Preterm birth (PTB) results in over 15 million early births annually and is the leading cause of neonatal deaths. There are no clinical methods currently available to evaluate risk of PTB at early stages in pregnancy; thus, a rapid diagnostic to analyze PTB risk would be beneficial. Microfluidic immunoaffinity extraction is a promising platform for preparing complex samples, such as maternal serum with PTB risk biomarkers. 3D printed microfluidic devices have advantages over conventional microfluidic systems including simple fabrication and potential for iterative optimization to improve designs. In this work, I developed immunoaffinity monoliths in 3D printed microfluidic devices modified with antibodies to enrich PTB biomarkers from human blood serum. I retained and eluted a peptide PTB biomarker in both buffer and blood serum using an immunoaffinity column. An additional three PTB biomarkers were also successfully extracted either from buffer or blood serum on single-antibody columns. Both polyclonal and monoclonal antibodies to PTB biomarkers were characterized by dot blots, biolayer interferometry, and surface plasmon resonance to determine their specificity and dissociation constants. I created multiplexed immunoaffinity columns to simultaneously enrich three PTB biomarkers from depleted human blood serum in a single extraction. This is the first demonstration of multiplexed immunoaffinity columns for PTB biomarkers in a 3D printed microfluidic device. My work is a key step towards the future development of 3D printed microfluidic devices for rapid PTB testing.

Wavelength division multiplexed (WDM) optical networks are rapidly becoming the technology of choice in network infrastructure and next-generation Internet architectures. WDM networks have the potential to provide unprecedented bandwidth, reduce processing cost, achieve protocol transparency, and enable efficient failure handling. This dissertation addresses the important issues of improving the performance and enhancing the reliability of WDM networks as well as modeling and evaluating the performance of these networks. Optical wavelength conversion is one of the emerging WDM enabling technologies that can significantly improve bandwidth utilization in optical networks. A new approach for the sparse placement of full wavelength converters based on the concept of the k-Dominating Set (k-DS) of a graph is presented. The k-DS approach is also extended to the case of limited conversion capability using three scalable and cost-effective switch designs: flexible node-sharing, strict node-sharing and static mapping. Compared to full search algorithms previously proposed in the literature, the K-DS approach has better blocking performance, has better time complexity and avoids the local minimum problem. The performance benefit of the K-DS approach is demonstrated by extensive simulation. Fiber delay line (FDL) is another emerging WDM technology that can be used to obtain limited optical buffering capability. A placement algorithm, k-WDS, for the sparse placement of FDLs at a set of selected nodes in Optical Burst Switching (OBS) networks is proposed. The algorithm can handle both uniform and non-uniform traffic patterns. Extensive performance tests have shown that k-WDS provides more efficient placement of optical fiber delay lines than the well-known approach of placing the resources at nodes with the highest experienced burst loss. Performance results that compare the benefit of using FDLs versus using optical wavelength converters (OWCs) are presented. A new algorithm, A-WDS, for the placement of an arbitrary numbers of FDLs and OWCs is introduced and is evaluated under different non-uniform traffic loads. This dissertation also introduces a new cost-effective optical switch design using FDL and a QoS-enhanced JET (just enough time) protocol suitable for optical burst switched WDM networks. The enhanced JET protocol allows classes of traffic to benefit from FDLs and OWCs while minimizing the end-to-end delay for high priority bursts. Performance evaluation models of WDM networks represent an important research area that has received increased attention. A new analytical model that captures link dependencies in all-optical WDM networks under uniform traffic is presented. The model enables the estimation of connection blocking probabilities more accurately than previously possible. The basic formula of the dependency between two links in this model reflects their degree of adjacency, the degree of connectivity of the nodes composing them and their carried traffic. The usefulness of the model is illustrated by applying it to the sparse wavelength converters placement problem in WDM networks. A lightpath containing converters is divided into smaller sub-paths such that each sub-path is a wavelength continuous path and the nodes shared between these sub-paths are full wavelength conversion capable. The blocking probability of the entire path is obtained by computing the blocking probabilities of the individual sub-paths. The analytical-based sparse placement algorithm is validated by comparing it with its simulation-based counterpart using a number of network topologies. Rapid recovery from failure and high levels of reliability are extremely important in WDM networks. A new Fault Tolerant Path Protection scheme, FTPP, for WDM mesh networks based on the alarming state of network nodes and links is introduced. The results of extensive simulation tests show that FTPP outperforms known path protection schemes in terms of loss of service ratio and network throughput. The simulation tests used a wide range of values for the load intensity, the failure arrival rate and the failure holding time. The FTPP scheme is next extended to the differentiated services model and its connection blocking performance is evaluated. Finally, a QoS-enhanced FTPP (QEFTPP) routing and path protection scheme in WDM networks is presented. QEFTPP uses preemption to minimize the connection blocking percentage for high priority traffic. Extensive simulation results have shown that QEFTPP achieves a clear QoS differentiation among the traffic classes and provides a good overall network performance.

