The field of microbial ecology has just entered a new era of rapid technological development and generation of big data. The high-throughput sequencing techniques presently available provide an opportunity to extensively inventorize the blueprints of life. Now, millions of microbes of natural microbial communities can be studied simultaneously without prior cultivation. New species and new functions (genes) can be discovered just by mining sequencing data. However, there is still a tremendous number of microorganisms not yet examined, nor are the ecosystem functions these carry out. The modern genomic technologies can contribute to solve environmental problems and help us understand ecosystems, but to most efficiently do so, methods need to be continuously optimised.   During my Ph. D. studies, I developed a method to survey eukaryotic microbial diversity with a higher accuracy, and applied various sequencing-based approaches in an attempt to answer questions of importance in environmental research and ecology. In PAPER-I, we developed a set of 18S rRNA gene PCR primers with high taxonomic coverage, meeting the requirements of currently popular sequencing technologies and matching the richness of 18S rRNA reference sequences accumulated so far. In PAPER-II, we conducted the first sequencing-based spatial survey on the combined eukaryotic and bacterial planktonic community in the Baltic Sea to uncover the relationship of microbial diversity and environmental conditions. Here, the 18S primers designed in PAPER-I and a pair of broad-coverage 16S primers were employed to target the rRNA genes of protists and bacterioplankton for amplicon sequencing. In PAPER-III, we integrated metagenomic, metabarcoding, and metatranscriptomic data in an effort to scrutinise the protein synthesis potential (i.e., activity) of microbes in the sediment at a depth of 460 m in the Baltic Sea and, thus, disclosing microbial diversity and their possible ecological functions within such an extreme environment. Lastly, in PAPER-IV, we compared the performance of E. coli culturing, high-throughput sequencing, and portable real-time sequencing in tracking wastewater contamination in an urban stormwater system. From the aspects of cost, mobility and accuracy, we evaluated the usage of sequencing-based approaches in civil engineering, and for the first time, validated the real-time sequencing device in use within water quality monitoring.   In summary, these studies demonstrate how DNA sequencing of microbial communities can be applied in environmental monitoring and ecological research. / <p>Yue Hu was supported by a scholarship from the China Scholarship Council (CSC #201206950024)</p><p>Yue Hu has been publishing papers under the name "Yue O. O. Hu".</p><p>QC 20170403</p>

Les poly(triazole amide)s sont une classe de polymères à séquences définies synthétisés par une approche « AB+CD » itérative, chimiosélective et supportée. Cette stratégie permet de contrôler parfaitement la séquence des monomères, puisque les unités constitutives sont ajoutées une à une. De plus, la chimiosélectivité des réactions de couplage permet de s’affranchir d’étapes de déprotection. En outre, l’utilisation d’un support solide minimise également le temps d’expérimentation et facilite les étapes de lavage, réduisant donc le temps total de synthèse. Cette voie de synthèse a été utilisée pour la préparation de différents types de polymères fonctionnels. Premièrement, plusieurs oligomères comme des structures composées de chaînes alkyles ou PEG ont été préparées, contenant des séquences de monomères non naturels qui forment un code binaire. Ces produits ont été analysés grâce à deux techniques de séquençage : la spectrométrie de masse en tandem et l’analyse de chaînes uniques par les nanopores. Une synthèse sans cuivre de ce type d’oligomères a également été considérée. Enfin, une nouvelle classe d’acides xénonucléiques (XNAs), les peptide triazole nucleic acids (PTzNAs), a été synthétisée et étudiée. En particulier, les propriétés d’hybridation de ces polymères contenant des séquences génétiques ont été examinées. / Poly(triazole amide)s are a class of sequence-defined polymers synthesized via a chemoselective iterative “AB+CD” approach on a solid support. This strategy allows to perfectly control the sequence of monomers, since the building blocks are added one by one. Moreover, the chemoselectivity of the coupling reactions enables to avoid the use of deprotection steps and to save time. In addition, the use of a solid support also minimizes the experiment time and facilitates the cleaning steps, thus reducing the total synthesis time. This synthesis pathway was used for the synthesis of different types of functional polymers. First of all, several oligomers such as structures based on alkyl or PEG chains were prepared, containing sequences of non-natural monomers that form a binary code. Those products were analyzed with two sequencing techniques: tandem mass spectrometry and nanopore single-chain analysis. A copper-free synthesis of this type of oligomers was also considered. Then, a new class of xeno nucleic acids (XNAs), peptide triazole nucleic acids (PTzNAs) was synthesized and studied. In particular, the hybridization properties of those natural sequence-containing polymers were investigated.

