Materiaalimallin määrityksestä kylmämuovattujen putkipalkkien K-liitosten FE-analyysissä

Työssä on tutkittu kylmämuovattujen nelikulmaisten putkipalkkien K-liitosten mallinnusta epälineaarisella elementtimenetelmällä. Työn tärkeimpänä tavoitteena on ollut kehittää putkipalkin osien materiaalimalleja siten, että liitosten kestävyyttä voidaan tutkia laboratoriokokeiden ohella luotettavasti myös elementtimenetelmällä. Toisena tavoitteena on ollut tutkia, voidaanko putkipalkkien liitosten mitoitusohjeita turvallisesti soveltaa kylmämuovatuille putkipalkeille, joissa valmistusprosessi aiheuttaa muutoksia materiaaliominaisuuksiin, erityisesti muodonmuutoskykyyn. Työssä tehtyjen laboratoriokokeiden ja elementtianalyysien perusteella elementti-menetelmä on käyttökelpoinen työkalu putkipalkkiliitosten staattista kestävyyttä määritettäessä, kun materiaalimallit on määritetty oikein. Erityisesti liitoksen käyttö-rajatilan mukaisen kestävyyden laskennassa elementtimenetelmällä saadaan hyvin laboratoriokokeita vastaavia tuloksia. Tehdyt laboratoriokokeet osoittavat myös, että Eurocode 3:n mukaisia putkipalkkien liitosten mitoitusohjeita voi turvallisesti käyttää kylmämuovatuille putkipalkeille.

Päällystyskoneiden runkojen värähtelyiden FE-analysoinnin kehittäminen

Työssä kehitettiin päällystyskoneen runkojen FE-analysointia erityisesti vastelaskennan kannalta, joskin myös ominaistaajuuslaskennan tarkentamiseksi esitettiin parannuksia. Työssä rajoitutaan harmonisten telaherätteiden aiheuttamien vasteiden analysointiin. . Työssä käsitellään värähtelyjen teoriaa ja tarkastellaan Abaqus-ohjelmiston (versio 5.8) laskentamenetelmiä vakiotilan värähtelyvasteen laskemiseksi. FEM-mallin rakenteeseen liittyen käsitellään perustuksien ja maaperän, konepalkin ja telaherätteiden mallintamista ja mallinnuksen laajuutta. Telaherätteitä käsitellään yksittäisen telan ja telojen samanvaiheisuuden kannalta. Samanvaiheisuutta tutkitaan työssä kehitetyllä summafunktiolla. Värähtelyjen mittaukseen FEM-mallien verifioimiseksi esitetään parannuksia. Nykyinen mallinnustapa käsitellään lyhyesti. Parannusehdotuksia kokeiltiin mallintamalla Rauma 400-päällystyskone ja vertaamalla tuloksia mitattuihin. Laskennan tulokset vastasivat vaihtelevasti mittaustuloksia, mittaustulosten puutteellisuus vaikeutti vertailua. Tulosten perusteella herätetiedon parantaminen on perusteltua ja mallin laajennus lisää todenmukaisuutta. Maaperän huomiointi vaikuttaa ennen kaikkea ominaistaajuuksiin ja muotoihin ja suora ratkaisutapa on käyttökelpoinen vasteen laskentamenetelmä otettaessa maaperä huomioon.

