In the past decade, the field of biosensing has experienced an incredible pace of development, due to the compelling need for accurate and reliable tools for characterization of biomolecular kinetics. Specifically, label-free kinetic measurements are the most direct method for studying molecular binding, for example to establish the efficacy of drug-receptor interactions. For this reason, researchers in the pharmaceutical industry rely heavily on label-free detection for drug and antibody screening. Meanwhile, in the biosafety industry and healthcare, there is great demand for screening tools that can target biothreats, in order to accurately recognize the presence of toxins and pathogens with high sensitivity in diverse samples, such as bodily fluids, food and drinking water. This research topic has become particularly relevant during the recent pandemic, where vaccine development was carried out side by side with quantification and characterization of single viral particles. Here, we introduce a versatile biosensing platform capable of characterizing virtually any type of target compound, down to the single molecule level. For this work, we have improved the Interferometric Reflectance Imaging Sensor (IRIS) to perform accurate measurements of the binding kinetics of analytes ranging in molecular weight from less than 1kDa (small molecules) to more than 1MDa (biological nanoparticles). For the first time, we demonstrate multiplexed kinetic binding characterization of small molecules to surface immobilized antibody probes, as well as detection and phenotyping of large and complex analytes, on the same platform. The IRIS platform utilizes the optical interference signal produced by thinly layered substrates in order to precisely measure the thickness of a transparent film atop a silicon chip. In the context of this work, dynamic characterization of a wide range of biomolecular and nanoparticle targets was made possible by a multidimensional optimization, in order to improve both the sensitivity and the dynamic range of the instrument. Analysis of low molecular weight compounds required a significant increase in signal to noise ratio, which was achieved through averaging, as well as complete elimination of background solution effects ('bulk effect’). Additionally, the best surface chemistry for each application was identified by a new technique which consists of immobilizing capture probes on a multiplexed array of active polymers functionalized on the same sensor surface, allowing for simultaneous side-by-side comparison of their performance. Surface chemistry plays a huge role in kinetic measurements, in terms of probe functionality, steric hindrance, charge distribution and diffusion effects. Finally, imaging optics, illumination wavelength, and thickness of the silicon dioxide film were optimized to perform detection and phenotyping of large analytes, such as extracellular vesicles (EVs) and antibody-conjugated gold nanoparticles (mAb-GNPs). Results obtained from numerical simulations allowed for selection of the best experimental parameters for each application. Experimentally, mAb-GNPs were utilized to produce a real-time sandwich lateral flow assay. In this context, we demonstrated how the improved IRIS platform can bridge the gap between single-particle detection ('digital’ configuration) and bulk reflectance measurements ('analog’ configuration), creating a new 'hybrid' system (h-IRIS), which only requires minimal hardware adjustments to easily switch from one modality to the other. This brought a substantial improvement in sensitivity, improving the limit of detection by three orders of magnitude and enabling single-molecule level measurements. Finally, future system optimization ideas are presented to achieve even higher accuracy and further extend the range of target analytes.

No description available.

DNA chip technology has drawn tremendous attention since it emerged in the mid 90 s as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges.. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same intensity of excitation light.. The fluorescence contrast is used to quantify the amount of probe-target hybridization. A mathematical model that considers multiple reflections and scattering is developed to explain the mechanism of the fluorescence contrast which depends on the thickness of the PS film. Scattering is the dominant factor that contributes to the contrast. The potential of this assay to detect single nucleotide polymorphism is also tested. / Ph. D.