Ce travail porte sur l'étude expérimentale de deux mécanismes de translocations d'acides nucléiques au travers d'une membrane lipidique : la translocation, forcée électrophorétiquement, d'oligomères au travers d'un pore d'alpha-hémolysine et la translocation passive d'un ADN génomique hors de la capside du bactériophage T5. La première partie de la thèse porte sur l'ouverture de molécules d'ADN double brin à travers le nanopore d'alpha hémolysine. Les temps de passage individuels de molécules d'ADN à travers le pore sont mesurés expérimentalement en fonction de la séquence, de la longueur et de la force appliquée sur l'ADN. Les distributions obtenues sont confrontées à un modèle décrivant le passage de l'ADN par la diffusion d'une fourche d'ouverture dans un paysage énergétique unidimensionnel, déterminé par la séquence de la molécule. La deuxième partie porte sur un système in vitro reconstituant les étapes initiales d'infection du bactériophage T5. L'interaction de T5 avec son récepteur membranaire FhuA purifié en détergent, génère une séquence d'événements qui conduit à l'éjection du génome viral hors de la capside : (i) fixation du récepteur ; (ii) activation conduisant à l'ouverture d'un canal d'ADN ; (iii) éjection de l'ADN. La dynamique des trois étapes est mesurée à l'aide d'expériences en population et en virus unique. La dernière étape est comparée à un modèle physique qui révèle une dynamique fortement hors d'équilibre à l'initiation de l'éjection. Enfin, FhuA est reconstitué dans des vésicules lipidiques géantes afin de suivre l'éjection par microscopie de fluorescence et par électrophysiologie à travers une membrane lipidique.

Most biological experiments are performed on an ensemble of cells under the assumption that all cells are identical. However, recent evidence from single cells studies reveals that this assumption is incorrect. Individual cells within the same generation may differ dramatically, and these differences have important consequences for the health and function of the entire living body. I have used Scanning Ion Conductance Microscopy (SICM) for imaging and analysis of topographical change of single cell membrane, which is difficult to be revealed by optical microscopes. Morphological change in the fixed and live HeLa cell membrane during endocytosis of conjugated polymer nanoparticles was studied. Results demonstrated SICM is a powerful tool to study the interaction between nanoparticle and cell membrane during internalization of nanoparticles through the membrane. This research can improve our fundamental understanding of cellular behavior and will be helpful for drug delivery applications. Based on conventional SICM, we have developed a novel method to simultaneous map the topography and potential distributions of the single living cells membranes. At the first step, multifunctional nanopipettes (nanopore/nanoelectrode) have been fabricated and characterized. To demonstrate the potential sensing capability and understand the mechanism, I measured the ionic current and local electric potential change during translocation of 40 nm charged gold nanoparticles. Our results reveal the capability of the multifunctional probe for the highly sensitive detection of the ionic current and local electrical potential changes during the translocation of the charged entity through the nanopore. From the potential change, we revealed the dynamic assembly of GNPs before entering the nanopore. The experimental results are also nicely explained by the finite element method based numerical simulation results. At the second step, I have measured the surface potential of living cell membrane at selected locations. Very recently, I have obtained results to show that we can map the extracellular membrane potential distribution of the complicated living cell membrane with sub-micron spatial resolution.This new imaging technique can help biologist to explore the extracellular potential distribution of varieties of cells quantitatively.These studies will have impacts on several biomedical applications such as regenerative repair and cancer treatment.

