Calibration of a desktop scanner and digital image analysis procedure for quantification of a root morphology

Selostus: Tasoskannerin ja digitaalisen kuva-analyysimenetelmän kalibrointi juurten morfologian kvantifioimiseksi

Quantification of fine root responses to selenium toxicity

Selostus: Seleenin myrkytysoireet juurissa

Determination of sugars and organic acids in pulp samples by capillary electrophoresis with UV detection

This MSc work was done in the project of BIOMECON financed by Tekes. The prime target of the research was, to develop methods for separation and determination of carbohydrates (sugars), sugar acids and alcohols, and some other organic acids in hydrolyzed pulp samples by capillary electrophoresis (CE) using UV detection. Aspen, spruce, and birch pulps are commonly used for production of papers in Finland. Feedstock components in pulp predominantly consist of carbohydrates, organic acids, lignin, extractives, and proteins. Here in this study, pulps have been hydrolyzed in analytical chemistry laboratories of UPM Company and Lappeenranta University in order to convert them into sugars, acids, alcohols, and organic acids. Foremost objective of this study was to quantify and identify the main and by-products in the pulp samples. For the method development and optimization, increased precision in capillary electrophoresis was accomplished by calculating calibration data of 16 analytes such as D-(-)-fructose, D(+)-xylose, D(+)-mannose, D(+)-cellobiose, D-(+)-glucose, D-(+)-raffinose, D(-)-mannitol, sorbitol, rhamnose, sucrose, xylitol, galactose, maltose, arabinose, ribose, and, α-lactose monohydratesugars and 16 organic acids such as D-glucuronic, oxalic, acetic, propionic, formic, glycolic, malonic, maleic, citric, L-glutamic, tartaric, succinic, adipic, ascorbic, galacturonic, and glyoxylic acid. In carbohydrate and polyalcohol analyses, the experiments with CE coupled to direct UV detection and positive separation polarity was performed in 36 mM disodium hydrogen phosphate electrolyte solution. For acid analyses, CE coupled indirect UV detection, using negative polarity, and electrolyte solution made of 2,3 pyridinedicarboxylic acid, Ca2+ salt, Mg2+ salts, and myristyltrimethylammonium hydroxide in water was used. Under optimized conditions, limits of detection, relative standard deviations and correlation coefficients of each compound were measured. The optimized conditions were used for the identification and quantification of carbohydrates and acids produced by hydrolyses of pulp. The concentrations of the analytes varied between 1 mg – 0.138 g in liter hydrolysate.

Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

Capillary electrophoresis for monitoring of carboxylic, phenolic and amino acids in bioprocesses

Bioprocess technology is a multidisciplinary industry that combines knowledge of biology and chemistry with process engineering. It is a growing industry because its applications have an important role in the food, pharmaceutical, diagnostics and chemical industries. In addition, the current pressure to decrease our dependence on fossil fuels motivates new, innovative research in the replacement of petrochemical products. Bioprocesses are processes that utilize cells and/or their components in the production of desired products. Bioprocesses are already used to produce fuels and chemicals, especially ethanol and building-block chemicals such as carboxylic acids. In order to enable more efficient, sustainable and economically feasible bioprocesses, the raw materials must be cheap and the bioprocesses must be operated at optimal conditions. It is essential to measure different parameters that provide information about the process conditions and the main critical process parameters including cell density, substrate concentrations and products. In addition to offline analysis methods, online monitoring tools are becoming increasingly important in the optimization of bioprocesses. Capillary electrophoresis (CE) is a versatile analysis technique with no limitations concerning polar solvents, analytes or samples. Its resolution and efficiency are high in optimized methods creating a great potential for rapid detection and quantification. This work demonstrates the potential and possibilities of CE as a versatile bioprocess monitoring tool. As a part of this study a commercial CE device was modified for use as an online analysis tool for automated monitoring. The work describes three offline CE analysis methods for the determination of carboxylic, phenolic and amino acids that are present in bioprocesses, and an online CE analysis method for the monitoring of carboxylic acid production during bioprocesses. The detection methods were indirect and direct UV, and laser-induced frescence. The results of this work can be used for the optimization of bioprocess conditions, for the development of more robust and tolerant microorganisms, and to study the dynamics of bioprocesses.

