Extremely small pore size, low porosity, and ultra-low permeability are among the characteristics of shale rocks. In tight shale reservoirs, the nano-confinement effects that include large gas-oil capillary pressure and critical property shifts could alter the phase behaviors, thereby affecting the oil or gas production. In this research, two in-house simulation models, i.e., a compositionally extended black-oil model and a fully composition model are developed to examine the nano-pore confinement effects on convective and diffusive multicomponent multiphase fluid transport. Meanwhile, the effect of nano-confinement and rock intrinsic properties (porosity and tortuosity factor) on predicting effective diffusion coefficient are investigated. First, a previously developed compositionally extended black-oil simulation approach is modified, and extended, to include the effect of large gas-oil capillary pressure for modeling first contact miscible (FCM), and immiscible gas injection. The simulation methodology is applied to gas flooding in both high and very low permeability reservoirs. For a high permeability conventional reservoir, simulations use a five-spot pattern with different reservoir pressures to mimic both FCM and immiscible displacements. For a tight oil-rich reservoir, primary depletion and huff-n-puff gas injection are simulated including the effect of large gas-oil capillary pressure in flow and in flash calculation on recovery estimations. A dynamic gas-oil relative permeability correlation that accounts for the compositional changes owing to the produced gas injection is introduced and applied to correct for changes in interfacial tension (IFT), and its effect on oil recovery is examined. The results show that the simple modified black-oil approach can model well both immiscible and miscible floods, as long as the minimum miscibility pressure (MMP) is matched. It provides a fast and robust alternative for large-scale reservoir simulation with the purpose of flaring/venting reduction through reinjecting the produced gas into the reservoir for EOR. Molecular diffusion plays an important role in oil and gas migration in tight shale formations. However, there are insufficient reference data in the literature to specify the diffusion coefficients within porous media. Another objective of this research is to estimate the diffusion coefficients of shale gas, shale condensate, and shale oil at reservoir conditions with CO2 injection for EOR/EGR. The large nano-confinement effects including large gas-oil capillary pressure and critical property shifts could alter the phase behaviors. This study estimates the diffusivities of shale fluids in nanometer-scale shale rock from two perspectives: 1) examining the shift of diffusivity caused by nanopore confinement effects from phase change (phase composition and fluid property) perspective, and 2) calculating the effective diffusion coefficient in porous media by incorporating rock intrinsic properties (porosity and tortuosity factor). The tortuosity is obtained by using tortuosity-porosity relations as well as the measured tortuosity of shale from 3D imaging techniques. The results indicated that nano-confinement effects could affect the diffusion coefficient through altering the phase properties, such as phase compositions and densities. Compared to bulk phase diffusivity, the effective diffusion coefficient in porous shale rock is reduced by 102 to 104 times as porosity decreases from 0.1 to 0.03. Finally, a fully compositional model is developed, which enables us to process multi-component multi-phase fluid flow in shale nano-porous media. The validation results for primary depletion, water injection, and gas injection show a good match with the results of a commercial software (CMG, GEM). The nano-confinement effects (capillary pressure effect and critical property shifts) are incorporated in the flash calculation and flow equations, and their effects on Bakken oil production and Marcellus shale gas production are examined. The results show that including oil-gas capillary pressure effect could increase the oil production but decrease the gas production. Inclusion of critical property shift could increase the oil production but decrease the gas production very slightly. The effect of molecular diffusion on Bakken oil and Marcellus shale gas production are also examined. The effect of diffusion coefficient calculated by using Sigmund correlation is negligible on the production from both Bakken oil and Marcellus shale gas huff-n-puff. Noticeable increase in oil and gas production happens only after the diffusion coefficient is multiplied by 10 or 100 times. / Doctor of Philosophy / Shale reservoir is one type of unconventional reservoir and it has extremely small pore size, low porosity, and ultra-low permeability. In tight shale reservoirs, the pore size is in nanometer scale and the oil-gas capillary pressure reaches hundreds of psi. In addition, the critical properties (such as critical pressure and critical temperature) of hydrocarbon components will be altered in those nano-sized pores. In this research, two in-house reservoir simulation models, i.e., a compositionally extended black-oil model and a fully composition model are developed to examine the nano-pore confinement effects on convective and diffusive multicomponent multiphase fluid transport. The large nano-confinement effects (large gas-oil capillary pressure and critical property shifts) on oil or gas production behaviors will be investigated. Meanwhile, the nano-confinement effects and rock intrinsic properties (porosity and tortuosity factor) on predicting effective diffusion coefficient are also studied.