Submitted by ERNANE DE FREITAS MARTINS (ernanefmg@hotmail.com) on 2018-06-21T18:31:22Z No. of bitstreams: 1 Tese_Ernane_FINAL.pdf: 73762259 bytes, checksum: 783c569159077630257fc1df333452da (MD5) / Approved for entry into archive by Hellen Sayuri Sato null (hellen@ift.unesp.br) on 2018-06-22T17:57:30Z (GMT) No. of bitstreams: 1 martins_ef_dr_ift.pdf: 73762259 bytes, checksum: 783c569159077630257fc1df333452da (MD5) / Made available in DSpace on 2018-06-22T17:57:30Z (GMT). No. of bitstreams: 1 martins_ef_dr_ift.pdf: 73762259 bytes, checksum: 783c569159077630257fc1df333452da (MD5) Previous issue date: 2018-06-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nanotechnology is an important and very active area of research contributing to many different fields. The development of new devices applied to personalized medicine is one of its applications. When we desire to develop new devices many effort are done, including experimental and theoretical investigations. The theoretical/computational physics can enormously contribute to this area, since the simulations can reveal the working mechanism in these systems being possible to understand and propose new devices with improved performance. We present an extensive theoretical investigation of the electronic transport properties of graphene-based devices for DNA sensing. We have used a hybrid methodology which combines quantum mechanics and molecular mechanics, the so called QM/MM method, coupled to electronic transport calculations using non-equilibrium Green’s functions. First, we studied graphene in solution in order to understand the effects of polarization on the electronic and transport properties under different salt concentrations. We also stud- ied graphene with Stone-Wales defect in pure water. For these systems we tested a simple polarization model based on rigid rods. Our analysis were also done over different QM/MM partitions including explicit water molecules in the quantum part. Our results showed that the inclusion of the solvent in the electronic transport calculations for graphene decreases the total transmission, showing the important role played by the water. Our results also showed that the electronic transport properties of graphene do not suffer significant changes as we increase the salt concentration in the solution. The inclusion of polarization effects in graphene, despite changing the structuring of water molecules that make up the first solvation shell of graphene, do not significantly affect the electronic transport through graphene. We then studied DNA sequencing devices. First we focused on sequencing using a nanopore between topological line defects in graphene. Our results showed that sequencing DNA with high selectivity and sensitivity using these devices appears possible. We also address nanogap in graphene. For this we looked at the effects of water on electronic transport by using different setups for the QM/MM partition. We showed that the inclusion of water molecules in the quantum part increases the electronic transmission in several orders of magnitude, also showing the fundamental role played by water in tunneling devices. The electronic transport simulations showed that the proposed device has the potential to be used in DNA sequencing, presenting high selectivity and sensitivity. We propose an graphene-based biochip for sequence-specific detection of DNA strands. The main idea of this sort of device is to detect hybridization of single-stranded DNA, forming double-stranded DNA. We showed that the vertical DNA adsorption, either through an anchor molecule (pyrene) or using the nucleotide itself as anchor, do not present good results for detection, since the signals for the single and double strands are quite similar. For the case of horizontal DNA adsorption on graphene our results indicated that the two signals can be distinguishable, showing promising potential for sensitivity and selectivity. / Nanotecnologia é uma importante e muito ativa área de pesquisa contribuindo para muitos campos diferentes. O desenvolvimento de novos dispositivos aplicados à medicina personalizada é uma de suas aplicações. Quando desejamos desenvolver novos dispositivos muitos esforços são feitos, incluindo investigações experimentais e teóricas. A Física teórica/computacional pode contribuir enormemente com esta área, já que simulações podem revelar o mecanismo de