Eye Fundus Image Analysis for Automatic Detection of Diabetic Retinopathy

Diabetes is a rapidly increasing worldwide problem which is characterised by defective metabolism of glucose that causes long-term dysfunction and failure of various organs. The most common complication of diabetes is diabetic retinopathy (DR), which is one of the primary causes of blindness and visual impairment in adults. The rapid increase of diabetes pushes the limits of the current DR screening capabilities for which the digital imaging of the eye fundus (retinal imaging), and automatic or semi-automatic image analysis algorithms provide a potential solution. In this work, the use of colour in the detection of diabetic retinopathy is statistically studied using a supervised algorithm based on one-class classification and Gaussian mixture model estimation. The presented algorithm distinguishes a certain diabetic lesion type from all other possible objects in eye fundus images by only estimating the probability density function of that certain lesion type. For the training and ground truth estimation, the algorithm combines manual annotations of several experts for which the best practices were experimentally selected. By assessing the algorithm’s performance while conducting experiments with the colour space selection, both illuminance and colour correction, and background class information, the use of colour in the detection of diabetic retinopathy was quantitatively evaluated. Another contribution of this work is the benchmarking framework for eye fundus image analysis algorithms needed for the development of the automatic DR detection algorithms. The benchmarking framework provides guidelines on how to construct a benchmarking database that comprises true patient images, ground truth, and an evaluation protocol. The evaluation is based on the standard receiver operating characteristics analysis and it follows the medical practice in the decision making providing protocols for image- and pixel-based evaluations. During the work, two public medical image databases with ground truth were published: DIARETDB0 and DIARETDB1. The framework, DR databases and the final algorithm, are made public in the web to set the baseline results for automatic detection of diabetic retinopathy. Although deviating from the general context of the thesis, a simple and effective optic disc localisation method is presented. The optic disc localisation is discussed, since normal eye fundus structures are fundamental in the characterisation of DR.

Smooth height/age curves from stem analysis with linear programming

[Abstract]

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.

Cascade hydropower capacity increase profitability analysis with optimisation modelling

The electricity market and climate are both undergoing a change. The changes impact hydropower and provoke an interest for hydropower capacity increases. In this thesis a new methodology was developed utilising short-term hydropower optimisation and planning software for better capacity increase profitability analysis accuracy. In the methodology income increases are calculated in month long periods while varying average discharge and electricity price volatility. The monthly incomes are used for constructing year scenarios, and from different types of year scenarios a long-term profitability analysis can be made. Average price development is included utilising a multiplier. The method was applied on Oulujoki hydropower plants. It was found that the capacity additions that were analysed for Oulujoki were not profitable. However, the methodology was found versatile and useful. The result showed that short periods of peaking prices play major role in the profitability of capacity increases. Adding more discharge capacity to hydropower plants that initially bypassed water more often showed the best improvements both in income and power generation profile flexibility.

Measurement of Bioactivity of Immunomodulatory Treatment of Relapsing Multiple Sclerosis. With Emphasis on MxA Protein

Background Multiple sclerosis (MS) is a demyelinating disease of the central nervous system, which mainly affects young adults. In Finland, approximately 2500 out of 6000 MS patients have relapsing MS and are treated with disease modifying drugs (DMD): interferon- β (INF-β-1a or INF-β-1b) and glatiramer acetate (GA). Depending on the used IFN-β preparation, 2 % to 40 % of patients develop neutralizing antibodies (NAbs), which abolish the biological effects of IFN-β, leading to reduced clinical and MRI detected efficacy. According to the Finnish Current Care Guidelines and European Federation of Neurological Societis (EFNS) guidelines, it is suggested tomeasure the presence of NAbs during the first 24 months of IFN-β therapy. Aims The aim of this thesis was to measure the bioactivity of IFN-β therapy by focusing on the induction of MxA protein (myxovirus resistance protein A) and its correlation to neutralizing antibodies (NAb). A new MxA EIA assay was set up to offer an easier and rapid method for MxA protein detection in clinical practice. In addition, the tolerability and safety of GA were evaluated in patients who haddiscontinued IFN-β therapy due to side effects and lack of efficacy. Results NAbs developed towards the end of 12 months of treatment, and binding antibodies were detectable before or parallel with them. The titer of NAb correlated negatively with the amount of MxA protein and the mean values of preinjection MxA levels never returned to true baseline in NAb negative patients, but tended to drop in the NAb positive group. The test results between MxA EIA and flow cytometric analysis showed significant correlation. GA reduced the relapse rate and was a safe and well-tolerated therapy in IFN-β-intolerant MS patients. Conclusions NAbs inhibit the induction of MxA protein, which can be used as a surrogate marker of the bioactivity of IFN-β therapy. Compared to flow cytometricanalysis and NAb assay, MxA-EIA seemed to be a sensitive and more practical method in clinical use to measure the actual bioactivity of IFN-β treatment, which is of value also from a cost-effective perspective.