[ES] La presente tesis muestra la utilización de las lecturas largas para resolver las limitaciones asociadas al ARN-Seq habitual, presentando innovaciones significativas en este campo. Las lecturas largas permiten capturar transcritos completos y detectar nuevas variantes de splicing, mejorando los resultados obtenidos con lecturas cortas en términos de precisión ya que no existe la necesidad de realizar un ensamblado de lecturas que podría dar lugar a isoformas quiméricas. En el marco de este trabajo, se ha desarrollado la herramienta SQANTI3, diseñada para la evaluación y filtrado de transcriptomas. SQANTI3 clasifica modelos de transcripción de lecturas largas según categorías estructurales basadas en sus splice junctions (SJ) y anota diversas características de calidad, tales como la presencia de SJ no canónicas o la fiabilidad de las anotaciones de los sitios de inicio y término de transcripción (TSS y TTS, por sus siglas en inglés) utilizando datos ortogonales. También ofrece un módulo de filtrado de artefactos basado en aprendizaje automático y reglas definidas por el usuario, así como un módulo de "rescate" para evitar la pérdida de genes completos por un filtrado excesivo. Por último, SQANTI3 integra la anotación funcional de los transcriptomas con isoAnnot Lite, facilitando el análisis de cambios en la expresión de isoformas y sus implicaciones funcionales. SQANTI3 se utilizó en los retos 1 y 3 del proyecto LRGASP (Long-read RNA-seq Genome Annotation Assessment Project), un esfuerzo internacional y multicéntrico para el benchmarking de herramientas bioinformáticas de lecturas largas en ARN-Seq. Ambos retos se centraron en la identificación correcta de transcritos en organismos altamente anotados (reto 1) y en organismos no modelo con limitaciones de información a priori (reto 3). LRGASP proporcionó datos de diferentes tecnologías y protocolos a los participantes para que presentaran los resultados obtenidos sus herramientas bioinformáticas. Estos resultados se evaluaron y compararon utilizando SQANTI3, dejando patente las diferencias de transcriptomas obtenidos para una misma muestra dependiendo de los datos y métodos empleados. En resumen, el trabajo en esta tesis resalta la importancia que la utilización de lecturas largas para ARN-Seq puede tener en el futuro y como SQANTI3 es y será una herramienta clave para la evaluación y mejora de la calidad de los transcriptomas. / [CA] La present tesi mostra la utilització de les lectures llargues per resoldre les limitacions associades a l'ARN-Seq habitual, presentant innovacions significatives en aquest camp. Les lectures llargues permeten capturar transcrits complets i detectar noves variants de splicing, millorant els resultats obtinguts amb lectures curtes en termes de precisió, ja que no és necessari realitzar un assemblatge de lectures que podria donar lloc a isoformes quimèriques. En el marc d'aquest treball, s'ha desenvolupat l'eina SQANTI3, dissenyada per a l'avaluació i filtratge de transcriptomes. SQANTI3 classifica models de transcripció de lectures llargues segons categories estructurals basades en les seues splice junctions (SJ) i anota diverses característiques de qualitat, com la presència de SJ no canòniques o la fiabilitat de les anotacions dels llocs d'inici i terme de transcripció (TSS i TTS, per les seues sigles en anglés) utilitzant dades ortogonals. També ofereix un mòdul de filtratge d'artefactes basat en aprenentatge automàtic o regles definides per l'usuari, així com un mòdul de "rescat" per a evitar la pèrdua de gens complets per un filtratge excessiu. Finalment, SQANTI3 integra l'anotació funcional dels transcriptomes amb isoAnnot Lite, facilitant l'anàlisi de canvis en l'expressió d'isoformes i les seues implicacions funcionals. SQANTI3 es va utilitzar en els reptes 1 i 3 del projecte LRGASP (Long-read RNA-seq Genome Annotation Assessment Project), un esforç internacional i multicèntric per al benchmarking d'eines bioinformàtiques de lectures llargues en ARN-Seq. Ambdós reptes es van centrar en la identificació correcta de transcrits en organismes altament anotats (repte 1) i en organismes no model amb limitacions d'informació a priori (repte 3). LRGASP va proporcionar dades de diferents tecnologies i protocols als participants perquè presentaren els resultats obtinguts amb les seues eines bioinformàtiques. Aquests resultats es van avaluar i comparar utilitzant SQANTI3, deixant patent les diferències de transcriptomes obtinguts per a una mateixa mostra depenent de les dades i mètodes emprats. En resum, aquesta tesi ressalta la importància que la utilització de lectures llargues per a ARN-Seq pot tindre en el futur i com SQANTI3 és i serà una eina clau per a l'avaluació i millora de la qualitat dels transcriptomes. / [EN] This thesis presents the usage of long-read sequencing to overcome the limitations associated with conventional RNA-Seq, introducing significant innovations in this field. Long-read sequencing enables the capture of full-length transcripts and the detection of novel splicing variants, improving the accuracy of results compared to short-read sequencing, as there is no need for assembly, which could otherwise lead to chimeric isoforms. As part of this work, the SQANTI3 tool has been designed and developed for the evaluation and filtering of transcriptomes. SQANTI3 classifies long-read transcription models into structural categories based on their splice junctions (SJ) and annotates a wide variety of quality features, such as the presence of non-canonical SJs or the reliability of Transcription Start and Termination Sites (TSS and TTS) detected using orthogonal data. It also includes an artifact filtering module based on machine learning or user-defined rules, as well as a "rescue" module to prevent the loss of complete genes due to excessive filtering. Finally, SQANTI3 integrates the functional annotation of transcriptomes with isoAnnot Lite, facilitating the analysis of isoform expression changes and their functional implications. SQANTI3 was used in challenges 1 and 3 of the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP), an international and multicenter effort to benchmark bioinformatic tools for long-read RNA-Seq data. Both challenges focused on the correct identification of transcripts in well-annotated organisms (challenge 1) and in non-model organisms with limited prior information (challenge 