Lungcancer är en heterogen sjukdom som kräver många histopatologiska analyser för att diagnostisera och behandla effektivt. Dessa analyser utförs för närvarande med singelplex immunohistokemi (IHC) vilket kräver mycket vävnadsprovmaterial om flera markörer behöver färgas för. I den här studien testades två tillvägagångssätt för multiplex immunfluorescens (IF) (indirekt sekventiell IF och multiplex tyramide signal amplification med spektral avblandning) för att se om en panel av 7 markörer, som rutinmässigt används kliniskt, kunde färgas i ett enda experiment på samma vävnadsprov. För att göra detta användes COMET- och LabSat-instrumenten från Lunaphore och de utvärderades utifrån kvalité och användarvänlighet. På COMETen kunde alla sju markörer inkluderas i panelen medan tillvägagångssättet med TSA på LabSat var begränsat till sex markörer på grund av protokollet för spektral separation. Panelen optimerades på COMET och sedan gjordes multiplexa färgningar med båda instrumenten. Färgningarna från LabSat avbildades med mikroskopet på PhenoImager Fusion-instrumentet. Alla färgningar på COMET var av god kvalité medan LabSat hade behövt vidare optimering för att prestera på samma nivå. COMETen fanns också vara det mer användarvänliga instrumentet pga det integrerade mikroskopet och mindre behov av användarintervention. Denna studie visade att det är möjligt att översätta singelplexa IHC-analyser till multiplex IF med vissa optimeringar och byte av några antikroppskloner. / Lung cancer is a heterogeneous group of malignancies that requires many histopathological analyses to diagnose and treat efficiently. These analyses are currently performed with singleplex immunohistochemistry (IHC) which needs a lot of tissue sample material if multiple markers have to be stained for. In this study, two different multiplexed immunofluorescence (IF) approaches (indirect sequential IF and multiplexed tyramide signal amplification with spectral unmixing) were tested to see if a panel of 7 markers routinely used in clinic to could be stained in a single experiment on the same tissue sample. To do this, the COMET and LabSat instruments from Lunaphore were used and evaluated in regards to quality and ease of use. With the COMET all seven markers could be included in the panel while the TSA approach on LabSat was limited to six markers due to the spectral unmixing protocol. The panel was optimized on COMET and then multiplexed stainings were performed on both instruments. The LabSat staining was imaged on the PhenoImager Fusion microscope. All the stainings were of good quality on the COMET while the LabSat would have needed further optimization to perform at the same level. The COMET was also found to be the more user-friendly instrument due to its integrated microscope and lesser need for user intervention. This study showed that it is possible to translate singleplex IHC analyses into multiplexed IF with some optimizations and change of a few antibody clones.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22448号 / 工博第4709号 / 新制||工||1735(附属図書館) / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 竹内 繁樹, 教授 白石 誠司, 准教授 浅野 卓 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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[ES] Los sistemas CRISPR-Cas se basan en un mecanismo del sistema inmune adaptativo que se encuentra en bacterias y arqueas. Funcionan utilizando endonucleasas guiadas por ARN (proteínas Cas) para localizar y reconocer secuencias específicas de ácidos nucleicos. Los sistemas CRISPR-Cas son revolucionarios en el campo de la ingeniería genética, permitiendo la edición específica del genoma, ensayos de regulación genética y, desarrolladas en los últimos años, aplicaciones de diagnóstico. Los métodos de detección de ácidos nucleicos son la técnica de diagnóstico de referencia en la clínica debido a su alta sensibilidad y especificidad. Sin embargo, de cara al futuro, debemos desarrollar métodos versátiles de detección de virus que permitan diagnósticos rápidos y fiables, que superen las limitaciones de las técnicas actuales y puedan usarse fácilmente fuera del laboratorio para aplicaciones en puntos de atención (POC). Los sistemas CRISPR-Cas se han reconvertido en herramientas de diagnóstico de ácidos nucleicos gracias a la capacidad de algunas proteínas Cas