funcionamento nesses sistemas tornando possível entender e propor novos dispositivos com desempenho melhorado. Nós apresentamos uma extensa investigação teórica das propriedades de transporte eletrônico de dispositivos baseados em grafeno para sensoriamento de DNA. Utilizamos uma metodologia híbrida que combina mecânica quântica e mecânica molecular, o chamado método QM/MM, acoplado a cálculos de transporte eletrônico utilizando funções de Green fora do equilíbrio. Primeiramente nós estudamos grafeno em solução de modo a entender os efeitos de polarização nas propriedades eletrônica e de transporte em diferentes concentrações de sal. Também estudamos grafeno com defeito Stone-Wales em água pura. Para esses sistemas, testamos um modelo de polarização simples baseado em bastões rígidos. Nossas análises também foram feitas em diferentes partições QM/MM incluindo moléculas de água explícitas na parte quântica. Nossos resultados mostraram que a inclusão do solvente nos cálculos de transporte eletrônico para o grafeno diminui a transmissão total, mostrando o papel fundamento desempenhado pelo água. Nossos resultados também mostraram que as propriedades de transporte eletrônico do grafeno não sofrem mudanças significativas na medida em que aumentamos a concentração de sal na solução. A inclusão de efeitos de polarização em grafeno, apesar de mudar a estruturação das moléculas de água que compõem a primeira camada de solvatação do grafeno, não afeta significativamente o transporte eletrônico através do grafeno. Nós, então, estudamos dispositivos para sequenciamento de DNA. Focamos primeira- mente no sequenciamento usando nanoporo entre defeitos de linha topológicos no grafeno. Nossos resultados mostraram que o sequenciamento de DNA com alta seletividade e sensitividade usando esses dispositivos se mostra possível. Nós também abordamos nanogap em grafeno. Para tal, avaliamos os efeitos da água no transporte eletrônico utilizando diferentes configurações para a partição QM/MM. Mostramos que a inclusão de moléculas de água na parte quântica aumenta a transmissão eletrônica em várias ordens de grandeza, também mostrando o papel fundamental desempenhado pela água em dispositivos de tunelamento. As simulações de transporte eletrônico mostraram que o dispositivo proposto tem o potencial de ser usado em sequenciamento de DNA, apresentando alta seletividade e sensitividade. Propusemos um biochip baseado em grafeno para detecção de sequências específicas de fitas de DNA. A ideia principal desta classe de dispositivos é detectar a hibridização da fita simples de DNA, formando a fita dupla de DNA. Mostramos que a adsorção vertical de DNA, seja utilizando uma molécula âncora (pireno) ou utilizando o próprio nucleotídio como âncora, não apresenta bons resultados para detecção, já que os sinais para as fitas simples e dupla são bem próximos. Para o caso da adsorção horizontal de DNA em grafeno nossos resultados indicaram que os dois sinais podem ser distinguíveis, mostrando potencial promissor para sensitividade e seletividade.

Le transport d’ions et de molécules à l’intérieur de canaux nanométriques diffère du transport à l’échelle micro- ou macroscopique du fait de rapports surface/volume bien plus élevés conduisant à de nouveaux phénomènes de transport. Les nanotubes de carbone avec leurs propriétés uniques apparaissent comme des canaux exceptionnellement intéressants pour mieux comprendre le transport ionique et fluidique à l’échelle nanométrique et pour d’éventuelles applications nanofluidiques. Ce travail est dédié à l’étude et la compréhension des mécanismes de transport des ions en phase aqueuse à l’intérieur de nanotubes de carbone, un sujet particulièrement important pour le développement d’applications dans le domaine du séquençage de l’ADN ou de l’analyse biochimique de petites molécules.Durant ce travail, un protocole a été développé pour la fabrication de dispositifs microfluidiques intégrant des nanotubes de carbone et permettant des mesures à la fois électriques et optiques. Les propriétés de transport à l’intérieur de nanotubes de carbone mono-feuillets ont été étudiées en combinant mesures de courant ionique sous application d’un champ électrique, spectroscopie Raman et modélisation théorique. Les résultats obtenus par cette étude démontrent la forte influence de l’environnement du nanotube sur la densité et la distribution des charges de surface