Evaluation of the spot urine sampling technique to assess urinary pseudouridine excretion in lactating dairy cows

Selostus: Mahdollisuus lyhytaikaisen virtsankeruun käyttöön lypsylehmien virtsan pseudouridiinin erityksen määrittämisessä

Multivariable regression analysis and hot-spot stress approach into fatigue life estimations of welded joints

Kolmen eri hitsausliitoksen väsymisikä arvio on analysoitu monimuuttuja regressio analyysin avulla. Regression perustana on laaja S-N tietokanta joka on kerätty kirjallisuudesta. Tarkastellut liitokset ovat tasalevy liitos, krusiformi liitos ja pitkittäisripa levyssä. Muuttujina ovat jännitysvaihtelu, kuormitetun levyn paksuus ja kuormitus tapa. Paksuus effekti on käsitelty uudelleen kaikkia kolmea liitosta ajatellen. Uudelleen käsittelyn avulla on varmistettu paksuus effektin olemassa olo ennen monimuuttuja regressioon siirtymistä. Lineaariset väsymisikä yhtalöt on ajettu kolmelle hitsausliitokselle ottaen huomioon kuormitetun levyn paksuus sekä kuormitus tapa. Väsymisikä yhtalöitä on verrattu ja keskusteltu testitulosten valossa, jotka on kerätty kirjallisuudesta. Neljä tutkimustaon tehty kerättyjen väsymistestien joukosta ja erilaisia väsymisikä arvio metodeja on käytetty väsymisiän arviointiin. Tuloksia on tarkasteltu ja niistä keskusteltu oikeiden testien valossa. Tutkimuksissa on katsottu 2mm ja 6mm symmetristäpitkittäisripaa levyssä, 12.7mm epäsymmetristä pitkittäisripaa, 38mm symmetristä pitkittäisripaa vääntökuormituksessa ja 25mm/38mm kuorman kantavaa krusiformi liitosta vääntökuormituksessa. Mallinnus on tehty niin lähelle testi liitosta kuin mahdollista. Väsymisikä arviointi metodit sisältävät hot-spot metodin jossa hot-spot jännitys on laskettu kahta lineaarista ja epälineaarista ekstrapolointiakäyttäen sekä paksuuden läpi integrointia käyttäen. Lovijännitys ja murtumismekaniikka metodeja on käytetty krusiformi liitosta laskiessa.

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.

Analysis of outliers in electricity spot prices with exampleof New England and New Zealand markets

Electricity spot prices have always been a demanding data set for time series analysis, mostly because of the non-storability of electricity. This feature, making electric power unlike the other commodities, causes outstanding price spikes. Moreover, the last several years in financial world seem to show that ’spiky’ behaviour of time series is no longer an exception, but rather a regular phenomenon. The purpose of this paper is to seek patterns and relations within electricity price outliers and verify how they affect the overall statistics of the data. For the study techniques like classical Box-Jenkins approach, series DFT smoothing and GARCH models are used. The results obtained for two geographically different price series show that patterns in outliers’ occurrence are not straightforward. Additionally, there seems to be no rule that would predict the appearance of a spike from volatility, while the reverse effect is quite prominent. It is concluded that spikes cannot be predicted based only on the price series; probably some geographical and meteorological variables need to be included in modeling.

Highly functional binding surface for miniaturised solid-phase immunoassays. - A spot story -