Tesis por compendio / [ES] En esta tesis se ha abordado el desarrollo de biosensores ópticos label-free, basados en tecnología de disco compacto, permitiendo así abaratar y simplificar su fabricación. El trabajo llevado a cabo ha consistido en estudiar diferentes propiedades físico-químicas desarrolladas por diversos materiales. Ello ha permitido obtener sistemas compactos y accesibles, capaces de sensar con buenas prestaciones analíticas en diferentes escenarios. En el capítulo 1 se presenta un estudio de inhibición enzimática sobre discos Blu-ray como plataforma de ensayo para el cribado de fármacos. Para ello, se inmoviliza orientadamente una glicoenzima, de la familia de las peroxidasas sobre la superficie del disco, cuya actividad se relaciona con los compuestos a cribar. Después de ensayar cada compuesto, se determina el grado de inhibición enzimática mediante la adición de un sustrato. La cantidad de producto obtenido, inversamente proporcional al potencial inhibitorio del compuesto en estudio, es cuantificado con un lector de discos que registra las variaciones en la intensidad del haz laser reflejado debidas al producto enzimático. Ello permite realizar más de 1700 ensayos simultáneos en un único disco Blu-ray lo que muestra su potencial en análisis masivo de alto rendimiento. Además, se plantea una estrategia basada en hipersuperficies para el análisis de la elevada cantidad de datos que se generan en las etapas del proceso de descubrimiento de fármacos. En el capítulo 2 se aborda el estudio de una metodología para reducir el ruido generado en la lectura de resultados obtenidos con biosensores ópticos, mejorando así su sensibilidad. Para ello, se plantea la estructuración del ensayo en forma de franjas en lugar del tradicional microarray, generando una señal periódica sinusoidal al ser escaneadas. Al analizar dicha señal en dominio de frecuencias, ésta se concentra en un pico a la frecuencia del ensayo, mientras que la mayor parte del ruido aparece a frecuencias mucho más altas. Inicialmente se quería reducir al máximo el ruido, para diferenciar las interacciones moleculares en formato label free. Sin embargo, los ensayos realizados con discos DVD no generaron suficiente señal, teniendo que recurrir en esta ocasión al marcaje para la cuantificación de inmunoglobulinas G y de caseína. Pese a ello, la metodología desarrollada también se puede aplicar en biosensores tipo label-free, reduciendo el ruido y mejorando su sensibilidad. El capítulo 3 se centra en el desarrollo de sustratos interferométricos multicapa que varían la intensidad de la luz reflejada al realizar un ensayo analítico en su superficie. Los sustratos fueron fabricados utilizando los materiales que componen los DVD-RW, depositados en capas de espesor controlado con el fin de obtener la máxima respuesta. A su vez, se diseñaron de tal forma que uno de ellos disminuya la intensidad del haz reflejado como respuesta a las interacciones moleculares, mientras que el otro la aumenta. El trabajo incluye la utilización de principios y materiales de la tecnología de disco compacto para el desarrollo del sistema de detección. Para ello, se emplea el cabezal de un lector de DVD, ya que dispone de un láser y de todos los elementos ópticos necesarios para el escaneado vertical. Con este sistema se cuantifican con éxito y sin marcaje inmunoglobulinas G y sulfasalazina, una macromolécula y un fármaco de masa molecular reducida. El capítulo 4 consiste en la fabricación de un cristal fotónico utilizando la estructura de los discos compactos cubiertos con una película de óxido de titanio. Se han estudiado las propiedades físico-químicas de estos sustratos y se han caracterizado sus propiedades fotónicas. Todo ello está en concordancia con los resultados obtenido mediante simulaciones. Para interrogar los cristales fotónicos fueron necesarios una fuente de luz blanca y un espectrofotómetro, además de los elementos ópticos necesarios / [CA] En aquesta tesi s'ha abordat el desenvolupament de biosensors òptics label-free, basats en tecnologia de disc compacte, permetent així abaratir i simplificar la seua fabricació. El treball dut a terme ha consistit en estudiar diferents propietats fisicoquímiques desenvolupades per diversos materials. Això ha permès obtenir sistemes compactes i accessibles, capaços de sensar, amb bones prestacions analítiques, en diferents escenaris. En el capítol 1 es presenta un estudi d'inhibició enzimàtica sobre discos Blu-ray com a plataforma