et donc sur les propriétés de transport à l’intérieur de ces nano-canaux dont les parois sont d’épaisseur atomique. Les ordres de grandeur des courants ioniques mesurés expérimentalement sont en bon accord avec les modèles standards de transport ionique dans un nanocanal en considérant des densités de charge de surface et des longueurs de glissement physiquement raisonnables. De manière importante, ce travail a permis de mettre en évidence un transport ionique activé par champ électrique à l’intérieur de nanotubes de carbone, qui peut être expliqué en considérant un modèle de transport plus élaboré intégrant une ou plusieurs barrières d’énergie le long du nanotube. Les résultats de la caractérisation Raman suggèrent que ces barrières d’énergie résultent d’un dopage hétérogène le long du nanotube induit par la matrice polymère. / Ionic and molecular transport inside nanometer scale geometries is distinct from micro- and macroscale transport due to the large surface-to-volume ratios which lead to unique transport phenomena. Carbon nanotubes with their peerless properties appear as exceptional channels for understanding fluidic and ionic transport at the nanoscale and for developing nanofluidics-based applications. This work is devoted at studying and understanding the transport mechanisms of ions in aqueous phase through carbon nanotubes, which is especially important for various applications such as DNA sequencing or biochemical analysis of small molecules.During this work, a protocol was developed for the fabrication of carbon nanotubes-based microfluidic devices which are suitable for both electrical and optical measurements. The transport properties through single-walled carbon nanotubes were investigated by combining ion current measurements under an applied voltage, Raman spectroscopy and theoretical modelling. The results obtained from this study highlight the strong influence of the nanotube environment on their surface charge density and distribution and hence on the ionic transport properties through these nanochannels having walls of atomic thickness. The orders of magnitude of the ionic currents experimentally measured are in good agreement with the standard models of ion transport through nanochannels when considering physically reasonable values of surface charge densities and slip lengths. Importantly, this work allowed us to evidence a novel voltage-activated transport of ions through carbon nanotubes which can be accounted for by considering a more elaborate transport model including the presence of one or more energy barriers along the nanotube. Raman characterization results support that these energy barriers result from a heterogeneous doping along the nanotubes induced by the polymer matrix.

Nanopore sensing is a versatile technique that employs very small openings, known as nanopores, to study biomolecules. The use of nanopores on solid-state membranes has gained attention due to its potential for low-cost and high-throughput sensing of single molecules in liquids. Controlled dielectric breakdown (CBD) is a method for fabricating nanopores in a suspended membrane using computer control, and can be performed in liquid, making it a more practical alternative to traditional techniques that require specialized equipment and high vacuum. Multilevel Pulse-Voltage Injection (MPVI) is a variant of CBD that allows for better control over the size and shape of the nanopore being fabricated. The main focus of this research is to develop electrical techniques for fabricating sub-10 nm solid-state nanopores in silicon nitride and graphene membranes, and to study the characteristics of the resulting nanopores. Two different MPVI schemes were implemented for fabricating nanopores in silicon nitride. The MPVI technique for Scheme 1 sets two thresholds to check if a pore is formed or not. Scheme 2 was developed by adding a threshold in order to avoid extra pore enlargement. For nanopores on a silicon nitride membrane with a 23 nm deep hole, the ratio of sub-20 nm pores improved from 20 % (Scheme 1) to around 90 % (Scheme 2). Additionally, the ratio of sub-10 nm nanopores via Scheme 2 was around 70 %. For nanopores on a silicon nitride membrane damaged by a single femtosecond laser pulse, 50 % of the fabricated nanopores via Scheme 2 were sub-10 nm. For bi-layer graphene membranes, the electrochemical reaction (ECR) technique was used to