de realizar cortes colaterales no específicos al reconocer su diana. Nuestro objetivo es ampliar el conjunto de herramientas de diagnóstico CRISPR-Cas mediante el desarrollo de una nueva estrategia de detección de ácidos nucleicos basada en CRISPR-Cas9, cuyo modo de acción se basa en el desplazamiento de hebra en lugar de en la catálisis colateral. Demostramos que los amplicones de ADN del SARS-CoV-2 generados a partir de muestras de pacientes pueden detectarse con CRISPR-Cas9. También demostramos la capacidad de realizar la detección simultánea de diferentes amplicones de ADN con la misma nucleasa, ya sea para identificar diferentes regiones de SARS-CoV-2 o diferentes virus respiratorios. Para avanzar hacia aplicaciones POC, acoplamos el paso de preamplificación isotérmica con oligos biotinilados para producir amplicones marcados. Cuando se combina con CRISPR-Cas9 y sondas marcadas con FAM, esto permite la visualización colorimétrica de la detección mediante ensayos de flujo lateral (LFA) en tiras disponibles en el mercado. La lectura colorimétrica permite la interpretación de los resultados a simple vista, superando la necesidad de un fluorímetro. Detectamos con éxito SARS-CoV-2 a partir de muestras de pacientes en la configuración LFA, y cuando se combinó con RT-RPA multiplexado, se logró la detección de dos regiones distintas del virus en una sola prueba. Además, la ausencia de actividad colateral en nuestra metodología permite el procesamiento de secuencias posteriores adicionales. Por lo tanto, también pretendemos explorar la capacidad de integración de señales de nuestro método de detección basado en CRISPR-Cas9. Demostramos que diseños de circuitos lógicos de ADN pueden procesar diferentes señales de SARS-CoV-2 detectadas por los complejos CRISPR. También nos propusimos mejorar nuestro sistema CRISPR-Cas9 para detectar tanto proteínas como ácidos nucleicos. Nos propusimos diseñar un ARN guía condicional que respondiera a la presencia de la proteína spike del SARS-CoV-2. Para ello, insertamos un aptámero en el ARN guía para bloquear la actividad de Cas9 en ausencia de la proteína. En presencia de la proteína spike, la interacción aptámero-proteína hace que el motivo de bloqueo sufra un cambio conformacional, liberando el ARN guía y activando Cas9. Aunque logramos con éxito el escenario de bloqueo utilizando las secuencias reguladoras, es necesario seguir trabajando para identificar una secuencia que pueda inducir el cambio conformacional específico necesario para restaurar la actividad de Cas9 en presencia de la proteína spike. En conjunto, esta plataforma de diagnóstico CRISPR-Cas9 permite una detección multiplexada en un solo tubo, complementando los métodos existentes basados en CRISPR, y ofreciendo un sistema extensible para aplicaciones en diagnóstico en el punto de atención y biocomputación. / [CA] Els sistemes CRISPR-Cas es basen en un mecanisme del sistema immunitari adaptatiu que es troba en bacteris i arqueus. Funcionen utilitzant endonucleases guiades per ARN (proteïnes Cas) per tal de dirigir-se i reconéixer seqüències específiques d'àcids nucleics. Els sistemes CRISPR-Cas siguen revolucionaris en el camp de l'enginyeria genètica, permetent l'edició específica del genoma, assajos de regulació gènica i, desenvolupades en els darrers anys, aplicacions de diagnòstic. Els mètodes de detecció d'àcids nucleics són la tècnica de diagnòstic de referència a la clínica degut a la seua alta sensibilitat i especificitat. No obstant, de cara al futur, hem de desenvolupar mètodes versàtils de detecció de virus que permeten diagnòstics ràpids i fiables, que superen les limitacions de les tècniques actuals i puguen utilitzar-se fàcilment fora del laboratori per a aplicacions en punts d'atenció (POC). Els sistemes CRISPR-Cas han estat reorientats com a eines de diagnòstic d'àcids nucleics, fet possible per la capacitat d'algunes proteïnes Cas de realitzar un tall col·lateral no específic en reconèixer un objectiu. El nostre objectiu és ampliar el conjunt d'eines de diagnòstic CRISPR-Cas desenvolupant una nova estratègia de detecció d'àcids nucleics basada en CRISPR-Cas9, el mode d'acció del qual es basa en el desplaçament de cadena en lloc de la catàlisi col·lateral. Mostrem que es poden detectar amplicons de DNA de SARS-CoV-2 generats a partir de mostres de