3). LRGASP provided participants with data from different sequencing technologies and protocols to submit the results obtained by their bioinformatics tools. These results were evaluated and compared using SQANTI3, highlighting the differences in transcriptomes obtained from the same sample depending on the data and methods used. In summary, the work in thesis emphasizes the importance that long-read RNA-Seq can have in the future and how SQANTI3 is and will continue to be a key tool for the evaluation and improvement of transcriptome quality. / The project is supported by the following grants: Pew Charitable Trust, NIGMS R35GM138122, NHGRI R21HG011280, Spanish Ministry of Science PID2020-119537RB-10, NIGMS R35GM142647, NIGMS R35GM133569, NHGRI U41HG007234, NHGRI F31HG010999, and UM1 HG009443, NHGRI R01HG008759 and R01HG011469, NHGRI R01HG007182, NHGRI UM1HG009402, NHMRC Investigator Grant GNT2017257, Comunitat Valenciana Grant ACIF/2018/290, Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation, Grant No. 2019-002443, an institutional fund from the Department of Biomedical Informatics, The Ohio State University, an institutional fund from the Department of Computational Medicine and Bioinformatics, University of Michigan, SPBU 73023672, AMED 22kk0305013h9903, 23kk0305024h0001, Wellcome Trust [WT222155/Z/20/Z] , and European Molecular Biology Laboratory. We acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, Centro de Excelencia Severo Ochoa, and CERCA Programme / Generalitat de Catalunya and the support of the German Federal Ministry of Education and Research with the grant 161L0242A. This work has been also funded by NIH grant R21HG011280, by the Spanish Ministry of Science grants BES-2016-076994 and PID2020-119537RB-100, and by the Comunitat Valenciana grant ACIF/2018/290. / Pardo Palacios, FJ. (2024). Long-Read RNA-Seq: Quality Control and Benchmarking [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/212027

Le diamant est un matériau fascinant avec d'exceptionnelles propriétés physiques. Son application à divers domaines reste limitée parce que sa fabrication est difficile et nécessite des substrats et conditions spécifiques. En outre, les dispositifs de diamant tels que les capteurs nécessitent généralement la structuration et l'échelle micro ou nanométrique, et l'inertie chimique du diamant rend ce processus technologique plus difficile que celui des semiconducteurs réguliers. Il s'agit d'un besoin évident de la recherche fondamentale d’explorer de nouvelles façons de fabriquer des nanostructures de diamant, ce qui permet de nouvelles formes de capteurs et dispositifs. Dans ce contexte, le travail présenté est d'une grande importance pour la communauté de diamant et pour le développement futur de la technologie du diamant.Le manuscrit est divisé en huit parties: une introduction; 6 chapitres, une conclusion générale. Dans l'introduction le contexte de l'étude est brièvement présenté avec les deux objectifs. Le premier consistait à étudier la croissance des nanofils de diamant et à trouver des conditions appropriées pour obtenir des nanofils de façon reproductible. Le deuxième objectif était la mise au point du procédé de gravure du diamant avec des particules de catalyseur et de l'optimisation des paramètres du procédé.Le premier chapitre de ce manuscrit présente tout d'abord l'état de l’art en mat ière de propriétés et des technologies de croissance du diamant. Puis, dans le deuxième chapitre, en vue de la croissance des nanofils et des études de gravure de nanostructures utilisées catalyseurs métalliques, la base de l'interaction métal-carbone est présenté.Le chapitre trois contient l'instrumentation et principe de fonctionnement des techniques expérimentales et analytiques utilisées dans cette étude. Le chapitre suivant se concentre sur la recherche de conditions favorables à la croissance des nanofils de diamant, d'abord en étudiant en détail un processus signalé en 2005 qui a conduit à la nucléation des nanocristaux sur des nanotubes de carbone, puis la croissance de nanofils.Les conditions de croissance ont été soigneusement reproduites, sans succès reproductible. Il en est déduit déduit que d'un élément non a contribué à la croissance, comme une contamination du catalyseur. La combinaison avec le fait que le processus publiée en 2005 n'a jamais été reproduite, en dépit de son importance technologique élevé, ce qui suggère que la contamination s'est produite également dans cette oeuvre originale.Puis, à partir de cette première observation, l'effet d'un catalyseur a été étudié, et des résultats intéressants ont été obtenus. Les nanofils ont été obtenus de façon reproductible, mais le point important est que les nanofils à base de silicium sont très faciles à cultiver, et qu'un environnement deCarbone pur était nécessaire d'étudier la croissance de nanofils de carbone. Dans ces conditions, un continuum allant de diamant de gravure pour la croissance du diamant a été obtenue en fonction de l'apport de carbone, très intéressant pour la technologie du diamant. Dans le cinquième chapitre du mécanisme de gravure de diamant par des particules de catalyseur est explorée. La gravure à motifs a été proposée pour la fabrication de nano-ou micro-structures dans le diamant, et il est présenté dans la dernière partie de ce chapitre. Le chapitre 6 présente deux applications intéressantes du processus dedéveloppement. Les premières membranes poreuses préoccupations utilisés comme bio-capteurs, et les nanotubes de carbone second concerne la base neuro-capteurs.Malgré l'étude infructueuse de la croissance du diamant nanofil, le travail fait des progrès significatifs à la science de la croissance matérielle nanocarbone. Et elle a conduit à l'étude approfondie de gravure diamant, qui est également très important pour la technologie. / One-dimensional structures with nanometre diameters, such as nanotubes and nanowires, have attracted extensive interest in recent years and form new family of materials that have characteristic of low weight with sometimes exceptional mechanical, electrical and thermal properties. Without any change in chemical composition, fundamental properties of bulk materials can be enhanced at the nanometre scale leading to extraordinary nanodevices.Since a few years, nanowires of different semiconducting materials have been grown. To mention few of these, Si, GaN, SnO, SiC and ZnO