fabricate nanopores, resulting in three nanopores with diameters of 6.4, 5.9, and 1.2 nm. The nanopores on all types of membranes were enlarged using MPVI of Scheme 1, resulting in a successful increase in pore size by 0.1 to 1 nm. Finally, DNA translocation experiments were conducted to verify the suitability of the fabricated nanopores. DNA translocation events were observed using fabricated nanopores on two types of silicon nitride membranes. They are not observed for the graphene nanopore. / Nanopor-avkänning är en mångsidig teknik som använder mycket små öppningar, så kallade nanoporer, för att studera biomolekyler. Användningen av nanoporer på fasta membran har fått uppmärksamhet tack vare dess potential för detektering av enstaka molekyler i vätskor, till lågt pris och med kort genomloppstid. Kontrollerad dielektrisk nedbrytning (CBD) är en metod för att tillverka nanoporer i ett suspenderat membran med hjälp av datorstyrning som kan utföras i vätska, vilket gör den till ett mer praktiskt alternativ till traditionella tekniker som kräver specialiserad utrustning och högt vakuum. Multilevel Pulse-Voltage Injection (MPVI) är en variant av CBD som möjliggör bättre kontroll över storleken och formen på nanoporen som tillverkas. Huvudfokus för denna forskning är att utveckla elektriska tekniker för att tillverka sub-10 nm fasta nanoporer i kiselnitrid och grafenmembran, och att studera egenskaperna hos de resulterande nanoporerna. Två olika MPVI-metoder implementerades för tillverkning av nanoporer i kiselnitrid. MPVI-tekniken i Metod 1 sätter två tröskelvärden för att: kontrollera om en por bildas eller inte, samt för att kontrollera porstorleken. Metod 2 utvecklades genom att lägga till ytterligare ett tröskelvärde för att undvika extra porförstoring. För nanoporer på ett kiselnitridmembran med ett 23 nm djupt hål förbättrades förhållandet mellan porer under 20 nm från 20 % (Metod 1) till cirka 90 % (Metod 2). Dessutom var förhållandet mellan nanoporer under 10 nm med Metod 2 cirka 70 %. För nanoporer på ett kiselnitridmembran som skadats av en femtosekundlaserpuls, även om en nanopor med en diameter under 5 nm inte tillverkades, var 50 % av de tillverkade nanoporerna via Metod 2 under 10 nm. För tvåskiktsgrafenmembran användes den elektrokemiska reaktionstekniken (ECR) för att tillverka nanoporer, vilket resulterade i tre nanoporer med diametrar på 6,4; 5,9 och 1,2 nm. Nanoporerna på alla typer av membran förstorades med MPVI i Metod 1, vilket resulterade i en framgångsrik förstoring av porstorleken med 0,1 till 1 nm. Slutligen genomfördes experiment med DNA-translokation för att verifiera lämpligheten av de tillverkade nanoporerna. DNA-translokationshändelser observerades med hjälp av tillverkade nanoporer på två typer av kiselnitridmembran. De observeras inte för grafen-nanoporen.

Nanopore sequencing is a relatively new DNA sequencing method which measures the current over a nanopore in a membrane as each nucleotide of the DNA passes through the nanopore. From the resulting current signal it is possible to determine the sequence of nucleotides in the DNA by using a base caller. The goal of this project was to create a machine learning model which could estimate the accuracy rate (identity score) of the sequenced DNA using the electric current signal and other data available through nanopore sequencing. The dataset that the machine learning models were trained on were samples from E. coli bacteria that had been sequenced through nanopore sequencing. In this project a linear regression model was created as well as several neural networks. The best performing model was a neural network which had a mean square error (MSE) of 6.12 ∙ 10-4, compared to a variance in the dataset of 2.11 ∙ 10-3. The low MSE indicates that the model can effectively predict identity scores. / Nanopore sequencing är en relativt ny DNA-sekvenseringsmetod som mäter strömmen över en nanoskopisk por i ett membran samtidigt som varje DNA-nukleotid passerar genom poren. Från den resulterande elektriska signalen så är det möjligt att bestämma sekvensen av nukleotider i DNA:t genom att använda en base caller. Målet med det här projektet var att skapa en maskininlärningsmodell som kunde bestämma graden av noggrannhet av det sekvenserade DNA:t genom att använda den elektriska strömsignalen och andra typer av data tillgängliga av Nanopore sequencing. Datamängden som maskininlärningsmodellerna använde för träning bestod av samples från en E. coli bakterie som sekvenserats med nanopore sequencing. I det här projektet har en linjär regressions-modell skapats samt flera olika neurala nätverk. Den bäst presterande modellen var ett neuralt nätverk, som hade ett minstakvadratfel (MSE) på 6.12 ∙ 10-4, jämfört med datamängdens varians på 2.11 ∙ 10-3. Det låga MSE-värdet visar på att modellen effektivt kan skatta noggrannhetsgraden av den avlästa DNA-sekvensen. / Kandidatexjobb i elektroteknik 2022, KTH, Stockholm