pacients amb CRISPR-Cas9. També demostrem la capacitat de realitzar la detecció simultània de diferents amplicons de DNA amb la mateixa nucleasa, ja siga per identificar diferents regions de SARS-CoV-2 o diferents virus respiratoris. Per avançar cap a aplicacions de punt d'atenció (POC), vam combinar el pas de preamplificació isotèrmica amb oligos biotinilats per produir amplicons etiquetats. Quan es combinen amb l'orientació CRISPR-Cas9 i sondes etiquetades amb FAM, això permet la visualització colorimètrica de la detecció mitjançant assajos de flux lateral (LFA) en tires comercials disponibles. La lectura colorimètrica permet la interpretació dels resultats a simple vista, superant la necessitat d'un fluorímetre. Vam detectar amb èxit el SARS-CoV-2 a partir de mostres de pacients en el sistema LFA, i quan es va combinar amb RT-RPA multiplexada, es va aconseguir la detecció de dues regions diferents del virus en una sola prova. A més, l'absència d'activitat col·lateral en la nostra metodologia permet el processament de seqüències addicionals. Per tant, també ens proposem explorar la capacitat d'integració de senyals del nostre mètode de detecció basat en CRISPR-Cas9. Demostrem que els circuits lògics d'ADN dissenyats poden processar diferents senyals de SARS-CoV-2 detectats pels complexos CRISPR. També vam intentar millorar el nostre sistema CRISPR-Cas9 per detectar tant proteïnes com àcids nucleics. Ens vam proposar dissenyar un ARN guia condicional que respon a la presència de la proteïna spike del SARS-CoV-2. Per aconseguir-ho, vam inserir una seqüència d'aptàmer dins de l'ARN guia per bloquejar l'activitat de Cas9 en absència de la proteïna. En presència de la proteïna spike, la interacció aptàmer-proteïna fa que el motiu de bloqueig experimente un canvi conformacional, alliberant l'ARN guia i activant Cas9. Tot i que vam aconseguir amb èxit el bloqueig utilitzant les seqüències reguladores, es necessita més treball per identificar una seqüència que puga induir el canvi conformacional específic necessari per restaurar l'activitat de Cas9 en presència de la proteïna spike. Col·lectivament, aquesta plataforma de diagnòstic CRISPR-Cas9 permet una detecció multiplexada en un sol tub, complementant els mètodes existents basats en CRISPR i oferint un sistema extensible per a aplicacions en diagnòstics de punt d'atenció i biocomputació. / [EN] CRISPR-Cas systems are based on an adaptive immune system mechanism found in bacteria and archaea. They function using RNA-guided endonucleases (Cas proteins), to target and recognize specific nucleic acid sequences. CRISPR-Cas systems are revolutionary in the field of genetic engineering, enabling specific genome editing, gene regulation assays and, developed in recent years, diagnostic applications. Nucleic acid detection methods are the gold-standard diagnostic technique in the clinic due to their high sensitivity and specificity. However, going forward, we must develop versatile virus detection methods that enable fast and reliable diagnostics and that can overcome the limitations of the current techniques and can be easily used outside the laboratory for point-of-care (POC) applications. CRISPR-Cas systems have been repurposed as nucleic acid diagnostic tools made possible by the ability of some Cas proteins to perform non-specific collateral cleavage upon target recognition. We aim to expand the CRISPR-Cas diagnostic toolkit by developing a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also demonstrate the ability to perform simultaneous detection of different DNA amplicons with the same nuclease, either to identify different SARS-CoV-2 regions or different respiratory viruses. To advance toward POC applications, we coupled the isothermal preamplification step with biotinylated oligos to produce labelled amplicons. When combined with CRISPR-Cas9 targeting and FAM-labelled probes, this allows for colorimetric visualization of detection using lateral flow assays (LFA) on commercially available strips. The colorimetric readout enables interpretation of the results with the naked eye, surpassing the need for a fluorimeter. We successfully detected SARS-CoV-2 from patient samples in the LFA setup, and when combined with multiplexed RT-RPA, the detection of two distinct regions of the virus was achieved in a single