Development of Machine Learning Techniques for Diabetic Retinopathy Risk Estimation

La retinopatia diabètica (DR) és una malaltia crònica. És una de les principals complicacions de diabetis i una causa essencial de pèrdua de visió entre les persones que pateixen diabetis. Els pacients diabètics han de ser analitzats periòdicament per tal de detectar signes de desenvolupament de la retinopatia en una fase inicial. El cribratge precoç i freqüent disminueix el risc de pèrdua de visió i minimitza la càrrega als centres assistencials. El nombre dels pacients diabètics està en augment i creixements ràpids, de manera que el fa difícil que consumeix recursos per realitzar un cribatge anual a tots ells. L’objectiu principal d’aquest doctorat. la tesi consisteix en construir un sistema de suport de decisions clíniques (CDSS) basat en dades de registre de salut electrònic (EHR). S'utilitzarà aquest CDSS per estimar el risc de desenvolupar RD. En aquesta tesi doctoral s'estudien mètodes d'aprenentatge automàtic per constuir un CDSS basat en regles lingüístiques difuses. El coneixement expressat en aquest tipus de regles facilita que el metge sàpiga quines combindacions de les condicions són les poden provocar el risc de desenvolupar RD. En aquest treball, proposo un mètode per reduir la incertesa en la classificació dels pacients que utilitzen arbres de decisió difusos (FDT). A continuació es combinen diferents arbres, usant la tècnica de Fuzzy Random Forest per millorar la qualitat de la predicció. A continuació es proposen diverses tècniques d'agregació que millorin la fusió dels resultats que ens dóna cadascun dels arbres FDT. Per millorar la decisió final dels nostres models, proposo tres mesures difuses que s'utilitzen amb integrals de Choquet i Sugeno. La definició d’aquestes mesures difuses es basa en els valors de confiança de les regles. En particular, una d'elles és una mesura difusa que es troba en la qual l'estructura jeràrquica de la FDT és explotada per trobar els valors de la mesura difusa. El resultat final de la recerca feta ha donat lloc a un programari que es pot instal·lar en centres d’assistència primària i hospitals, i pot ser usat pels metges de capçalera per fer l'avaluació preventiva i el cribatge de la Retinopatia Diabètica.La retinopatía diabética (RD) es una enfermedad crónica. Es una de las principales complicaciones de diabetes y una causa esencial de pérdida de visión entre las personas que padecen diabetes. Los pacientes diabéticos deben ser examinados periódicamente para detectar signos de diabetes. desarrollo de retinopatía en una etapa temprana. La detección temprana y frecuente disminuye el riesgo de pérdida de visión y minimiza la carga en los centros de salud. El número de pacientes diabéticos es enorme y está aumentando rápidamente, lo que lo hace difícil y Consume recursos para realizar una evaluación anual para todos ellos. El objetivo principal de esta tesis es construir un sistema de apoyo a la decisión clínica (CDSS) basado en datos de registros de salud electrónicos (EHR). Este CDSS será utilizado para estimar el riesgo de desarrollar RD. En este tesis doctoral se estudian métodos de aprendizaje automático para construir un CDSS basado en reglas lingüísticas difusas. El conocimiento expresado en este tipo de reglas facilita que el médico pueda saber que combinaciones de las condiciones son las que pueden provocar el riesgo de desarrollar RD. En este trabajo propongo un método para reducir la incertidumbre en la clasificación de los pacientes que usan árboles de decisión difusos (FDT). A continuación se combinan diferentes árboles usando la técnica de Fuzzy Random Forest para mejorar la calidad de la predicción. Se proponen también varias políticas para fusionar los resultados de que nos da cada uno de los árboles (FDT). Para mejorar la decisión final propongo tres medidas difusas que se usan con las integrales Choquet y Sugeno. La definición de estas medidas difusas se basa en los valores de confianza de las reglas. En particular, uno de ellos es una medida difusa descomponible en la que se usa la estructura jerárquica del FDT para encontrar los valores de la medida difusa. Como resultado final de la investigación se ha construido un software que puede instalarse en centros de atención médica y hospitales, i que puede ser usado por los médicos de cabecera para hacer la evaluación preventiva y el cribado de la Retinopatía Diabética.Diabetic retinopathy (DR) is a chronic illness. It is one of the main complications of diabetes, and an essential cause of vision loss among people suffering from diabetes. Diabetic patients must be periodically screened in order to detect signs of diabetic retinopathy development in an early stage. Early and frequent screening decreases the risk of vision loss and minimizes the load on the health care centres. The number of the diabetic patients is huge and rapidly increasing so that makes it hard and resource-consuming to perform a yearly screening to all of them. The main goal of this Ph.D. thesis is to build a clinical decision support system (CDSS) based on electronic health record (EHR) data. This CDSS will be utilised to estimate the risk of developing RD. In this Ph.D. thesis, I focus on developing novel interpretable machine learning systems. Fuzzy based systems with linguistic terms are going to be proposed. The output of such systems makes the physician know what combinations of the features that can cause the risk of developing DR. In this work, I propose a method to reduce the uncertainty in classifying diabetic patients using fuzzy decision trees. A Fuzzy Random forest (FRF) approach is proposed as well to estimate the risk for developing DR. Several policies are going to be proposed to merge the classification results achieved by different Fuzzy Decision Trees (FDT) models to improve the quality of the final decision of our models, I propose three fuzzy measures that are used with Choquet and Sugeno integrals. The definition of these fuzzy measures is based on the confidence values of the rules. In particular, one of them is a decomposable fuzzy measure in which the hierarchical structure of the FDT is exploited to find the values of the fuzzy measure. Out of this Ph.D. work, we have built a CDSS software that may be installed in the health care centres and hospitals in order to evaluate and detect Diabetic Retinopathy at early stages