Several bioaffinity assays are based on the detection of an analyte which is bound on a solid substrate via biochemical interaction. These so called solid phase assays are based on the adhesion of the primary binding partner on a solid surface, which then binds the analyte to be detected. In this thesis work a novel solid phase based assay technology, known as spot technology, was developed. The spot technology is based on combination of high-capacity solid phases, concentrated in a spot format, utilising modified streptavidin molecules and recombinant antibody fragments. The reduction of the solid phase binding surface to a size of a spot enabled denser binding of the target molecules, providing improved signal intensities and signal-to-background ratio when applied in different solid phase immunoassays. Streptavidin-biotin interactions are commonly utilised in numerous different bioaffinity assays and the ultimate nature of streptavidin to bind biotin is among the strongest non-covalent interaction reported between two biomolecules. In this study native core streptavidin was chemically modified to provide polymerised streptavidin molecules with altered adsorption properties. These streptavidin conjugates, when coated onto polystyrene surface, provided enhanced biotin binding capacity and surface stability when compared to a reference coating constructed with native streptavidin. Furthermore, the combination of chemically modified streptavidin, sitespecifically biotinylated antibody fragments and the spot coating technology provided highly dense solid phase coating with improved binding properties. The performance of the spot assay technology was further demonstrated in different immunoassay configurations. Human thyroid stimulating hormone (TSH) and human cardiac troponin I (cTnI) were used as model analytes to show the applicability of the highly sensitive spot-based solid-phase immunoassay for detection of very low levels of analytes. It was demonstrated that the spot technology provided an assay concept with enhanced sensitivity and short turn-around times, characteristics that are highly suitable for point-of-care applications.

Prognosis of after spot volumes at the Scandinavian electricity market

The Thesis is dedicated to development of an operative tool to support decision making in after spot trading on the Nordic electricity market. The basics of the Nordic electricity market, trading mechanisms on the spot and after spot markets are presented in the Thesis. Mathematical equations that describe electricity balance condition in the power system are offered. The main driving factors that impact deviation of actual electricity balance from the scheduled one (object) in the power system have been explored and mathematically defined. The behavioral model of the object and principal trends in change of state of the object under an impact of the driving factors are determined with the help of regression analysis made in Microsoft Office Excel. The behavioral model gives an indication for the total regulation volume (Elbas trades volume, volume of regulation market, balance power) for a certain hour that serves as the base input in estimating prices on the after spot markets. Proposals for development of methodologies of forecasting the after spot electricity prices are offered.

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.

A comparison of electricity spot prices simulation using ARMA-GARCH and mean-reverting models

The aim of this work is to compare two families of mathematical models for their respective capability to capture the statistical properties of real electricity spot market time series. The first model family is ARMA-GARCH models and the second model family is mean-reverting Ornstein-Uhlenbeck models. These two models have been applied to two price series of Nordic Nord Pool spot market for electricity namely to the System prices and to the DenmarkW prices. The parameters of both models were calibrated from the real time series. After carrying out simulation with optimal models from both families we conclude that neither ARMA-GARCH models, nor conventional mean-reverting Ornstein-Uhlenbeck models, even when calibrated optimally with real electricity spot market price or return series, capture the statistical characteristics of the real series. But in the case of less spiky behavior (System prices), the mean-reverting Ornstein-Uhlenbeck model could be seen to partially succeeded in this task.

Analysis of Patterns in Electricity Spot Market Time Series

Due to its non-storability, electricity must be produced at the same time that it is consumed, as a result prices are determined on an hourly basis and thus analysis becomes more challenging. Moreover, the seasonal fluctuations in demand and supply lead to a seasonal behavior of electricity spot prices. The purpose of this thesis is to seek and remove all causal effects from electricity spot prices and remain with pure prices for modeling purposes. To achieve this we use Qlucore Omics Explorer (QOE) for the visualization and the exploration of the data set and Time Series Decomposition method to estimate and extract the deterministic components from the series. To obtain the target series we use regression based on the background variables (water reservoir and temperature). The result obtained is three price series (for Sweden, Norway and System prices) with no apparent pattern.

A cell spot microarray method for high-throughput biology

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for the conventional multi-well based high-throughput screening -platforms, here the development of a novel cell spot microarray method for production of high density siRNA reverse transfection arrays is described. The cell spot microarray platform is distinguished from the majority of other transfection cell microarray techniques by the spatially confined array layout that allow highly parallel screening of large-scale RNAi reagent libraries with assays otherwise difficult or not applicable to high-throughput screening. This study depicts the development of the cell spot microarray method along with biological application examples of high-content immunofluorescence and phenotype based cancer cell biological analyses focusing on the regulation of prostate cancer cell growth, maintenance of genomic integrity in breast cancer cells, and functional analysis of integrin protein-protein interactions in situ.

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.

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.