test. Furthermore, the absence of collateral activity in our methodology allows for the processing of additional downstream sequences. Therefore, we also aim to explore the signal integration capability of our CRISPR-Cas9-based detection method. We demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. We also aimed to enhance our CRISPR-Cas9 system to sense both proteins and nucleic acids. We aimed to engineer a conditional guide RNA that responds to the presence of the SARS-CoV-2 spike protein. To achieve this, we inserted an aptamer sequence within the guide RNA to block Cas9 activity in the absence of the protein. Upon the presence of the spike protein, aptamer-protein interaction makes the blocking motif undergo a conformational change, releasing the guide RNA and activating Cas9. While we successfully achieved the blocking scenario using the regulatory sequences, further work is needed to identify a sequence that can induce the specific conformational change required to restore Cas9 activity upon spike protein presence. Collectively, this CRISPR-Cas9 diagnostic platform allows a multiplexed detection in a single tube, complementing the existing CRISPR-based methods, and offering an extensible system for applications in point-of-care diagnostics and biocomputing. / This work was supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation (PRE2019-088531). / Márquez Costa, R. (2024). Engineering of CRISPR-Cas9-Based Systems for Diagnostics and Biocomputing [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/210634

O problema estudado relaciona-se ao planejamento de redes ópticas de acesso TWDM (Time and Wavelength Division Multiplexing), tendo como meta prover meios para atender ao consumo crescente de banda e, ao mesmo tempo, associar o mesmo ao menor consumo de energia possível e disponibilizar serviços de qualidade aos usuários da rede. Neste contexto, após a apresentação de uma revisão bibliográfica, alguns trabalhos foram selecionados como referências base para o estudo. Neste, foram analisadas técnicas de gerenciamento do comprimento de onda numa possível migração tecnológica dos equipamentos de acesso à rede pelo lado do usuário, as unidades de rede óptica (ONUs), considerando ser possível aproveitar de forma eficiente a distribuição de ONUs em cada par de comprimentos de onda, e como resultado, desligar interfaces de gerenciamento para economia de energia. Em paralelo a este objetivo, foram agregados métodos de \"registro adaptativo\" para melhorar o desempenho das ONUs que estão migrando e/ou entrando na rede, evitando colisões entre seus sinais e aumentando as chances de registro no terminal de linha óptico (OLT). Além do mais, o trabalho aborda o cumprimento do contrato de serviços (SLA) por parte das operadoras de telecomunicações, bem como oferece meios de estruturar uma rede óptica passiva (PON) para oferecer serviços com garantia de métricas de qualidade (QoS). Diferentes cenários foram tratados ao variar a porcentagem de usuários ativos na rede, e percebeu-se que, quando a PON opera com baixo índice de atividade (média 10% de ONUs ativas), foi possível economizar mais de 80% de energia em interfaces de gerenciamento. Por outro lado, ao envolver cenários mais realistas em redes ópticas (média de 50% de ONUs ativas), o TWDM-PON economizou quase 50% de energia nestas interfaces ao ser configurado com baixo período de migração (5 minutos). Além disso, alcançou um SLA de aproximadamente 100%, um valor considerado acima do usual quando se trata de cumprimento de serviços. Como uma tecnologia emergente, o TWDM-PON destaca-se num cenário que deve ser planejado para ser compatível com tecnologias implantadas, e além de disso, superar o legado de redes PON na economia de energia e qualidade de serviços oferecidos. / The problem studied is related to the planning of optical access networks TWDM (Time and Wavelength Division Multiplexing), aiming to provide means to meet the increasing bandwidth consumption and, at the same time, associating it to the lowest possible power consumption and providing quality services to the network users. In this context, following the presentation of a literature review, some studies were selected as a reference basis for the study. In this, some wavelength management techniques were analyzed aiming a possible