Application of calorimetry and thermal analysis to determine the liquid range and the environmental toxicity of ionic liquids

This PhD Thesis is focused on the applicability of thermal analysis and calorimetry techniques to determine phase transitions (through Differential Scanning Calorimetry (DSC)), thermal stability (measured by Thermogravimetric Analysis (TGA)) and ecotoxicity (trhough Thermal Activity Monitor (TAM) and seed germination response tests) of ionic liquids (ILs) to be used as lubricants and/or absorbents in heat pumps. The studied compounds are based on different anions (imide, triflate, sulphonate, phosphate) and cations (phosphonium, pyridinium, pyrrolidinium, imidazolium, choline) in order to determine possible dependences of these properties according to a selected ion. From the results of DSC and TGA techniques, the liquid range of the selected ILs was estimated using as limits of this interval tm and tonset/t´0.10/10h

Carbonate based ionic liquid synthesis: development, supported by quantum chemical computation and technical application

Die Carbonat basierende Synthese Ionischer Flüssigkeiten (CBILS®) ist ein grünes Produktionsverfahren und basiert auf der Verwendung von Alkylcarbonaten als Alkylierungsmittel. In der vorliegenden Arbeit wurde die Entwicklung von CBILS® bis zum Multi-100kg Maßstab behandelt. Kritische Nebenprodukte konnten unterdrückt oder erfolgreich entfernt werden. Es wurden eine Vielzahl von diversen Ausgangsmaterialien experimentell gescreent und quantenchemische Berechnungsmethoden entwickelt, um thermodynamische Funktionen für die Optimierung und Anwendung des CBILS®-Prozesses modellieren zu können.The Carbonate Based Ionic Liquid Synthesis (CBILS®) is a green manufacturing process and is based on the use of alkylcarbonates as alkylating agents. In this work the development of CBILS® up to a multi-100kg scale is presented. Critical side products could be suppressed or removed successfully. A large number of diverse starting materials have been screened experimentally and quantum-chemical calculation methods have been established, in order to model thermodynamic functions for the development and application of the CBILS® process