nanowires were all successfully demonstrated. However, the growth of diamond nanowires has not yet been demonstrated, despite the strong interest for this material. Bulk diamond combines various exceptional properties for a wide range of applications: Chemical inertness, radiation hardness, biocompatibility, high hole/electron mobility (2000/1000 cm2/V/s), high thermal conductivity (22 W/cm/K), wide bandgap (5.5 eV), and wide electric potential window (3.25 eV H-O evolutions).Since about 30 years, the growth of diamond thin film is well controlled either as insulator or as semiconductor with p- and n- type dopants. Fabrication of 25x25 mm2 monocrystalline diamond wafer has already been reported, and two inches wafers are expected in a couple of years demonstrating the growing interest for this material. Among present or short-term applications one can mention alpha-particle detectors, solar-blind UV sensors, high voltage electronic devices, bio-sensors and single photon source. The realization of nanowires should improve the performance of some of these devices and also open a range of new high performance applications.The stability of 0D (nanocrystals) and 1D (nanowires) diamond nanostructures has been extensively studied using ab initio modelling and indicates that for specific crystallographic orientations clusters of nanometric size are thermodynamically stable. One experimental indication for diamond nanowire growth has been published by Sun et al. in 2005, based on nanocrystal nucleation and growth on carbon nanotubes followed by 1D growth. This particular nucleation process on carbon nanotube has furthermore been explained theoretically in 2009.Based on these experimental and theoretical results, the first objective of this thesis was to explore the growth of diamond nanowire and find suitable conditions to obtain nanowires in a reproducible way. A wide range of process conditions were explored, first without any catalyst, then with metallic catalyst in order to promote Vapour-Liquid-Solid (VLS) growth. Although a comprehensive knowledge regarding carbon nanotube stability in hydrogen atmosphere and diamond-catalyst interaction has been obtained and some carbon nanostuctures were grown, no diamond nanowires were obtained in a reproducible way.However, the careful study of the diamond-catalyst interaction revealed a very interesting etching process that could be very useful for the fabrication of diamond nanostructures. A second objective was then defined: development of the etching process for diamond using transition metal as catalyst and optimization of the process parameters for specific applications such as the fabrication of porous diamond membranes for bio-sensors.

Force est de constater que les dépérissements forestiers augmentent. Ces observations vont de pairs avec l’accroissement des événements climatiques extrêmes. Aussi dans ce contexte, il est nécessaire d’identifier denouveaux caractères de resistance à la sécheresse. La résistance à la cavitation est actuellement le meilleurmarqueur de la survie d’une espèce à la sécheresse.Cette thèse avait deux objectifs : (i) comprendre le mécanisme de propagation de la cavitation dans le xylèmechez les gymnospermes. (ii) Quantifier la variation phénotypique in situ de ce caractère chez Pinus pinaster ainsique (iii) quantifier la variation génétique, et sa plasticité phénotypique.La démarché a été la suivante (i) une étude interspécifique de la résistance à la cavitation a été couplé à desmesures micro-anatomiques. (ii) Pour le volet intraspécifique, nous avons phénotypé 6 populations dans deuxtest de populations-descendances, ainsi qu’en population naturelles in situ.La propagation de l’embolie chez les Pinaceae et les ex-Taxodiaceae pourrait être due au passage du germe d’air(rupture capillaire) à travers des nanopores dans le torus. En effet, la pression de rupture d’un ménisque air-sèveest corrélée à l’entrée de l’air dans le xylème (P12). Alors que la variation interspécifique est grande, la résistanceà la cavitation varie faiblement au sein d’une espèce. Ainsi les populations provenant de climat contrasté neprésentent pas ou peu de différence génétique (en test de provenance) ou en populations naturelles in situ. Cecaractère présente une plasticité phénotypique mais faible comparée à celle de la croissance en hauteur parexemple. La comparaison entre la variation génétique entre populations et la variation des marqueurs neutresentre ces mêmes populations montrent que la variation de ce caractère semble réduite par l’architecturegénétique sous-jacente. La resistance à la cavitation est vraisemblablement un trait canalisé. / Several review reported global forest die-back that are caused, directly or indirectly, by extreme climatic events(like heat waves or prolonged drought). In this context, there is an urgent need to identify new traits to tracedrought tolerance. Resistance to cavitation is one of the best proxy for survival during extreme drought.The aim of this work was (i) to understand how spreads cavitation in the vascular pathway of gymnosperms (ii)to quantify the phenotypic variation of resistance to cavitation for Pinus pinaster species, (iii) to determine theamount of the genetic variation and phenotypic plasticity available for this trait.A micro-anatomy study was coupled to measurement of resistance to cavitation for various species to foundwhere air-seeding occurs in the bordered pit. To quantify the variability of resistance to cavitation, wephenotyped 506 genotypes using to replicated provenance-progeny trials and on natural in situ populations.The spread of embolism for Pinaceae and ex-Taxodiaceae could be due to minute pore in tori, which are remainsof secondary plasmodesmata. We found that the pressure needed to break a water/air meniscus in these minutepores is correlated with the xylem air entry (P12). Despite the great variability of resistance to cavitation betweenspecies, we found low variability within species. Most of the variability is within population, rather than betweenpopulations. The phenotypic plasticity of resistance to cavitation is low compare to growth traits. Comparisonbetween QST and FST shows that populations exhibit less variation compare to what it is expected under geneticdrift. The variation of resistance to cavitation seems to be narrowed by the genetic architecture, which is the signof canalisation.