d'assaig per al cribratge de fàrmacs. Per a això, s'immobilitza de manera orientada una glicoenzima de la família de les peroxidases sobre la superfície del disc, i la seua activitat es relaciona amb els compostos a garbellar. Després d'assajar cada compost, es determina el grau d'inhibició enzimàtica mitjançant l'addició d'un substrat. La quantitat de producte obtingut, inversament proporcional al potencial inhibitori del compost en estudi, és quantificat amb un lector de discos que registra les variacions en la intensitat del làser reflectit degudes al producte enzimàtic. Això permet realitzar més de 1700 assajos simultanis en un únic disc Blu-ray el que mostra el seu potencial en anàlisi massiva d'alt rendiment. A més, es planteja una estratègia basada en hipersuperficies per a l'anàlisi de l'elevada quantitat de dades que es generen en les etapes del procés de descobriment de fàrmacs. En el capítol 2 s'aborda l'estudi d'una metodologia per reduir el soroll generat en la lectura de resultats obtinguts amb biosensors òptics, millorant així la seua sensibilitat. Per a això, es planteja l'estructuració de l'assaig en forma de franges en lloc del tradicional microarray, generant un senyal periòdic sinusoïdal en ser escanejades. En analitzar aquest senyal en domini de freqüències, aquesta es concentra en un pic a la freqüència de l'assaig, mentre que la major part del soroll apareix a freqüències molt més altes. Inicialment es volia reduir al màxim el soroll, per diferenciar les interaccions moleculars en format label-free. No obstant això, els assajos realitzats amb discos DVD no van generar prou senyal, havent de recórrer en aquesta ocasió al marcatge per a la quantificació d'immunoglobulines G i de caseïna. Malgrat això, la metodologia desenvolupada també es pot aplicar en biosensors tipus label-free, reduint el soroll i millorant la seua sensibilitat. El capítol 3 es centra en el desenvolupament de substrats interferometrics multicapa que varien la intensitat de la llum reflectida en realitzar un assaig analític en la seua superfície. Els substrats van ser fabricats utilitzant els materials que componen els DVD-RW, dipositats en capes de gruix controlat per tal d'obtenir la màxima resposta. Al seu torn, es van dissenyar de tal manera que un d'ells disminueixi la intensitat del feix reflectit com a resposta a les interaccions moleculars, mentre que l'altre l'augmenta. El treball inclou la utilització de principis i materials de la tecnologia de disc compacte per al desenvolupament del sistema de detecció. Per a això, es va utilitzar el capçal d'un lector de DVD, ja que disposa d'un làser i de tots els elements òptics necessaris per a l'escanejat vertical. Amb aquest sistema es quantifiquen amb èxit i sense marcatge immunoglobulines G i sulfasalazina, un macromolècula i un fàrmac de massa molecular reduïda. El capítol 4 consisteix en la fabricació d'un cristall fotònic utilitzant l'estructura dels discos compactes coberts amb una pel·lícula d'òxid de titani. S'han estudiat les propietats fisicoquímiques d'aquests substrats i s'han caracteritzat les propietats fotòniques. Tot això està en concordança amb els resultats obtingut mitjançant simulacions. Per interrogar els cristalls fotònics van ser necessaris una font de llum blanca i un espectrofotòmetre, a més dels elements òptics necessaris per guiar la llum. / [EN] In this thesis the development of label-free optical biosensors, based on compact disc technology, has been approached, thus making their manufacture cheaper and simpler. The work carried out has consisted of studying different physical-chemical properties manifested with several materials. This has allowed to obtain compact and accessible systems, capable of sensing with a great analytical performance in different scenarios. Chapter 1 presents an enzymatic inhibition study on Blu-ray discs as a test platform for drug screening. For this purpose, a glycoenzyme of the peroxidase family is immobilized on the surface of the disc whose activity is related to the compounds to be screened. After testing each compound, the degree of enzymatic inhibition is determined by adding the enzymatic substrate. The amount of product obtained is inversely proportional to the inhibitory potential of the compound, and is quantified with a disk reader that records the variations in the intensity of the reflected laser beam due to the enzymatic product. In addition, more than 1700 tests are performed on a single Blu-ray disc as proof