Influence of the Conformation of Conotoxins on their Bioactivity

In the last decade peptides filled the gap between small molecule drugs and biologics as therapeutics. A rich source of such peptides represents the venom of different animals evolved over millions of years. Conotoxins are small, disulfide-rich neuropeptides isolated from cone snails of the genus Conus, which act on different biological targets like ion channels or receptors. These toxins contain a complex cocktail of bioactive substances and are utilized for self-defence or hunting prey. With over 700 species known, these marine invertebrates reveal a large potential of novel pharmacologically active molecules, e.g. for the treatment of severe and chronic pain. Although peptide synthesis is established nowadays, preparation and characterization of disulfide-rich peptides is still a challenge. The formation of different disulfide isomers is one main issue facilitating the elucidation of such disulfide-rich peptides and proteins. The aim of this work was to investigate the folding of small multiple disulfide-bridged peptides under different conditions and perform structure-activity relationship studies of the resulting products. Within the scope of this project different conotoxins were synthesized by solidphase peptide synthesis. Disulfide formation was achieved by different methods i.e. oxidative self-folding, oxidation in Ionic Liquids and successive oxidation in combination with an orthogonal protecting group strategy. The oxidized conotoxins were systematically investigated and characterized with different analytical methods. Elucidation of the final three-dimensional structure was performed by NMR spectroscopy. Furthermore, determination of the disulfide connectivity was investigated by different methods of mass spectrometric analysis (LC-ESI and MALDI) after partial reduction and derivatization. The results of all applied analytical methods revealed their advantages and limitations for the discrimination of different disulfide isomers within conotoxins depending on primary amino acid sequence. Biological testing of the synthesized peptides on ion channels was performed in parallel with computational studies to enlighten the structure-activity relationships. The work presented in this thesis significantly extends the knowledge about conotoxins and disulfide formation, their impact with respect to biological activity, and the challenging characterization and structure elucidation procedures.Auswirkung der Faltung von Conotoxinen auf ihre Biologische Aktivität In den letzten Jahrzehnten haben Peptidtherapeutika begonnen, die Lücke zwischen niedermolekularen Arzneimitteln und Biopharmazeutika zu schließen. Eine reiche Quelle für solche Peptide stellen die Giftstoffe verschiedener Tiere dar, die sich über Millionen von Jahren entwickeln konnten. Conotoxine sind kleine, disulfidverbrückte, neuroaktive Peptide, die verschiedene biologische Ziele wie Ionenkanäle und Rezeptoren beeinflussen und aus dem Gift der Kegelschnecke der Gattung Conus isoliert werden. Dieses Gift besteht aus einem komplexen Cocktail aus bioaktiven Substanzen und wird von der Schnecke zur Selbstverteidigung oder zur Jagd eingesetzt. Mit über 700 bekannten Spezies bergen diese wirbellosen Meerestiere ein großes Potential für neuartige, pharmakologisch wirksame Moleküle, z.B. zur Behandlung von akuten und chronischen Schmerzen. Obwohl die Peptidsynthese heutzutage etabliert ist, stellt die Herstellung und Charakterisierung von disulfidverbrückten Peptiden immer noch eine Herausforderung dar. Hierbei ist die Bildung von unterschiedlichen disulfidverbrückten Peptidisomeren ein Hauptproblem, welches die Aufklärung disulfidverbrückter Peptide und Proteine erschwert. Das Ziel dieser Arbeit war es, die Faltung von kleinen, mehrfach disulfidverbrückten Peptide unter verschiedenen Bedingungen zu analysieren und Struktur-Aktivitätsuntersuchungen der erhaltenen Produkte durchzuführen. Im Rahmen dieses Projektes wurden unterschiedliche Conotoxine mittels Festphasenpeptidsynthese hergestellt. Zur Ausbildung der Disulfidbrücken wurden verschiedene Methoden eingesetzt: oxidative Selbstfaltung, Oxidation in ionischen Flüssigkeiten und stufenweise Oxidation in Verbindung mit einer orthogonalen Schutzgruppenstrategie. Die oxidierten Conotoxine wurden anschließend funktionell untersucht und durch verschiedene analytische Methoden charakterisiert. Mittels NMR-Spektroskopie wurden die dreidimensionalen Strukturen der erhaltenen Peptide aufgeklärt. Weiterhin konnte nach partieller Reduktion und Derivatisierung die Disulfidverbrückung durch verschiedene massenspektrometrische Verfahren (LC-ESI und MALDI) bestimmt werden. Die Ergebnisse zeigen die jeweiligen Vorteile und Limitierungen der verwendeten analytischen Methoden hinsichtlich der Unterscheidung verschiedener disulfidverbrückter Conotoxinisomere in Abhängigkeit von ihrer primären Aminosäuresequenz. Parallel zu den biologischen Testungen der hergestellten Peptide wurden computerbasierte Untersuchungen durchgeführt um ihre Struktur-Aktivitätsbeziehungen zu analysieren. Die vorgestellte Dissertation erweitert das Wissen über Conotoxine und die Bildung von Disulfidbrücken mit ihren Auswirkungen auf die biologische Aktivität und demonstriert darüber hinaus die schwierige Strukturaufklärung und Charakterisierung