Le rythme impressionnant auquel les technologies de séquençage progressent est alimenté par leur promesse de révolutionner les soins de santé et la recherche biomédicale. Le séquençage par nanopores est devenu une technologie attrayante pour résoudre des lacunes des technologies précédentes, mais aussi pour élargir nos connaissances sur le transcriptome en générant des lectures longues qui simplifient l’assemblage et la détection de grandes variations structurelles. Au cours du processus de séquençage, les nanopores mesurent les signaux de courant électrique représentant les bases (A, C, G, T) qui se déplacent à travers chaque nanopore. Tous les nanopores produisent simultanément des signaux qui peuvent être analysés en temps réel et traduits en bases par le processus d’appel de bases. Malgré la réduction du coût de séquençage et la portabilité des séquenceurs, le taux d’erreur de l’appel de base entrave leur mise en oeuvre dans la recherche biomédicale. Le but de ce mémoire est de classifier des séquences d’ARNm individuelles en différents groupes d’isoformes via l’élucidation de motifs communs dans leur signal brut. Nous proposons d’utiliser l’algorithme de déformation temporelle dynamique (DTW) pour l’alignement de séquences combiné à la technologie nanopore afin de contourner directement le processus d’appel de base. Nous avons exploré de nouvelles stratégies pour démontrer l’impact de différents segments du signal sur la classification des signaux. Nous avons effectué des analyses comparatives pour suggérer des paramètres qui augmentent la performance de classification et orientent les analyses futures sur les données brutes du séquençage par nanopores. / The impressive rate at which sequencing technologies are progressing is fueled by their promise to revolutionize healthcare and biomedical research. Nanopore sequencing has become an attractive technology to address shortcomings of previous technologies, but also to expand our knowledge of the transcriptome by generating long reads that simplify assembly and detection of large structural variations. During the sequencing process, the nanopores measure electrical current signals representing the bases (A, C, G, T) moving through each nanopore. All nanopores simultaneously produce signals that can be analyzed in real time and translated into bases by the base calling process. Despite the reduction in sequencing cost and the portability of sequencers, the base call error rate hampers their implementation in biomedical research. The aim of this project is to classify individual mRNA sequences into different groups of isoforms through the elucidation of common motifs in their raw signal. We propose to use the dynamic time warping (DTW) algorithm for sequence alignment combined with nanopore technology to directly bypass the basic calling process. We explored new strategies to demonstrate the impact of different signal segments on signal classification. We performed comparative analyzes to suggest parameters that increase classification performance and guide future analyzes on raw nanopore sequencing data.

Nanopore technology has introduced new techniques for single particle detection and analysis. A nanopore consists of a small opening in a membrane on the nanometer scale. Nanopores are found in nature and are utilized for transporting molecules through biological membranes. Researchers have been able to mimic naturally forming biological nanopores and utilize them for a variety of sensing applications. Nanopores, fabricated either organically or inorganically, can be used for detecting biomarkers such as proteins, nucleic acids, and metabolites that translocate the membrane by way of the nanopore. Constant ionic current flow is measured through the nanopore by way of a sensitive ammeter. In the presence of a biomarker, the ionic current flow will be impeded, causing the electrical signal to drop. This drop uniquely corresponds to the type of particle passing through the nanopore. In this work, the thin membrane on which the nanopore resides is created through a newly developed meniscus shaped sacrificial technique. The sacrificial polymer material starts as a liquid and is confined to the microfluidic channel through the capillary effect, giving it the meniscus profile. It is used as a structural support on which a thin silicon dioxide layer is grown. The layer of oxide takes on the same natural meniscus shape as the sacrificial material. The polymer is subsequently etched, resulting in a hollow core liquid channel with a suspended meniscus membrane. This process allows a thin membrane to be fabricated on top of a microfluidic channel that ranges from 50-200 nm in thickness. The meniscus membrane is crucial to the success of nanopore formation. The nanoscale membrane allows for smaller, more precise nanopores to be created. Reduced nanopore dimensions are advantageous for the detection of smaller biomarkers. The platform described in this dissertation integrates solid-state naturally forming meniscus membranes with solid-core and optofluidic waveguides for nanopore detection applications. The waveguides allow for a particle trap to be introduced to the system. The ability to trap particles directly under the nanopore is critical to the speed of which the nanopore can operate. This dissertation focuses on the fabrication, characterization, and testing of an optofluidic platform that features a nanopore for rapid single molecule detection and analysis.