Improvements in the Assessment of Bacterial Viability and Killing

It is axiomatic that our planet is extensively inhabited by diverse micro-organisms such as bacteria, yet the absolute diversity of different bacterial species is widely held to be unknown. Different bacteria can be found from the depths of the oceans to the top of the mountains; even the air is more or less colonized by bacteria. Most bacteria are either harmless or even advantageous to human beings but there are also bacteria, which can cause severe infectious diseases or spoil the supplies intended for human consumption. Therefore, it is vitally important not only to be able to detect and enumerate bacteria but also to assess their viability and possible harmfulness. Whilst the growth of bacteria is remarkably fast under optimum conditions and easy to detect by cultural methods, most bacteria are believed to lie in stationary phase of growth in which the actual growth is ceased and thus bacteria may simply be undetectable by cultural techniques. Additionally, several injurious factors such as low and high temperature or deficiency of nutrients can turn bacteria into a viable but non-culturable state (VBNC) that cannot be detected by cultural methods. Thereby, various noncultural techniques developed for the assessment of bacterial viability and killing have widely been exploited in modern microbiology. However, only a few methods are suitable for kinetic measurements, which enable the real-time detection of bacterial growth and viability. The present study describes alternative methods for measuring bacterial viability and killing as well as detecting the effects of various antimicrobial agents on bacteria on a real-time basis. The suitability of bacterial (lux) and beetle (luc) luciferases as well as green fluorescent protein (GFP) to act as a marker of bacterial viability and cell growth was tested. In particular, a multiparameter microplate assay based on GFP-luciferase combination as well as a flow cytometric measurement based on GFP-PI combination were developed to perform divergent viability analyses. The results obtained suggest that the antimicrobial activities of various drugs against bacteria could be successfully measured using both of these methods. Specifically, the data reliability of flow cytometric viability analysis was notably improved as GFP was utilized in the assay. A fluoro-luminometric microplate assay enabled kinetic measurements, which significantly improved and accelerated the assessment of bacterial viability compared to more conventional viability assays such as plate counting. Moreover, the multiparameter assay made simultaneous detection of GFP fluorescence and luciferase bioluminescence possible and provided extensive information about multiple cellular parameters in single assay, thereby increasing the accuracy of the assessment of the kinetics of antimicrobial activities on target bacteria.

Kaupungin ääniä, Santa Fe

Kaupunki, Santa Fe. Katuhälyä. Englanninkielistä puhetta, miehiä, naisia ja lapsia.

Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.

Biogeochemistry in acid sulphate soil landscapes and small urban centres in Western Finland

The overall purpose of this thesis was to increase the knowledge on the biogeochemistry of rural acid sulphate (AS) soil environments and urban forest ecosystems near small towns in Western Finland. In addition, the potential causal relationship between the distribution of AS soils and geographical occurence of multiple sclerosis (MS) disease was assessed based on a review of existing literature and data. Acid sulphate soils, which occupy an area of approximately 17–24 million hectare worldwide, are regarded as the nastiest soils in the world. Independent of the geographical locality of these soils, they pose a great threat to their surrounding environment if disturbed. The abundant metal-rich acid drainage from Finnish AS soils, which is a result of sulphide oxidation due to artificial farmland drainage, has significant but spatially and temporally variable ecotoxicological impacts on biodiversity and community structure of fish, benthic invertebrates and macrophytes. This has resulted in mass fish kills and even eradication of sensitive fish species in affected waters. Moreover, previous investigations demonstrated significantly enriched concentrations of Co, Ni, Mn and Al, metals which are abundantly mobilised in AS soils, in agricultural crops (timothy grass and oats) and approximately 50 times higher concentrations of Al in cow milk originating from AS soils in Western Finland. Nevertheless, the results presented here demonstrate, in general, relatively moderate metal concentrations in oats and cabbage grown on AS soils in Western Finland, although some of the studied fields showed anomalous values of metals (e.g. Co and Ni) in both the soil and target plants (especially oats), similar to that of the previous investigations. The results indicated that the concentrations of Co, Ni, Mn and Zn in oats and Co and Zn in cabbage were governed by soil geochemistry as these metals were correlated with corresponding concentrations extracted from the soil by NH4Ac-EDTA and NH4Ac, respectively. The concentrations of Cu and Fe in oats and cabbage were uncorrelated to that of the easily soluble concentrations in the soils, suggesting that biological processes (e.g. plant-root processes) overshadow geochemical variation. The concentrations of K and Mg in cabbage, which showed a low spread and were strongly correlated to the NH4Ac extractable contents in the soil, were governed by both the bioavailable fractions in the topsoil and plant-uptake mechanisms. The plant´s ability to regulate its uptake of Ca and P (e.g. through root exudates) seemed to be more important than the influence of soil geochemistry. The distribution of P, K, Ca, Mg, Mn and S within humus, moss and needles in and around small towns was to a high degree controlled by biological cycling, which was indicated by the low correlation coefficients for P, K, Ca, Mg and S between humus and moss, and the low spread of these nutrients in moss and needles. The concentration variations of elements in till are mainly due to natural processes (e.g. intrusions, weathering, mineralogical variations in the bedrock). There was a strong spatial pattern for B in humus, moss and needles, which was suggested to be associated with anthropogenic emissions from nearby town centres. Geogenic dust affected the spatial distribution of Fe and Cr in moss, while natural processes governed the Fe anomaly found in the needles. The spatial accumulation patterns of Zn, Cd, Cu, Ni and Pb in humus and moss were strong and diverse, and related to current industry, the former steel industry, coal combustion, and natural geochemical processes. An intriguing Cu anomaly was found in moss. Since it was located close to a main railway line and because the railway line´s electric cables are made of Cu, it was suggested that the reason for the Cu anomaly is corrosion of these cables. In Western Finland, where AS soils are particularly abundant and enrich the metal concentrations of stream waters, cow milk and to some extent crops, an environmental risk assessment would be motivated to elucidate if the metal dispersion affect human health. Within this context, a topic of concern is the distribution of multiple sclerosis as high MS prevalence rates are found in the main area of AS soils. Regionally, the AS soil type in the Seinäjoki area has been demonstrated to be very severe in terms of metal leaching, this area also shows one of the highest MS rates reported worldwide. On a local scale, these severe AS soil types coincide well with the corresponding MS clustering along the Kyrönjoki River in Seinäjoki. There are reasons to suspect that these spatial correlations are causal, as multiple sclerosis has been suggested to result from a combination of genetic and environmental factors.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