Les eucaryotes sont des organismes chimériques issus de l’endosymbiose entre une archéobactérie et une α-protéobactérie. Au cours de ce processus, ces organismes ont évolué de sorte à obtenir un grand nombre de caractéristiques observées chez les eucaryotes modernes, notamment une mitochondrie, un noyau, un système endomembranaire, un système d’épissage ou encore des chromosomes linéaires terminés par un télomère. Bien que les caractéristiques du dernier ancêtre commun des eucaryotes aient majoritairement été identifié, la suite des évènements évolutifs ayant mené à l’apparition de cet organisme demeure peu compris. Afin de mieux reconstruire cette suite d’évènements, l’analyse des génomes d’organismes basals aux eucaryotes sera nécessaire pour identifier des traces de cette évolution. Ainsi, nous proposons que l’analyse d’une collection de génomes d’eucaryotes « primitifs », les jakobides et malawimonades, des eucaryotes unicellulaires flagellés se nourrissant de bactéries, pourrait permettre une meilleure compréhension de ce processus. De plus, il a été supposé que le génome d’un de ces organismes, Andalucia godoyi, pourrait posséder des chromosomes circulaires, une caractéristique atypique chez les eucaryotes, une caractéristique qui pourra être confirmée par la production d’assemblage génomique de haute contigüité. Afin d’obtenir des assemblages génomiques de haute qualité, les jakobides A. godoyi, Jakoba bahamiensis, Seculamonas ecuadoriensis, Stygiella incarcerata et le malawimonades Malawimonas californiana ont été séquencés par nanopore. Le séquençage nanopore a présenté des résultats mitigés et les organismes J. bahamiensis et M. californiana ont présentés un faible rendement de séquençage, possiblement dû à la contamination par des polysaccharides. Pour les autres organismes, nous avons développé un pipeline d’assemblage utilisant les assembleurs Flye et Shasta qui nous a permis de produire des assemblages génomiques. L’analyse du génome de A. godoyi a permis d’identifier la présence de quatre chromosomes circulaires, possiblement localisés dans le noyau, contenant plusieurs gènes liés au métabolisme, au transport et à la signalisation et qui constituent possiblement un type de chromosome circulaire différent de ceux observés précédemment chez les eucaryotes. Dans l’ensemble, ces travaux ont permis la mise en place d’une collection de génome d’eucaryotes « primitifs » qui pourront être utilisés pour des analyse de génomique comparative afin de mieux comprendre l’évolution des eucaryotes. / Eucaryotes are chimeric organisms that are the product of an endosymbiotic event between an archaebacteria and an α-proteobacteria. During the eukaryogenesis, these organisms have gained many characteristics that defines modern eucaryotes such as a mitochondrion, a nucleus, an endomembrane system, the splicing machinery, and linear chromosome with telomeres. While most characteristics of the last common eukaryote ancestor have mostly been identified, most of the evolutionary process that led to this organism is still unknown. To reconstruct this string of event, we must analyse the genome of “primitive” basal eukaryotes with a slow evolutionary rate and a lifestyle like that of the last common eukaryotes ancestor, and thus are most likely to contain remains of ancestral mechanisms that have been lost in most known eukaryotes. We propose that this analysis of the genome of the jakobids and malawimonads, two groups are free-living flagellate that feeds on bacteria, could provide such clues on the evolution of eukaryotes. Using nanopore sequencing, a collection of high-quality genomes has been built to help in this analysis. Furthermore, it has been supposed that the genome of the jakobid Andalucia godoyi could be composed to both linear and circular chromosomes, a genomic structure that have not been identified