Tesis por compendio / [ES] La detección y diagnóstico de resistencias en Mycobacterium tuberculosis (MTB) supone un reto para el control de la tuberculosis (TB). La técnica diagnóstica implica el cultivo de muestras diagnósticas, pero este proceso es lento y retrasa los resultados hasta meses demorando la prescripción del tratamiento óptimo. El cultivo también se utiliza para enriquecer muestras diagnósticas con fines de investigación, como la secuenciación del genoma de MTB. Sin embargo, se desconoce si cultivar reduce la diversidad genética de la muestra diagnóstica, sesgando los resultados. Actualmente, las técnicas moleculares permiten evitar el cultivo en el diagnóstico, detectando resistencia a antibióticos más rápidamente, pero se limitan a identificar las mutaciones comunes asociadas a la resistencia a la rifampicina (RIF) e isoniazida (INH), los fármacos de primera línea. No obstante, la composición compleja de las muestras diagnósticas como el esputo sigue siendo un desafío para la secuenciación. Esta tesis presenta técnicas y estrategias de secuenciación para recuperar MTB a partir de esputos, centrándose en la diversidad genética en esputos y cultivos y explorando el potencial de la secuenciación para determinar resistencias. En el Capítulo 1 comparamos la diversidad genética entre pares de esputos y cultivos en dos entornos diferentes. Desarrollamos una técnica de secuenciación de esputos, realizando una secuenciación directa o enriquecida según la cantidad de MTB presente. Además, implementamos un flujo de análisis para descartar artefactos del procesamiento de muestras. Los resultados muestran una alta concordancia en la diversidad entre esputos y cultivos, cuestionando la hipótesis inicial del cuello de botella en el cultivo. En el Capítulo 2 exploramos las limitaciones del Gene Xpert MTB/RIF Ultra (XpertUltra) para identificar mutaciones de resistencia a RIF, examinando las mutaciones de resistencia más prevalentes en el sur de Mozambique. Utilizamos la secuenciación del genoma completo para identificar variantes de resistencia en cepas recolectadas en Manhiça, Mozambique. Detectamos dos cepas con mutaciones de resistencia a RIF no identificadas por el XpertUltra y una alta prevalencia de cepas resistentes a INH sin resistencia a RIF (no multirresistentes, MDR). Los resultados sugieren que el XpertUltra podría no detectar algunos casos de resistencia a RIF y/o INH, destacando la necesidad de incluir nuevas mutaciones en las pruebas moleculares. En el Capítulo 3 evaluamos el potencial de la secuenciación de amplicones para predecir la resistencia a antibióticos e identificar el linaje de las cepas. Diseñamos un panel de nueve genes asociados a la resistencia a siete antibióticos y dos regiones filogenéticas, poniendo a punto una PCR multiplex para amplificar todas las regiones en una sola reacción. A diferencia de otros paneles, los cebadores diseñados cubren los genes completos para detectar todas las mutaciones presentes. La secuenciación se realiza con MinION (Nanopore), una plataforma portátil con análisis en tiempo real. Comparamos las variantes obtenidas con las de Illumina para calibrar los parámetros del llamado de variantes y evitar falsos positivos. Los resultados muestran una alta correlación entre MinION e Illumina en la detección de resistencia e identificación de linajes, independientemente del tipo de muestra. Estos resultados, junto con el análisis en tiempo real de MinION, son prometedores para su implementación en atención primaria. En conclusión, esta tesis contribuye a los avances en la genómica de muestras diagnósticas. Por un lado, demostrando que el cultivo refleja la diversidad genética del esputo, y por otro, revelando el potencial de la secuenciación del genoma y de amplicones para predecir resistencia a antibióticos. Las técnicas y resultados que se presentan contribuirán a promover la secuenciación directa de esputo como técnica de diagnóstico rápida y precisa, pero también su uso para la investigación. / [CA] La detecció i diagnòstic de resistències en Mycobacterium tuberculosis (MTB) suposa un repte per al control de la tuberculosi (TB). La tècnica de referència ha estat el cultiu de mostres diagnòstiques, però aquest procés és lent, retardant els resultats durant setmanes o mesos i dificultant la prescripció d'un tractament òptim. El cultiu també s'ha utilitzat per enriquir mostres diagnòstiques amb finalitats de recerca, com la seqüenciació del genoma de MTB. No obstant això, encara es desconeix si el cultiu redueix la diversitat genètica de la mostra diagnòstica, cosa que podria generar un biaix en els resultats. Actualment, les tècniques moleculars han permès evitar el cultiu en el diagnòstic, detectant resistència a antibiòtics més ràpidament, però es limiten a identificar les mutacions comunes associades a resistència a la rifampicina (RIF) i isoniazida (INH), els fàrmacs de primera línia. Tanmateix, la composició complexa de les mostres diagnòstiques com l'esput segueix sent un repte per a la seqüenciació. Aquesta tesi presenta tècniques i estratègies de seqüenciació per recuperar MTB a partir d'esputs, centrant-se en la diversitat genètica en esputs i cultius i explorant el potencial de la seqüenciació per determinar resistències. Al Capítol 1 