The Regulation of Skeletal and Cardiac Muscle Blood Flow in Humans. Studies by positron emission tomography with special reference to exercise, adenosine and nitric oxide

Virtually every cell and organ in the human body is dependent on a proper oxygen supply. This is taken care of by the cardiovascular system that supplies tissues with oxygen precisely according to their metabolic needs. Physical exercise is one of the most demanding challenges the human circulatory system can face. During exercise skeletal muscle blood flow can easily increase some 20-fold and its proper distribution to and within muscles is of importance for optimal oxygen delivery. The local regulation of skeletal muscle blood flow during exercise remains little understood, but adenosine and nitric oxide may take part in this process. In addition to acute exercise, long-term vigorous physical conditioning also induces changes in the cardiovasculature, which leads to improved maximal physical performance. The changes are largely central, such as structural and functional changes in the heart. The function and reserve of the heart’s own vasculature can be studied by adenosine infusion, which according to animal studies evokes vasodilation via it’s a_2A receptors. This has, however, never been addressed in humans in vivo and also studies in endurance athletes have shown inconsistent results regarding the effects of sport training on myocardial blood flow. This study was performed on healthy young adults and endurance athletes and local skeletal and cardiac muscle blod flow was measured by positron emission tomography. In the heart, myocardial blood flow reserve and adenosine A_2A receptor density, and in skeletal muscle, oxygen extraction and consumption was also measured. The role of adenosine in the control of skeletal muscle blood flow during exercise, and its vasodilator effects, were addressed by infusing competitive inhibitors and adenosine into the femoral artery. The formation of skeletal muscle nitric oxide was also inhibited by a drug, with and without prostanoid blockade. As a result and conclusion, it can be said that skeletal muscle blood flow heterogeneity decreases with increasing exercise intensity most likely due to increased vascular unit recruitment, but exercise hyperemia is a very complex phenomenon that cannot be mimicked by pharmacological infusions, and no single regulator factor (e.g. adenosine or nitric oxide) accounts for a significant part of exercise-induced muscle hyperemia. However, in the present study it was observed for the first time in humans that nitric oxide is not only important regulator of the basal level of muscle blood flow, but also oxygen consumption, and together with prostanoids affects muscle blood flow and oxygen consumption during exercise. Finally, even vigorous endurance training does not seem to lead to supranormal myocardial blood flow reserve, and also other receptors than A_2A mediate the vasodilator effects of adenosine. In respect to cardiac work, atheletes heart seems to be luxuriously perfused at rest, which may result from reduced oxygen extraction or impaired efficiency due to pronouncedly enhanced myocardial mass developed to excel in strenuous exercise.

Cuticular and Suberin Polymers of Edible Plants. Analysis by Gas Chromatographic-Mass Spectrometric and Solid State Spectroscopic Methods

Cutin and suberin are structural and protective polymers of plant surfaces. The epidermal cells of the aerial parts of plants are covered with an extracellular cuticular layer, which consists of polyester cutin, highly resistant cutan, cuticular waxes and polysaccharides which link the layer to the epidermal cells. A similar protective layer is formed by a polyaromatic-polyaliphatic biopolymer suberin, which is present particularly in the cell walls of the phellem layer of periderm of the underground parts of plants (e.g. roots and tubers) and the bark of trees. In addition, suberization is also a major factor in wound healing and wound periderm formation regardless of the plants’ tissue. Knowledge of the composition and functions of cuticular and suberin polymers is important for understanding the physiological properties for the plants and for nutritional quality when these plants are consumed as foods. The aims of the practical work were to assess the chemical composition of cuticular polymers of several northern berries and seeds and suberin of two varieties of potatoes. Cutin and suberin were studied as isolated polymers and further after depolymerization as soluble monomers and solid residues. Chemical and enzymatic depolymerization techniques were compared and a new chemical depolymerization method was developed. Gas chromatographic analysis with mass spectrometric detection (GC-MS) was used to assess the monomer compositions. Polymer investigations were conducted with solid state carbon-13 cross polarization magic angle spinning nuclear magnetic resonance spectroscopy (13C CP-MAS NMR), Fourier transform infrared spectroscopy (FTIR) and microscopic analysis. Furthermore, the development of suberin over one year of post-harvest storage was investigated and the cuticular layers from berries grown in the North and South of Finland were compared. The results show that the amounts of isolated cuticular layers and cutin monomers, as well as monomeric compositions vary greatly between the berries. The monomer composition of seeds was found to differ from the corresponding berry peel monomers. The berry cutin monomers were composed mostly of long-chain aliphatic ω-hydroxy acids, with various mid-chain functionalities (double-bonds, epoxy, hydroxy and keto groups). Substituted α,ω-diacids predominated over ω-hydroxy acids in potato suberin monomers and slight differences were found between the varieties. The newly-developed closed tube chemical method was found to be suitable for cutin and suberin analysis and preferred over the solvent-consuming and laborious reflux method. Enzymatic hydrolysis with cutinase was less effective than chemical methanolysis and showed specificity towards α,ω-diacid bonds. According to ₁₃C CP-MAS NMR and FTIR, the depolymerization residues contained significant amounts of aromatic structures, polysaccharides and possible cutan-type aliphatic moieties. Cultivation location seems to have effect on cuticular composition. The materials studied contained significant amounts of different types of biopolymers that could be utilized for several purposes with or without further processing. The importance of the so-called waste material from industrial processes of berries and potatoes as a source of either dietary fiber or specialty chemicals should be further investigated in detail. The evident impact of cuticular and suberin polymers, among other fiber components, on human health should be investigated in clinical trials. These by-product materials may be used as value-added fiber fractions in the food industry and as raw materials for specialty chemicals such as lubricants and emulsifiers, or as building blocks for novel polymers.