of concept for application in high performance analysis and a hypersurface based strategy is proposed for the analysis of the large amount of data generated in the stages of the drug discovery process. Chapter 2 deals with the study of a methodology to reduce noise generated in the reading of results obtained with optical biosensors, hence improving their sensitivity. For this purpose, the structure of the test is proposed in the form of stripes instead of the traditional microarray, generating a sinusoidal periodic signal when they are scanned. When analysing this signal in frequency domain, it is concentrated in a peak at the frequency of the test, while most of the noise appears at much higher frequencies. Initially, the aim was to reduce noise as much as possible in order to differentiate molecular interactions in a label-free format. However, the tests carried out on a DVD did not generate enough signal, having to resort to labelling on this occasion for the quantification of immunoglobulins G and casein. Nevertheless, the methodology developed can be applied to label-free biosensors, reducing noise and improving sensitivity. Chapter 3 focuses on the development of multilayer interferometric substrates that vary the intensity of reflected light when performing an analytical test on their surface. The substrates were manufactured using the materials that make up the DVD-RW, deposited in layers of controlled thickness in order to obtain maximum response. At the same time, they were designed in such a way that one of them decreased the intensity of the reflected beam as a response to molecular interactions, while the other increased it. The work includes the use of principles and materials from compact disc technology for the development of the detection system. For this, the head of a DVD reader is used, as it has a laser and all the optical elements necessary for vertical scanning. With this system, immunoglobulins G and sulfasalazine, a macromolecule and a drug with reduced molecular mass are successfully quantified without labelling. Chapter 4 consists of the fabrication of a photonic crystal using the structure of the compact discs covered with a titanium oxide layer. The physical-chemical properties of these substrates have been studied and their photonic properties have been characterized. All this is in accordance with the results obtained through simulations. To interrogate the photonic crystals, a white light source and a spectrophotometer were needed, as well as the optical elements necessary to guide the light. / Sancho Fornés, G. (2019). Integración de diferentes fenómenos fotónicos en tecnología de disco compacto para el desarrollo de biosensores label-free [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124819 / Compendio

O E. grandis x E. camaldulensis possui características favoráveis de adaptação à seca, conferidas pelo E. camaldulensis e qualidade da madeira para papel e celulose, conferida pelo E. grandis. Esta adaptação à seca está relacionada a fatores fisiológicos e também moleculares, expressos em sua proteoma e metaboloma, que se alteram na presença do estresse. Objetiva-se neste trabalho estudar as respostas fisiológicas, proteômicas e metabolômicas (metabólitos primários) diferencialmente expressos em folhas de Eucalyptus submetidas ao déficit hídrico. Dois genótipos de E. grandis x E. camaldulensis, sendo um tolerante (T) e um susceptível (S) ao déficit hídrico foram submetidos à 100% e 30% da capacidade de campo (CC), para as plantas bem irrigadas e as em déficit hídrico, respectivamente. Os tratamentos foram chamados de T100, T30, S100 e S30 para os diferentes genótipos, T e S, submetidos a diferentes CC, 100% e 30%. Estas plantas foram avaliadas fisiologicamente com auxílio do equipamento Infrared Gas Analyzer (IRGA). Foram empregadas técnicas de proteômica quantitativa, label-free e shotgun, através do uso de UPLC-MSE. O estudo de metabolômica ocorreu através da utilização do GC x GC-TOF/MS. Os dados de proteômica foram processados no programa Protein Lynx Global Server (PLGS) e ExpressionE, através das análises comparativas S100 vs S30 e T100 vs T30, e dos metabólitos primários nos programas ChromaTOF e MetaboAnalyst. Foi possível observar que o T100 apresentou menor taxa fotossintética e condutância estomática do que o S100. Ambos os genótipos apresentaram taxas fotossintéticas e condutância estomática muito menores a 30% da CC do que a 100% da CC. A análise proteômica identificou