comparem la diversitat genètica entre parelles d'esputs i cultius en dos entorns diferents. Desenvolupem una tècnica de seqüenciació d'esputs, realitzant una seqüenciació directa o enriquida segons la quantitat de MTB present. A més, implementem un flux d'anàlisi per descartar artefactes del processament de mostres. Els resultats mostren una alta concordança en la diversitat entre esputs i cultius, qüestionant la hipòtesi inicial d'un coll de botella al cultiu. Al Capítol 2 explorem les limitacions de la prova molecular Gene Xpert MTB/RIF Ultra (XpertUltra) per identificar mutacions de resistència a RIF, examinant les mutacions de resistència més prevalents al sud de Moçambic. Utilitzem la seqüenciació del genoma complet per identificar variants de resistència a antibiòtics en soques recol·lectades a Manhiça, Moçambic. Detectem dues soques amb mutacions de resistència a rifampicina no identificades per l'XpertUltra i una alta prevalença de soques resistents a INH sense resistència a RIF (no multiressistents, MDR). Els resultats sugereixen que l'XpertUltra pot no detectar alguns casos de resistència a RIF i/o INH, destacant la necessitat d'incloure noves mutacions en proves moleculars. Al Capítol 3 avaluem el potencial de la seqüenciació d'amplicons per predir resistència a antibiòtics i identificar el llinatge de les soques. Dissenyem un panell de nou gens associats a resistència a set antibiòtics i dues regions filogenètiques, posant a punt una PCR multiplex per amplificar totes les regions en una sola reacció. A diferència d'altres panells, els encebadors dissenyats cobreixen els gens complets, per detectar totes les mutacions presents. La seqüenciació es realitza amb MinION (Nanopore), una plataforma portàtil amb anàlisi en temps real. Comparem les variants obtingudes amb les d'Illumina per calibrar paràmetres per cridar de variants i evitar falsos positius. Els resultats mostren una alta correlació entre MinION i Illumina en la detecció de resistència i identificació de llinatges, independentment del tipus de mostra. Aquests resultats, juntament amb l'anàlisi en temps real de MinION, són prometedors per a la seva implementació en atenció primària. En conclusió, aquesta tesi contribueix als avanços en la genòmica directa de mostra diagnòstica. D'una banda, demostrant que el cultiu reflecteix la diversitat genètica de l'esput i, d'altra banda, revelant el potencial de la seqüenciació del genoma i d'amplicons per predir resistència a antibiòtics. Els resultats obtinguts superen les limitacions de les proves moleculars actuals, i les tècniques presentades podrien promoure la seqüenciació directa d'esput com a eina de diagnòstic ràpida i precisa, així com per a la investigació. / [EN] Mycobacterium tuberculosis (MTB) detection and diagnosis of drug resistance(DR) have been a challenge for tuberculosis(TB) control. Culturing mycobacteria from diagnostic samples has been the most commonly used diagnostic technique. However, culturing depends on MTB's slow growth, delaying results and the prescription of optimal treatment for weeks to months. Culture is also essential for research purposes such as MTB genomics. However, it remains unclear whether it causes a bottleneck in the sample's original genetic diversity, potentially biasing results. Skipping the culturing step has been possible with the introduction of molecular techniques in surveillance systems. These reduce turnaround time and allow MTB and DR detection through a single assay. However, such tests are limited to detecting common mutations associated with rifampicin(RIF) and isoniazid(INH) resistance. Moreover, the complex composition of diagnostic samples, like sputum, continues to challenge sequencing workflows in research. In this thesis, we explore different sequencing approaches to recover and sequence MTB from sputum samples, focusing on overcoming the hypothesized culture bottleneck and evaluating the potential of sequencing for DR prediction. In Chapter 1, we compare genetic diversity within sputum-culture paired samples in two settings. We implement a culture-free sequencing (cfWGS) approach for sputum, using both direct and enriched sequencing, depending on the initial amount of MTB DNA. We also develop an analysis pipeline to filter artifactual variants from cfWGS approaches. To generalize results, three additional datasets are reanalyzed. The results show high concordance in genetic diversity between sputum-culture pairs, both in terms of presence and frequency of variants. These results contradict the initial culture bottleneck assumption, emphasizing the importance of tailored filtering steps depending on sample complexity. In Chapter 2, we explore the limitations of the Gene Xpert MTB/RIF Ultra (XpertUltra) for detecting non-common mutations and screen for prevalent DR mutations in southern Mozambique. We use WGS to identify DR variants in strains collected in Manhiça (Mozambique) during two time periods. Two strains were found to harbor RIF resistance mutations beyond XpertUltra's detection scope. We also detect a high prevalence of INH-resistant strains without RIF resistance (non-MDR TB). This suggests that XpertUltra, the primary diagnostic tool in the region, sometimes misclassifies RIF-resistant cases, highlighting the need for