Application of capillary electrophoretic methods for determining carbohydrates and aliphatic carboxylic acids formed during wood processing

Knowledge of the behaviour of cellulose, hemicelluloses, and lignin during wood and pulp processing is essential for understanding and controlling the processes. Determination of monosaccharide composition gives information about the structural polysaccharide composition of wood material and helps when determining the quality of fibrous products. In addition, monitoring of the acidic degradation products gives information of the extent of degradation of lignin and polysaccharides. This work describes two capillary electrophoretic methods developed for the analysis of monosaccharides and for the determination of aliphatic carboxylic acids from alkaline oxidation solutions of lignin and wood. Capillary electrophoresis (CE), in its many variants is an alternative separation technique to chromatographic methods. In capillary zone electrophoresis (CZE) the fused silica capillary is filled with an electrolyte solution. An applied voltage generates a field across the capillary. The movement of the ions under electric field is based on the charge and hydrodynamic radius of ions. Carbohydrates contain hydroxyl groups that are ionised only in strongly alkaline conditions. After ionisation, the structures are suitable for electrophoretic analysis and identification through either indirect UV detection or electrochemical detection. The current work presents a new capillary zone electrophoretic method, relying on in-capillary reaction and direct UV detection at the wavelength of 270 nm. The method has been used for the simultaneous separation of neutral carbohydrates, including mono- and disaccharides and sugar alcohols. The in-capillary reaction produces negatively charged and UV-absorbing compounds. The optimised method was applied to real samples. The methodology is fast since no other sample preparation, except dilution, is required. A new method for aliphatic carboxylic acids in highly alkaline process liquids was developed. The goal was to develop a method for the simultaneous analysis of the dicarboxylic acids, hydroxy acids and volatile acids that are oxidation and degradation products of lignin and wood polysaccharides. The CZE method was applied to three process cases. First, the fate of lignin under alkaline oxidation conditions was monitored by determining the level of carboxylic acids from process solutions. In the second application, the degradation of spruce wood using alkaline and catalysed alkaline oxidation were compared by determining carboxylic acids from the process solutions. In addition, the effectiveness of membrane filtration and preparative liquid chromatography in the enrichment of hydroxy acids from black liquor was evaluated, by analysing the effluents with capillary electrophoresis.

Printed platforms for paper-based analytical applications

Paper-based analytical technologies enable quantitative and rapid analysis of analytes from various application areas including healthcare, environmental monitoring and food safety. Because paper is a planar, flexible and light weight substrate, the devices can be transported and disposed easily. Diagnostic devices are especially valuable in resourcelimited environments where diagnosis as well as monitoring of therapy can be made even without electricity by using e.g. colorimetric assays. On the other hand, platforms including printed electrodes can be coupled with hand-held readers. They enable electrochemical detection with improved reliability, sensitivity and selectivity compared with colorimetric assays. In this thesis, different roll-to-roll compatible printing technologies were utilized for the fabrication of low-cost paper-based sensor platforms. The platforms intended for colorimetric assays and microfluidics were fabricated by patterning the paper substrates with hydrophobic vinyl substituted polydimethylsiloxane (PDMS) -based ink. Depending on the barrier properties of the substrate, the ink either penetrates into the paper structure creating e.g. microfluidic channel structures or remains on the surface creating a 2D analog of a microplate. The printed PDMS can be cured by a roll-ro-roll compatible infrared (IR) sintering method. The performance of these platforms was studied by printing glucose oxidase-based ink on the PDMS-free reaction areas. The subsequent application of the glucose analyte changed the colour of the white reaction area to purple with the colour density and intensity depending on the concentration of the glucose solution. Printed electrochemical cell platforms were fabricated on paper substrates with appropriate barrier properties by inkjet-printing metal nanoparticle based inks and by IR sintering them into conducting electrodes. Printed PDMS arrays were used for directing the liquid analyte onto the predetermined spots on the electrodes. Various electrochemical measurements were carried out both with the bare electrodes and electrodes functionalized with e.g. self assembled monolayers. Electrochemical glucose sensor was selected as a proof-of-concept device to demonstrate the potential of the printed electronic platforms.