Quantification and Clinical Relevance of Cystatin C

An aging population and increasing rates of diabetes mellitus contribute to a high prevalence of kidney dysfunction – approximately 10 percent of adults in developed countries have chronic kidney disease (CKD). CKD is a progressive loss of kidney function and this remains permanent. Early recognition of this condition is important for prevention or impeding severe adverse cardiac and renal outcomes. Cystatin C is a low molecular weight cysteine protease inhibitor that has emerged as a biomarker of kidney function. The special potential of plasma cystatin C in this setting is related to its independency of muscle mass, which is a remarkable limitation of the traditional marker creatinine. Cystatin C is a sensitive marker in diagnosing mild and moderate CKD, especially in small children, in the elderly and in conditions where muscle mass is affected. Cystatin C is quantified with immunoassays, mainly based on particle-enhanced nephelometry (PENIA) or turbidimetry (PETIA). The aim of this study was to develop a rapid and reliable assay for quantification of human cystatin C in plasma or serum by utilizing time-resolved fluorescence-based immunoassay methods. This was accomplished by utilizing different antibodies, including polyclonal and 7 monoclonal antibodies against cystatin C. Different assay designs were tested and the best assay was further modified to a dry-reagent double monoclonal assay run on an automated immunonalyzer. This assay was evaluated for clinical performance in estimating reduced kidney function and in predicting risk of adverse outcomes in patients with non-ST elevation acute coronary syndrome. Of the tested assay designs, heterogeneous non-competitive assay had the best performace and was chosen to be developed further. As an automated double monoclonal assay, this assay enabled a reliable measurement of clinically relevant cystatin C concentrations. It also showed a stronger concordance with the reference clearance method than the conventional PETIA method in patients with reduced kidney function. Risk of all-cause mortality and combined events, defined by death and myocardial infarction, increased with higher cystatin C and cystatin C remained an independent predictor of death and combined events after adjustment to nonbiochemical baseline factors. In conclusion, the developed dry-reagent double monoclonal assay allows rapid and reliable quantitative measurement of cystatin C. As measured with the developed assay, cystatin C is a potential predictor of adverse outcomes in cardiac patients.

Quantitative Magnetic Resonance Imaging of the Liver and Heart In Iron Overload

Systemic iron overload (IO) is considered a principal determinant in the clinical outcome of different forms of IO and in allogeneic hematopoietic stem cell transplantation (alloSCT). However, indirect markers for iron do not provide exact quantification of iron burden, and the evidence of iron-induced adverse effects in hematological diseases has not been established. Hepatic iron concentration (HIC) has been found to represent systemic IO, which can be quantified safely with magnetic resonance imaging (MRI), based on enhanced transverse relaxation. The iron measurement methods by MRI are evolving. The aims of this study were to implement and optimise the methodology of non-invasive iron measurement with MRI to assess the degree and the role of IO in the patients. An MRI-based HIC method (M-HIC) and a transverse relaxation rate (R2*) from M-HIC images were validated. Thereafter, a transverse relaxation rate (R2) from spin-echo imaging was calibrated for IO assessment. Two analysis methods, visual grading and rSI, for a rapid IO grading from in-phase and out-of-phase images were introduced. Additionally, clinical iron indicators were evaluated. The degree of hepatic and cardiac iron in our study patients and IO as a prognostic factor in patients undergoing alloSCT were explored. In vivo and in vitro validations indicated that M-HIC and R2* are both accurate in the quantification of liver iron. R2 was a reliable method for HIC quantification and covered a wider HIC range than M-HIC and R2*. The grading of IO was able to be performed rapidly with the visual grading and rSI methods. Transfusion load was more accurate than plasma ferritin in predicting transfusional IO. In patients with hematological disorders, the prevalence of hepatic IO was frequent, opposite to cardiac IO. Patients with myelodysplastic syndrome were found to be the most susceptible to IO. Pre-transplant IO predicted severe infections during the early post-transplant period, in contrast to the reduced risk of graft-versus-host disease. Iron-induced, poor transplantation results are most likely to be mediated by severe infections.

Controlling and Assessment Indirect Spend – Case Study from Beverage Industry

Over the past decades, companies’ interest in controlling indirect sourcing process has increased. New indirect procurement strategies developed for the companies are needed in order to manage their indirect costs. New cost management strategies allow companies to improve their core competences. The research methodology and used in this thesis is qualitative. The theory is based on scientific publications. Empirical data given by the case organization, and was collected in the company’s own systems and in project steering meetings. The purpose of the study was to select a new electronic system for the company and give options for the company to reduce case organization’s indirect costs. The result showed that the most effective indirect cost management strategy was adopting a new electronic procurement system.

