish between stages of infection in cases where the patient presents with symptoms compatible with active TB and with a positive Interferon Gamma Release Assay (IGRA) result, but bacteriological confirmation is not yet available. In addition, methods are needed that enable us to predict the activation of infection or monitor the effects of treatment. The aim of this thesis was to investigate: a) the possible diagnostic use of heparin-binding haemagglutinin (HBHA), a surface protein of M. tuberculosis, in distinguishing between stages of infection in a vaccinated population; b) the effect of sample concentration on the properties of Clearview® TB ELISA, a urine antigen test measuring lipoarabinomannan (LAM), a M. tuberculosis glycolipid, in the diagnosis of active TB; c) the suitability of granzyme B (GrB), perforin (Prf), and interferon-gamma (IFN-γ)-producing and CD107a-degranulation factor-expressing cytotoxic T lymphocytes (CTLs) for differentiating between stages of infection; d) the suitability of IFN-γ, interleukin (IL) -17, IL-4, IL-4δ2 and Forkhead box P3 (FoxP3) mRNA expression levels for differentiation between stages of infection. The results showed that HBHA-specific cells producing IFN-γ were also found in the circulation of healthy subjects who had been given the Bacillus Calmette-Guérin vaccine. In view of this, use of the HBHA antigen for diagnostic purposes does not look promising in countries where a widespread vaccination programme has taken place. No differences were found in the numbers or phenotypic properties of CTLs between persons with active TB and those with LTBI, which is why they are not suited for the differential diagnosis of infection stages. Functional antigen-specific CTLs were found in the circulation of persons who had been treated for TB. This most significant finding of this thesis demonstrates that the T cell memory generated by active TB maintains functionally active CTL populations decades after infection and despite adequate treatment with rifampicin. Although no statistically significant differences were found between patients with active TB and LTBI in the quantitative detection of IFN-γ, IL-17, or IL-4 mRNA from cells stimulated with purified protein derivative (PPD), this approach revealed a trend towards discrimination. The usefulness of combined quantitative IFN-γ, IL-17, and IL-4 mRNA expression for the differential diagnosis of active TB and LTBI should be retested with a larger sample size enrolling more homogenous patients in the LTBI group. The findings indicating that LAM may be detected with moderate sensitivity (57%) in the urine samples of TB patients show the greatest promise from a diagnostic point of view. The 100-fold concentration of urine used in this study improved the sensitivity of the Clearview® TB ELISA test from 7% to 57%, although specificity was decreased somewhat from 98% to 89%. Based on the results, demonstrating the presence of LAM in urine samples may be considered a potential diagnostic tool for detecting active TB. The method does require further development, however.
  • Palgi, Mari (Helsingin yliopisto, 2012)
    Among neurotrophic factors MANF/CDNF family is unique as their protein sequences are evolutionarily conserved between multicellular organisms. Still, little is known about their mechanism of action and interacting molecules. At the time of initiation of this study there were no known neurotrophic factors in invertebrates. According to the protein sequence homology there was an uncharacterized homologue to the novel neurotrophic factor MANF in Drosophila melanogaster. We found that Drosophila Manf (DmManf) is an essential gene in a fruit fly development. DmManf represents a true orthologue to mammalian MANF as its mutant lethality is rescued by human MANF. We have generated DmManf deletion mutant surviving to second instar larval stage with maternal contribution. When the maternal contribution of DmManf is abolished, the mutants die at the end of embryogenesis before hatching. In DmManf mutant the dopaminergic neurites degenerate and the dopamine level is extremely low. Ultrastructural analysis reveals nonapoptotic cell death in the embryonic ventral nerve cord and neuropile decomposition together with cell body glia activation taking place. In secretory cells like gastric caeca or fat body the visible loss of rough endoplasmic reticulum and drastic accumulation of vesicles, some filled with cellular debris, occur. According to microarray expression analysis data, expression of genes involved in vesicular transport and metabolism were altered in DmManf mutants. The expression of several genes implicated in pathology of Parkinson s disease (PD) was also altered. The degeneration of dopaminergic neurons is the hallmark for PD and this thesis work makes an effort to enlighten the mechanisms how the neurotrophic factor MANF protects these degenerating neurons.