um total de 397, 305, 366, 309 proteínas nos tratamentos S100, S30, T100 e T30 respectivamente. As análises comparativas por PLGS constataram que houve um aumento no número de proteínas diferencialmente expressas na presença do déficit hídrico. Cinco processos biológicos que apresentaram um aumento no número de proteínas diferencialmente expressas na presença do déficit hídrico foram: homeostase celular, fotossíntese, resposta ao estímulo abiótico, resposta ao estresse e morte celular. Três vias biológicas que apresentaram a participação de muitas enzimas identificadas, relacionadas a processos fotossintéticos, foram: fixação de carbono em organismos fotossintéticos, ciclo TCA e glicólise/gluconeogênese. O déficit hídrico diminuiu o número de proteínas diferencialmente expressas relacionadas ao processo metabólico de compostos contendo bases nucleares, regulação biológica e processo biossintético, que estão relacionados ao crescimento, desenvolvimento e manutenção dos processos vitais das plantas. Em relação à análise metabolômica foram identificados um total de 93, 94, 90 e 91 metabólitos primários nos tratamentos S100, S30, T100 e T30, respectivamente. Utilizando o programa Metaboanalyst, foi possível identificar os 15 metabólitos que mais contribuíram para a separação dos tratamentos, com maiores \"VIP scores\", sendo alguns responsivos ao déficit hídrico. A via da purina e arginina foi identificada como a mais frequente dentre os metabólitos identificados com VIP score ≥ 1,5. / The E. grandis x E. camaldulensis has favourable characteristics of adaptation to drought, conferred by E. camaldulensis and quality of wood for pulp and paper, conferred by E. grandis. This adaptation to drought is related to physiological factors and also expressed in their molecular proteome and metabolome, which change in the presence of stress. The aim of this work was to study the physiological responses, proteomics and metabolomics (primary metabolites) differentially expressed in leaves of Eucalyptus under drought. Two genotypes of E. grandis x E. camaldulensis, a tolerant (T) and a susceptible (S) to drought stress, were subjected to 100% and 30% of field capacity (FC), for the well-watered plants and drought stressed plants, respectively. The treatments were called T100, T30, S100 and S30 for different genotypes, T and S, submitted to different FC, 100% and 30%. These plants were evaluated physiologically using the Infrared Gas Analyzer (IRGA). Label-free and shotgun quantitative proteomics were realized using UPLC-MSE. The metabolomics study was carried out using GC x GC-TOF/MS mass spectrometer. The proteomics data were processed using the Protein Lynx Global Server program (PLGS) and ExpressionE program, through comparative analyses S100 vs S30 and T100 vs S30, and primary metabolites in ChromaTOF and MetaboAnalyst programs. It was observed that T100 had lower photosynthetic rate and stomatal conductance than S100. Both genotypes showed stomatal conductance and photosynthetic rates lower at 30% of FC than at 100% of FC. The proteomic analysis identified a total of 397, 305, 366, 309 proteins in the treatments S100, S30, T100 and T30 respectively. Comparative PLGS analyses showed an increase in the number of differentially expressed proteins under drought stress. The five biological processes that showed an increase in the number of differentially expressed proteins under drought stress were: cellular homeostasis, photosynthesis, response to abiotic stimulus, response to stress and cell death. The three biological pathways that had the participation of many identified enzymes, which are related to photosynthetic processes, were: carbon fixation in photosynthetic organisms, TCA cycle and glycolysis / gluconeogenesis. The drought reduced the number of differentially expressed proteins related to the metabolism of compounds containing nuclear bases, biological regulation and biosynthetic process, which were related to growth, development and maintenance of the vital processes of plants. The metabolomic analysis identified a total of 93, 94, 90 and 91 primary metabolites in the treatments S100, S30, T100 and T30, respectively. Using Metaboanalyst program, it was possible to identify 15 metabolites that contributed to the separation of treatments with higher \"VIP scores\", some of these are responsive to drought. The purine and arginine pathway was identified as the most frequent among the metabolites identified with VIP score ≥ 1.5.