INTERFACIAL PROPERTIES OF IONIC LIQUIDS:ELECTRIC PROPERTIES OF THIN FILMS AND INTERACTION WITH MODEL MEMBRANES AND LIVING CELLS

Room-Temperature Ionic Liquids (ILs) have attracted considerable interest in recent years. This interest is motivated by the physico-chemical properties of these systems, tunable modifying the chemical structure of ions. Generally, ILs show chemical and thermal stability, i.e. they do not easily decompose or react. Furthermore, these compounds remain liquid over an extended range of temperatures, in which they show also a remarkably low volatility. The low vapor pressure of ILs, promote them as good solvents for the growing field of the \u201dGreen Chemistry\u201d, in substitution of the volatile organic compounds. The fact that ILs are composed solely by ions, and can have a quite wide electrochemical window, make them very interesting as electrolytes. For these purposes, this PhD thesis is devoted to the investigation of ILs in contact with solid interfaces, primary targets of interaction. To deepen the analysis of electric properties at the solid interface, thin layers of ILs deposited on conductive substrates were investigated by means of AFM. The \u201dGreen\u201d character of these compounds was investigated studying their interaction with biomembrane models and external membranes of living cells, by means of AFM and electrochemical methods. Because of their ionic nature, ILs can be used as electrolytes in several devices aimed at conversion and storage of energy, such as electrochemical supercapacitors, Graetzel solar cells and batteries. In these devices a key role is played by the interface between the surface of the electrodes and the electrolyte; in particular, structural-morphological and electrical properties of the first few nanometers of IL interacting with the solid electrode surface are expected to have the strongest impact on device performance. AFM morphological analysis of small quantity of [C 4 MIM] [NTf 2 ] IL, deposited on various insulating surfaces revealed a population of nanodroplets and new structures. Remarkably, the solid surfaces induce the organization of the ionic liquid into regular, lamellar solid-like nanostructures presenting a high degree of vertical structural order and high mechanical resistance to normal compressive stresses. Nanomechanical investigation reveals that the structures resist to normal compressive loads up to 1.5 MPa; beyond that limit, indentation, in discrete steps, occurs. Furthermore, lamellar [C 4 MIM] [NTf 2 ] islands are not affected when scanned by a biased AFM tip under the influence of an electric field as intense as 10 8 V/m, while the liquid nano-and micro-droplets are easily swept away. These results confirm the solid-like character of the ordered lamellar nanostructures observed when thin films of [C 4 MIM] [NTf 2 ] are deposited on solid surfaces, and suggest that these films may possess an insulating, dielectric behavior, at odd with the case of the bulk ionic liquids. Nanoscale impedance measurements (capacitance vs. distance) and electrostatic force spectroscopy (electric force vs. distance) between a conductive AFM tip and the IL structures confirmed that values of the dielectric constant (\u3b5 r = 3-5) are significantly smaller than those measured in the bulk liquid (\u3b5 r = 9-15). These results support the picture of solid-like ordered domains where the ion mobility is significantly inhibited with respect to the bulk liquid phase. These findings also highlight the potentialities of scanning probe techniques for the quantitative investigation of the interfacial electrical properties of thin ionic liquid films, suggesting that ILs at electrified solid surfaces may possess unexpected electrical and structural properties, thus influencing the behavior of photo-electrochemical devices. The \u201dgreen\u201d character of ionic liquids (ILs) is dependent on their negligible vapor pressure but in contrast to their environmental behavior their intrinsic toxicity is not at present completely understood. Accordingly, although ILs will not evaporate which alleviates air pollution problems, a potential hazard of Ils to living organisms via aqueous media cannot be ruled out. A rigorous investigation on the interaction of ILs with biomaterials is required to provide information about their