Quantification of Toxin Biosynthesis Genes In Cyanobacteria and Dinoflagellates - Genetic Factors as Predictors of Toxin Production in the Environment

Harmful algal blooms (HABs) are events caused by the massive proliferation of microscopic, often photosynthetic organisms that inhabit both fresh and marine waters. Although HABs are essentially a natural phenomenon, they now cause worldwide concern. Recent anthropogenic effects, such as climate change and eutrophication via nutrient runoff, can be seen in their increased prevalence and severity. Cyanobacteria and dinoflagellates are often the causative organisms of HABs. In addition to adverse effects caused by the sheer biomass, certain species produce highly potent toxic compounds: hepatotoxic microcystins are produced exclusively by cyanobacteria and neurotoxic saxitoxins, also known as paralytic shellfish toxins (PSTs), by both cyanobacteria and dinoflagellates. Specific biosynthetic genes in the cyanobacterial genomes direct the production of microcystin and paralytic shellfish toxins. Recently also the first paralytic shellfish toxin gene sequences from dinoflagellate genomes have been elucidated. The public health risks presented by HABs are evident, but the monitoring and prediction of toxic events is challenging. Characterization of the genetic background of toxin biosynthesis, including that of microcystins and paralytic shellfish toxins, has made it possible to develop highly sensitive molecular tools which have shown promise in the monitoring and study of potentially toxic microalgae. In this doctoral work, toxin-specific genes were targeted in the developed PCR and qPCR assays for the detection and quantification of potentially toxic cyanobacteria and dinoflagellates in the environment. The correlation between the copy numbers of the toxin biosynthesis genes and toxin production were investigated to assess whether the developed methods could be used to predict toxin concentrations. The nature of the correlation between gene copy numbers and amount of toxin produced varied depending on the targeted gene and the producing organism. The combined mcyB copy numbers of three potentially microcystin-producing cyanobacterial genera showed significant positive correlation to the observed total toxin production. However, the presence of PST-specific sxtA, sxtG, and sxtB genes of cyanobacterial origin was found to be a poor predictor of toxin production in the studied area. Conversely, the dinoflagellate sxtA4 was a good qualitative indicator of a neurotoxic bloom both in the laboratory and in the field, and population densities reflected well the observed toxin concentrations. In conclusion, although the specificity of each potential targeted toxin biosynthesis gene must be assessed individually during method development, the results obtained in this doctoral study support the use of quantitative PCR -based approaches in the monitoring of toxic cyanobacteria and dinoflagellates.

Opportunity Analysis and Category Management as a Base for Effective Indirect Sourcing

Hankintojen johtamisen kirjallisuus korostaa tehokkaan hankinnan olevan käypä keino tehostaa organisaation tulosta kokonaisvaltaisesti. Myös kasvava tietoisuus erityisesti epäsuorista hankintamenetelmistä ja työkaluista toimivat kannustimina tälle tutkimukselle. Tämän Pro Gradu -tutkimuksen päätarkoituksena on rakentaa kokonaisvaltainen ymmärrys epäsuorasta hankinnasta sekä löytää keinoja sen tehostamiseksi. Tutkimuksen tavoitteena on selvittää, miten globaali, monikansal- linen organisaatio voi parantaa kannattavuuttaan epäsuorissa hankinnoissa, sekä mitkä tekijät hankintastrategiassa vaikuttavat siihen. Tutkimus toteutettiin yksittäisenä tapaustutkimuksena suuren globaalin, monikan- sallisen yrityksen työntekijän näkökulmasta, Pääosa datasta pohjautuu vuonna 2015 toteutettuun Opportunity -analyysi projektiin, joka toteutettiin yhteistyössä ulkoisen konsulttifirman kanssa. Osa datasta pohjautuu puolistrukturoituihin haas- tatteluihin organisaation hankintajohtajan kanssa. Datan keruussa hyödynnettiin lisäksi henkilökohtaista havainnointia ja sekundääristä aineistoa organisaatiosta. Tämä Pro Gradu tutkimus on toteutettu kvalitatiivisella otteella, sisältäen joitakin kvantitatiivisia metodin piirteitä.