expanded drug and mutation coverage in molecular diagnostic tests. In Chapter 3, we assess the potential of targeted sequencing for DR prediction and lineage identification by designing a gene panel of 9 genes associated with resistance to 7 drugs and two phylogenetic regions. We develop a multiplex PCR to amplify all target regions in one reaction. Unlike other commercial panels, our primers cover entire genes allowing the identification of all mutations, not just those in hotspot regions. We employ the portable MinION (Nanopore) platform, which provides real-time analysis. A key aim is to calibrate parameters for MinION variant calling by comparing its results to Illumina sequencing, establishing a frequency cut-off to avoid false positives due to MinION's error rate. Our results show high concordance between MinION and Illumina in detecting DR and genotyping, regardless of the sample type. These findings present a significant advantage for future implementation in point-of-care systems. This thesis advances the field of cfWGS by confirming that culture mirrors sputum's genetic diversity and demonstrating the potential of whole genome and targeted sequencing to overcome the limitations of current molecular diagnostic tests in predicting DR. The techniques and findings presented could help promote cfWGS as a front-line approach for faster, more accurate diagnosis, and contribute to further research. / This work has been supported by the following: European Research Council (ERC): H2020-ERC-COG/0800; Ministerio Español de Ciencia e Innovación: PID2022-137607OB-I00; Fundació La Caixa: HR21-00415, International Science and Technology Center (ISTC): Project #G-2143 and National Institute of Allergy and Infectious Diseases (NIH). This work was also supported by STOP TB partnership [STBP/TBREACH/GSA/W5-30: CA-3-D000920001], the TB Portals programme of the National Institutes of Health, the European Research Council [101001038-TB-RECONNECT: H2020-ERC-COG/0800 and 638553-TB-ACCELERATE], Generalitat Valenciana [AICO/2018/113], the Spanish Ministry of Science and Innovation [PID2019-104477RB-I00] and the Ministerio de Economía, Indústria y Competividad (SAF2016-77346-R) / Mariner Llicer, C. (2024). Culture-Free Sequencing of Mycobacterium Tuberculosis for Diagnosis and Genetic Diversity Identification [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/213332 / Compendio

Today, the growing and aging population, and the rise of new global threats on human health puts an increasing demand on the healthcare system and calls for preventive actions. To make existing medical treatments more efficient and widely accessible and to prevent the emergence of new threats such as drug-resistant bacteria, improved diagnostic technologies are needed. Potential solutions to address these medical challenges could come from the development of novel lab-on-chip (LoC) for point-of-care (PoC) diagnostics. At the same time, the increasing demand for sustainable energy calls for the development of novel approaches for energy conversion and storage systems (ECS), to which micro- and nanotechnologies could also contribute. This thesis has for objective to contribute to these developments and presents the results of interdisciplinary research at the crossing of three disciplines of physics and engineering: electrokinetic transport in fluids, manufacturing of micro- and nanofluidic systems, and surface control and modification. By combining knowledge from each of these disciplines, novel solutions and functionalities were developed at the macro-, micro- and nanoscale, towards applications in PoC diagnostics and ECS systems. At the macroscale, electrokinetic transport was applied to the development of a novel PoC sampler for the efficient capture of exhaled breath aerosol onto a microfluidic platform. At the microscale, several methods for polymer micromanufacturing and surface modification were developed. Using direct photolithography in off-stoichiometry thiol-ene (OSTE) polymers, a novel manufacturing method for mold-free rapid prototyping of microfluidic devices was developed. An investigation of the photolithography of OSTE polymers revealed that a novel photopatterning mechanism arises from the off-stoichiometric polymer formulation. Using photografting on OSTE surfaces, a novel surface modification method was developed for the photopatterning of the surface energy. Finally, a novel method was developed for single-step microstructuring and micropatterning of surface energy, using a molecular self-alignment process resulting in spontaneous mimicking, in the replica, of the surface energy of the mold. At the nanoscale, several solutions for the study of electrokinetic transport toward selective biofiltration and energy conversion were developed. A novel, comprehensive model was developed for electrostatic gating of the electrokinetic transport in nanofluidics. A novel method for the manufacturing of electrostatically-gated nanofluidic membranes was developed, using atomic layer deposition (ALD) in deep anodic alumina oxide (AAO) nanopores. Finally, a preliminary investigation of the nanopatterning of OSTE polymers was performed for the manufacturing of polymer nanofluidic devices. / <p>QC 20140509</p> / Rappid / NanoGate / Norosensor