intrinsic toxicity. In order to test the biocompatible character of ILs, as a first objective, the interaction of various ILs with biological membrane (biomembrane) models was studied using electrochemical methods. A series of imidazolium based ILs were investigated whose interactions highlighted the role of anion and lateral side chain of cation during the interaction with dioleoyl phosphatidylcholine (DOPC) monolayer. It was shown that the hydrophobic and lipophilic character of the IL cations is a primary factor responsible for this interaction. The modifications of the Hg supported monolayer caused by ILs were simultaneously monitored electrochemically in a well controlled manner using rapid cyclic voltammetry (RCV), alternating current voltammetry (ACV), and electrochemical impedance spectroscopy (EIS). Hg supported monolayers provide an accurate analysis of the behavior of ILs at the interface of a biomembrane leading to a comprehensive understanding of the interaction mechanisms involved. At the same time, these experiments show that the Hg-phospholipid model is an effective toxicity sensing technique as shown by the correlation between literature in vivo toxicity data and the data from this study. Cell membrane is the main target of ILs interaction, depending on the lipophilicity of hydrophobic lateral chain of cation. In order to test the biocompatible character of ILs, the interaction of various imidazolium-based ILs with supported DOPC phospholipid bilayers (as models of the cell membrane) and living MDA-MB-231 cells (@37 \u25e6C) was investigated. Atomic Force Microscopy (AFM) was used to carry on a combined topographic and mechanical analysis of supported DOPC bilayers as well as of living cells. During the analysis of DOPC bilayers we have observed modifications in breakthrough force and membrane elasticity related to the ingress of lateral chains of cations in the bilayer, demonstrating agreement with electrochemical results. The parallel nanomechanical analysis performed on living cells interacting with ILs at various concentrations showed modifications of elasticity (effective Young\u2019s modulus) and morphology of cells after exposure to ILs dispersed in their culture medium. The measurements confirmed the primary action of ILs on membrane and actin cytoskeleton, highlighting a subtoxic/toxic effect dependent on ILs concentration and chemical nature of cation. Our results may be helpful for filling existing gaps of knowledge about ionic liquids toxicity and their impact on living organisms. From these evidences, interaction of ILs with micro-organisms and single cells is an important step to assess the environmental sustainability of this novel and promising class of solvents and to attribute a \u201dgreen\u201d label to it. Studying the interaction of ionic liquids, it has been recognized that the interface is a vital component. When the bulk symmetry of IL is broken by surfaces, the electrical properties are greatly affected, leading from a ion conductor to an insulator behavior. Also the interaction with biological entity is driven, in first instance, by surface interaction. Biomembrane models and cell membranes are affected by ILs that accumulate/penetrate the surface interface, leading to structural reorganization/damage of external membrane

Peptide mimics targeting bacterial membranes

In recent years, society has been increasingly concerned with bacteria that are no longer susceptible to commercial antibiotics. Faced with a lack of tools, medical practitioners today are forced to prescribe medicines that, although effective, cause as much harm to the patient as the principal infection. The purpose of this research project is to develop novel antibacterials that remain potent against bacterial infections without being toxic to the patient

Electrochemical studies toward proteomic analysis

This thesis provides the basis for a label-free bioanalytical platform using electrochemical analysis at liquid –liquid interfaces. The possibility to detect biomolecules such as proteins in a label-free manner via adsorption and ion-transfer was achieved. Several pre-treatment steps used in proteome analysis, such as protein pre-concentration and digestion, were studied. The results demonstrate the promise of this strategy for the detection and identification of proteins