O E. grandis x E. camaldulensis possui características favoráveis de adaptação à seca, conferidas pelo E. camaldulensis e qualidade da madeira para papel e celulose, conferida pelo E. grandis. Esta adaptação à seca está relacionada a fatores fisiológicos e também moleculares, expressos em sua proteoma e metaboloma, que se alteram na presença do estresse. Objetiva-se neste trabalho estudar as respostas fisiológicas, proteômicas e metabolômicas (metabólitos primários) diferencialmente expressos em folhas de Eucalyptus submetidas ao déficit hídrico. Dois genótipos de E. grandis x E. camaldulensis, sendo um tolerante (T) e um susceptível (S) ao déficit hídrico foram submetidos à 100% e 30% da capacidade de campo (CC), para as plantas bem irrigadas e as em déficit hídrico, respectivamente. Os tratamentos foram chamados de T100, T30, S100 e S30 para os diferentes genótipos, T e S, submetidos a diferentes CC, 100% e 30%. Estas plantas foram avaliadas fisiologicamente com auxílio do equipamento Infrared Gas Analyzer (IRGA). Foram empregadas técnicas de proteômica quantitativa, label-free e shotgun, através do uso de UPLC-MSE. O estudo de metabolômica ocorreu através da utilização do GC x GC-TOF/MS. Os dados de proteômica foram processados no programa Protein Lynx Global Server (PLGS) e ExpressionE, através das análises comparativas S100 vs S30 e T100 vs T30, e dos metabólitos primários nos programas ChromaTOF e MetaboAnalyst. Foi possível observar que o T100 apresentou menor taxa fotossintética e condutância estomática do que o S100. Ambos os genótipos apresentaram taxas fotossintéticas e condutância estomática muito menores a 30% da CC do que a 100% da CC. A análise proteômica identificou um total de 397, 305, 366, 309 proteínas nos tratamentos S100, S30, T100 e T30 respectivamente. As análises comparativas por PLGS constataram que houve um aumento no número de proteínas diferencialmente expressas na presença do déficit hídrico. Cinco processos biológicos que apresentaram um aumento no número de proteínas diferencialmente expressas na presença do déficit hídrico foram: homeostase celular, fotossíntese, resposta ao estímulo abiótico, resposta ao estresse e morte celular. Três vias biológicas que apresentaram a participação de muitas enzimas identificadas, relacionadas a processos fotossintéticos, foram: fixação de carbono em organismos fotossintéticos, ciclo TCA e glicólise/gluconeogênese. O déficit hídrico diminuiu o número de proteínas diferencialmente expressas relacionadas ao processo metabólico de compostos contendo bases nucleares, regulação biológica e processo biossintético, que estão relacionados ao crescimento, desenvolvimento e manutenção dos processos vitais das plantas. Em relação à análise metabolômica foram identificados um total de 93, 94, 90 e 91 metabólitos primários nos tratamentos S100, S30, T100 e T30, respectivamente. Utilizando o programa Metaboanalyst, foi possível identificar os 15 metabólitos que mais contribuíram para a separação dos tratamentos, com maiores \"VIP scores\", sendo alguns responsivos ao déficit hídrico. A via da purina e arginina foi identificada como a mais frequente dentre os metabólitos identificados com VIP score ≥ 1,5. / The E. grandis x E. camaldulensis has favourable characteristics of adaptation to drought, conferred by E. camaldulensis and quality of wood for pulp and paper, conferred by E. grandis. This adaptation to drought is related to physiological factors and also expressed in their molecular proteome and metabolome, which change in the presence of stress. The aim of this work was to study the physiological responses, proteomics and metabolomics (primary metabolites) differentially expressed in leaves of Eucalyptus under drought. Two genotypes of E. grandis x E. camaldulensis, a tolerant (T) and a susceptible (S) to drought stress, were subjected to 100% and 30% of field capacity (FC), for the well-watered plants and drought stressed plants, respectively. The treatments were called T100, T30, S100 and S30 for different genotypes, T and S, submitted to different FC, 100% and 