in other eukaryotes, which was investigated using the high quality nanopore assembly. To generate a collection of high-quality genome assemblies, we have sequenced the genomes of the jakobids A. godoyi, Jakoba bahamiensis, Seculamonas ecuadoriensis and Stygiella incarcerata as well as the malawimonad Malawimonas californiana by nanopore. While the yields were too low for J. bahamiensis and M. californiana, probably due to a contamination by polysaccharides, we were able to assemble chromosome level genome for A. godoyi and S. incarcerata and high-quality draft genome for S. ecuadoriensis et R. americana. Using this assembly, we were able to identify four circular chromosomes in the genome of A. godoyi. The circular chromosomes are likely to be located in the nucleus and encodes genes with functions related to the metabolism, ions and macromolecules transport as well as signaling. Furthermore, these molecules differ from known circular chromosome in eukaryotes as they are unlikely to be selfish DNA elements, such as known eucaryotes plasmids, or circular by-product of replication identified in other eukaryotes. Overall, this work sets the bases for larger scale comparative genomics of the jakobids and malawimonads, by generating a small collection of genomes that will be used in future studies to better understand the origin of the eukaryotes.

Many environmental compartments are acknowledged transmission routes for infectious diseases, antibiotic resistance, and anthropogenic pollution. The need for environmental sensing has consistently been stressed as a means to minimize public health threats caused by such contaminants. Many analytical detection techniques have been developed and applied for environmental sensing. However, these techniques are often reliant upon centralized facilities and require intensive resources. For these reasons their use can be challenging under resource-constrained conditions characterized by poor water, sanitation, and hygiene (WASH) services. In this dissertation, we developed biotechnology- and/or nanotechnology-advanced analytical tools for environmental sensing that have potential for future application in regions with poor WASH services. First, loop-mediated isothermal amplification (LAMP) and nanopore sequencing were applied to develop assays for the detection of SARS-CoV-2, the causative agent of COVID-19, in wastewater samples. Second, surface-enhanced Raman spectroscopy (SERS) was applied for environmental detection of a range of analytes. Gold nanoparticle (AuNP)-based SERS substrates were fabricated by droplet evaporation-induced aggregation on a hydrophobic substrate. These SERS substrates were then applied for the detection of antibiotic resistance genes (ARGs) and other environmental contaminants (e.g., dye or hydrophobic organic contaminants). In a separate study, Au nanostructured SERS substrates were fabricated and applied for pH sensing in a range of environmental media. Finally, the environmental impact of an AuNP-based colorimetric detection assay was assessed via life cycle assessment. / Doctor of Philosophy / Environmental sensing is an important means to intervene against public health threats of infectious diseases and environmental contaminants. However, currently available analytical tools for environmental samples often require intensive resources that are not available in low- and middle-income countries. In this dissertation, we developed biotechnology and/or nanotechnology advanced analytical tools for environmental sensing that have potential future application applied under resource-constrained conditions. First, we applied loop-mediated isothermal amplification (LAMP) and nanopore sequencing to develop detection assays for SARS-CoV-2, the causative agent of COVID-19, in wastewater samples. Second, we applied surface-enhanced Raman spectroscopy (SERS) to develop assays for environmental analytes. We fabricated SERS substrates by evaporation-induced aggregation of gold nanoparticles (AuNPs) on a hydrophobic substrate and applied these for the detection of antibiotic resistance genes (ARGs) and other environmental contaminants. In addition, Au nanostructured SERS substrates were fabricated and applied for pH sensing in a range of environmental media. Finally, we used life cycle assessment to quantitatively evaluate the environmental impacts of an AuNP-based sensing applications.