DPS-Like Peroxide Resistance Protein: Structural and Functional Studies on a Versatile Nanocontainer

Oxidative stress is a constant threat to almost all organisms. It damages a number of biomolecules and leads to the disruption of many crucial cellular functions. It is caused by reactive oxygen species (ROS), such as hydrogen peroxide (H2O₂), superoxide (•O₂ -), and hydroxyl radical (•OH). The most harmful of these compounds is •OH, which is only formed in cells in the presence of redox-cycling transition metals, such as iron and copper. Bacteria have developed a number of mechanisms to cope with ROS. One of the most widespread means employed by bacteria is the DNA-binding proteins from starved cells (Dps). Dps proteins protect the cells by binding and oxidizing Fe2+, thus greatly reducing the production of •OH. The oxidized iron is stored inside the protein as an iron core. In addition, Dps proteins bind directly to DNA forming a protective coating that shields DNA from harmful agents. Moreover, Dps proteins have been found to elicit other protective functions in cells and to participate in bacterial virulence. Dps proteins are of special importance to Streptococci owing to the lack of catalase in this genus of bacteria.This study was focused on structural and functional characterization of streptococcal Dpslike peroxide resistance (Dpr) proteins. Initially, crystal structures of Streptococcus pyogenes Dpr were determined. The data confirmed the presence of a di-metal ferroxidase center (FOC) in Dpr proteins and revealed the presence of a novel N-terminal helix as well as a surface metal-binding site. The crystal structures of Streptococcus suis Dpr complexed with transition metals demonstrated the metal specificity of the FOC. Solution binding studies also indicated the presence of a di-metal FOC. These results suggested a possible role for Dpr in the detoxification of various metals. Iron was found to mineralize inside the protein as ferrihydrite based on X-ray absorption spectroscopy data. The iron core was found to exhibit clear superparamagnetic behaviour using magnetic and Mössbauer measurements. The results from this study are expected to further increase our understanding on the binding, oxidation, and mineralization of iron and other metals in Dpr proteins. In particular, the structural and magnetic properties of the iron core can form a basis for potential new applications in nanotechnology. From the streptococcal viewpoint, the results would help in understanding better the complicated picture of bacterial pathogenesis. Dpr proteins may also provide a novel target for drug design due to their tight involvement in bacterial virulence.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Power cycling lifetime estimation of IGBT power modules based on chip temperature modeling