30%. These plants were evaluated physiologically using the Infrared Gas Analyzer (IRGA). Label-free and shotgun quantitative proteomics were realized using UPLC-MSE. The metabolomics study was carried out using GC x GC-TOF/MS mass spectrometer. The proteomics data were processed using the Protein Lynx Global Server program (PLGS) and ExpressionE program, through comparative analyses S100 vs S30 and T100 vs S30, and primary metabolites in ChromaTOF and MetaboAnalyst programs. It was observed that T100 had lower photosynthetic rate and stomatal conductance than S100. Both genotypes showed stomatal conductance and photosynthetic rates lower at 30% of FC than at 100% of FC. The proteomic analysis identified a total of 397, 305, 366, 309 proteins in the treatments S100, S30, T100 and T30 respectively. Comparative PLGS analyses showed an increase in the number of differentially expressed proteins under drought stress. The five biological processes that showed an increase in the number of differentially expressed proteins under drought stress were: cellular homeostasis, photosynthesis, response to abiotic stimulus, response to stress and cell death. The three biological pathways that had the participation of many identified enzymes, which are related to photosynthetic processes, were: carbon fixation in photosynthetic organisms, TCA cycle and glycolysis / gluconeogenesis. The drought reduced the number of differentially expressed proteins related to the metabolism of compounds containing nuclear bases, biological regulation and biosynthetic process, which were related to growth, development and maintenance of the vital processes of plants. The metabolomic analysis identified a total of 93, 94, 90 and 91 primary metabolites in the treatments S100, S30, T100 and T30, respectively. Using Metaboanalyst program, it was possible to identify 15 metabolites that contributed to the separation of treatments with higher \"VIP scores\", some of these are responsive to drought. The purine and arginine pathway was identified as the most frequent among the metabolites identified with VIP score ≥ 1.5.

Le Benzo(a)pyrène (BaP), appartenant à la famille des hydrocarbures aromatiques polycycliques (HAP) est cancérigène pour l’homme. Nous avons développé une approche protéomique quantitative nanoLC-MS/MS label-free pour identifier des biomarqueurs liés à l’exposition au BaP dans le sécrétome des cellules hépatiques humaines exposées au BaP vs. des cellules non exposées et exposées au Benzo(e)pyrène (BeP). Le BeP, agent non classifié comme cancérigène pour l’homme, est choisi comme contrôle négatif afin de distinguer les protéines spécifiques du BaP de celles des HAP. 847 protéines ont été identifiées et quantifiées, et 55 ont été fortement surexprimées avec un ratio supérieur à 5 : la plupart de ces protéinessurexprimées sont précoces et liées au cancer. Une validation ultérieure de l'expression de ces protéines dans le plasma de la population exposée au BaP aidera dans le développement de biomarqueurs qui permettront d'améliorer la détection précoce, le pronostic et prévention. / Benzo(a)pyrene (BaP) belongs to a class of polycyclic aromatic hydrocarbon and is reported as a potent human carcinogen. We performed a nanoLC-MS/MS-based label-free quantitative proteomics approach to identify potential biomarkers of exposure in the secretome of BaPtreated vs. non-treated and Benzo(e)pyrene (BeP)-treated human hepatoma cell line HepG2.BeP-treated cells were chosen as a negative control to distinguish the BaP-specific from the HAP-specific regulated proteins: BeP is not classifiable as to its carcinogenicity to humans. 847 proteins have been identified and quantified and 55 proteins were seen as being highly upregulated with a fold change of at least 5. Most of these up-regulated proteins were focused incancer-related activities. Further validation of expression of these proteins in the plasma of BaP-exposed population will assist in the development of biomarkers that will greatly improve early detection, prognosis, prediction of treatment response and prevention.

La borréliose de Lyme est une maladie à transmission vectorielle en forte progression ces dernières années. Après une infection par la bactérie appartenant au complexe Borrelia burgdorferi sensu lato via une piqûre de tique, de multiples troubles (cardiaques, rhumatologiques…) peuvent apparaître. Il n’existe à l’heure actuelle aucun vaccin contre la maladie chez l’homme. De plus, les méthodes actuelles de diagnostic souffrent d’un manque de sensibilité, de spécificité ou de rapidité. Nous avons développé différentes approches protéomiques pour l’étude de cette maladie. Dans un premier temps, nous avons mis en évidence de nouveaux candidats vaccinaux par une approche Ge-LC-MS/MS de type label free. Dans un second temps, nous avons mis au point une méthode de détection de la bactérie dans des biopsies cutanées par spectrométrie de masse ciblée SRM. Nous avons également caractérisé les modifications post-traductionnelles d’une protéine identifiée dans les glandes salivaires de tique, et capable de lyser les fibroblastes. Un dernier volet a concerné l’évaluation de deux instruments et de l’apport de modes d’acquisition originaux pour l’analyse protéomique / Lyme borreliosis has been rising strongly for the last twenty years. After an infection by the bacterium belonging to the Borrelia burgdorferi sensu lato complex through a tick bite, multiple disorders (cardiac, rhumatological…) may appear. There is no current vaccine available for human being. Moreover, actual diagnosis methods lack of sensitivity, specificity and quickness. We developed various proteomic approaches to study the Lyme disease. Firstly, we discovered new vaccine candidates by using a Ge-LC-MS/MS label free approach. Secondly, we set up the detection of the bacteria in human cutaneous biopsies by targeted SRM mass spectrometry. We also characterized the post-translational modifications of a lytic protein present in tick salivary glands. Finally, we evaluated the performances of two instruments and the contribution of original acquisition modes for proteomic analyses.

CHARLES UNIVERSITY and HASSELT UNIVERSITY / tUL Doctoral dissertation Collagen structures from cell culture to intact tendon ABSTRACT Author: Daniel Hadraba Promoters: Assoc. Prof. Karel Jelen | Charles University Prof. Marcel Ameloot | Hasselt University Co-promoters: Dr. Frantisek Lopot | Charles University Prof. Virginie Bito | Hasselt University Annotation Author: Ing. Mgr. Daniel Hadraba Doctoral thesis title: Collagen structures from cell culture to intact tendon Year: 2010 - 2017 Doctoral program: Doctor of Biomechanics at Charles University Doctor of Biomedical Science at Hasselt University / transnational University Limburg Departments: Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Dept. Biophysics | Hasselt University Promoters: Assoc. Prof. Karel Jelen | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Marcel Ameloot | Hasselt University / transnational University Limburg Co-promoters: Dr. Frantisek Lopot | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Virginie Bito | Hasselt University / transnational University Limburg Bibliography details: Pages 102 Figures 30 Tables 2 Equations 17 Keywords: tendon, collagen, crimps, orientation, aging,...

CHARLES UNIVERSITY and HASSELT UNIVERSITY / tUL Doctoral dissertation Collagen structures from cell culture to intact tendon ABSTRACT Author: Daniel Hadraba Promoters: Assoc. Prof. Karel Jelen | Charles University Prof. Marcel Ameloot | Hasselt University Co-promoters: Dr. Frantisek Lopot | Charles University Prof. Virginie Bito | Hasselt University Annotation Author: Ing. Mgr. Daniel Hadraba Doctoral thesis title: Collagen structures from cell culture to intact tendon Year: 2010 - 2017 Doctoral program: Doctor of Biomechanics at Charles University Doctor of Biomedical Science at Hasselt University / transnational University Limburg Departments: Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Dept. Biophysics | Hasselt University Promoters: Assoc. Prof. Karel Jelen | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Marcel Ameloot | Hasselt University / transnational University Limburg Co-promoters: Dr. Frantisek Lopot | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Virginie Bito | Hasselt University / transnational University Limburg Bibliography details: Pages 102 Figures 30 Tables 2 Equations 17 Keywords: tendon, collagen, crimps, orientation, aging,...