In this doctoral thesis, methods to estimate the expected power cycling life of power semiconductor modules based on chip temperature modeling are developed. Frequency converters operate under dynamic loads in most electric drives. The varying loads cause thermal expansion and contraction, which stresses the internal boundaries between the material layers in the power module. Eventually, the stress wears out the semiconductor modules. The wear-out cannot be detected by traditional temperature or current measurements inside the frequency converter. Therefore, it is important to develop a method to predict the end of the converter lifetime. The thesis concentrates on power-cycling-related failures of insulated gate bipolar transistors. Two types of power modules are discussed: a direct bonded copper (DBC) sandwich structure with and without a baseplate. Most common failure mechanisms are reviewed, and methods to improve the power cycling lifetime of the power modules are presented. Power cycling curves are determined for a module with a lead-free solder by accelerated power cycling tests. A lifetime model is selected and the parameters are updated based on the power cycling test results. According to the measurements, the factor of improvement in the power cycling lifetime of modern IGBT power modules is greater than 10 during the last decade. Also, it is noticed that a 10 C increase in the chip temperature cycle amplitude decreases the lifetime by 40%. A thermal model for the chip temperature estimation is developed. The model is based on power loss estimation of the chip from the output current of the frequency converter. The model is verified with a purpose-built test equipment, which allows simultaneous measurement and simulation of the chip temperature with an arbitrary load waveform. The measurement system is shown to be convenient for studying the thermal behavior of the chip. It is found that the thermal model has a 5 C accuracy in the temperature estimation. The temperature cycles that the power semiconductor chip has experienced are counted by the rainflow algorithm. The counted cycles are compared with the experimentally verified power cycling curves to estimate the life consumption based on the mission profile of the drive. The methods are validated by the lifetime estimation of a power module in a direct-driven wind turbine. The estimated lifetime of the IGBT power module in a direct-driven wind turbine is 15 000 years, if the turbine is located in south-eastern Finland.

FE-analyysin soveltaminen S960 QC teräksisen I-profiilin äärikestävyyden määrittämisessä

Tässä työssä tutkittiin FE-analyysin soveltamista S960 QC teräksisen I-profiilin kestävyyden määrittämisessä. Työn tavoitteena oli tarkastella nykyisten suunnitteluohjeiden soveltuvuutta ultralujille teräksille ja koota ohjemateriaali I-profiilin optimoimisesta sekä FE-analyysin hyö-dyntämisestä I-profiilin staattisen ja dynaamisen kestävyyden määrittämisessä. I-profiili mitoitettiin ja optimoitiin Eurokoodi 3:ssa esitettyjen PL3 mukaisten mitoitusohjeiden avulla. Rakenteelle suoritettiin Eurokoodi 3:n ja IIW:n mukaiset lommahdus-, kiepahdus- ja vä-symiskestävyystarkastelut. Väsymistarkastelussa sovellettiin nimellisen jännityksen, rakenteelli-sen jännityksen ja tehollisen lovijännityksen menetelmiä sekä murtumismekaniikkaa. Rakenteel-lisen jännityksen menetelmässä sovellettiin lisäksi lineaarista ja parabolista pintaa pitkin ekstra-polointia, paksuuden yli linearisointia sekä Dong:in menetelmää. Lommahdus-, kiepahdus- ja väsymistarkasteluissa hyödynnettiin analyyttistä laskentaa, FE-analyysiä sekä Frank2d sovellusta. Tarkastelujen perusteella voidaan todeta, että analyyttisillä menetelmillä saadaan numeerisia me-netelmiä varmemmalla puolella olevia tuloksia. Lommahdustarkastelussa ero tulosten välillä on suurimmillaan 8 % ja kiepahdustarkastelussa suurimmillaan 20 % mutta väsymistarkastelussa saadut tulokset eroavat keskenään huomattavasti. Väsymistarkastelussa tehollisen lovijännityksen menetelmällä sekä rakenteellisen jännityksen menetelmän Dong:in menetelmällä saadaan huo-mattavasti muita menetelmiä pidempiä kestoikiä, kun taas yksinkertaisemmilla menetelmillä saa-dut kestoiät ovat lyhyempiä. Rakenteen kestävyyden määrittäminen analyyttisillä menetelmillä on melko helppoa, mutta tu-lokset ovat monesti liian konservatiivisia. FE-analyysillä saadaan puolestaan hyvin tarkkoja tu-loksia mallin ollessa yksityiskohtainen. Mallintaminen on kuitenkin aikaa ja resursseja vievää ja vaatii käyttökokemusta. FE-analyysin mahdolliset hyödyt on aina arvioitava tapauskohtaisesti tarkasteltavan geometrian, kuormitusten ja reunaehtojen perusteella.