technological migration of the network access equipment at the user side, the optical network units (ONU), whereas it is possible to use efficiently the ONUs distribution in each pair of wavelength. As a result, it becomes possible to switch off some management interfaces thus assuring a degree of power consumption save. In parallel, methods for providing an \"adaptive record\" of ONUs have been aggregated to improve the performance of those ONUs that are migrating and/or entering the network, avoiding collisions between their signals and increasing the chance of their registration in the optical line terminal (OLT). Furthermore, this work deals with the fulfillment of the service agreement (SLA) signed by telecommunications operators and proposes ways to design a passive optical network (PON) in order to offer services with quality metrics assurance (QoS). Different scenarios were treated to vary the percentage of active users on the network, and it was noticed that when the PON operates with low rate of activity (average 10% of active ONUs), it was possible to save over 80% energy in the management interfaces. Moreover, by involving more realistic scenarios in optical networks (50%, in average, of active ONUs), the TWDM-PON saved almost 50% of energy at the interfaces to be configured with low migration period (5 minutes). Additionally, it reached an SLA of approximately 100%, a value considered above the usual when it comes to compliance services. As an emerging technology, TWDM-PON stands out in a scenario that should be designed to be compatible with deployed technologies, and beyond that, to overcome the legacy of PON networks in energy saving and quality of services offered.

O problema estudado relaciona-se ao planejamento de redes ópticas de acesso TWDM (Time and Wavelength Division Multiplexing), tendo como meta prover meios para atender ao consumo crescente de banda e, ao mesmo tempo, associar o mesmo ao menor consumo de energia possível e disponibilizar serviços de qualidade aos usuários da rede. Neste contexto, após a apresentação de uma revisão bibliográfica, alguns trabalhos foram selecionados como referências base para o estudo. Neste, foram analisadas técnicas de gerenciamento do comprimento de onda numa possível migração tecnológica dos equipamentos de acesso à rede pelo lado do usuário, as unidades de rede óptica (ONUs), considerando ser possível aproveitar de forma eficiente a distribuição de ONUs em cada par de comprimentos de onda, e como resultado, desligar interfaces de gerenciamento para economia de energia. Em paralelo a este objetivo, foram agregados métodos de \"registro adaptativo\" para melhorar o desempenho das ONUs que estão migrando e/ou entrando na rede, evitando colisões entre seus sinais e aumentando as chances de registro no terminal de linha óptico (OLT). Além do mais, o trabalho aborda o cumprimento do contrato de serviços (SLA) por parte das operadoras de telecomunicações, bem como oferece meios de estruturar uma rede óptica passiva (PON) para oferecer serviços com garantia de métricas de qualidade (QoS). Diferentes cenários foram tratados ao variar a porcentagem de usuários ativos na rede, e percebeu-se que, quando a PON opera com baixo índice de atividade (média 10% de ONUs ativas), foi possível economizar mais de 80% de energia em interfaces de gerenciamento. Por outro lado, ao envolver cenários mais realistas em redes ópticas (média de 50% de ONUs ativas), o TWDM-PON economizou quase 50% de energia nestas interfaces ao ser configurado com baixo período de migração (5 minutos). Além disso, alcançou um SLA de aproximadamente 100%, um valor considerado acima do usual quando se trata de cumprimento de serviços. Como uma tecnologia emergente, o TWDM-PON destaca-se num cenário que deve ser planejado para ser compatível com tecnologias implantadas, e além de disso, superar o legado de redes PON na economia de energia e qualidade de serviços oferecidos. / The problem studied is related to the planning of optical access networks TWDM (Time and Wavelength Division Multiplexing), aiming to provide means to meet the increasing bandwidth consumption and, at the same time, associating it to the lowest possible power consumption and providing quality services to the network users. In this context, following the presentation of a literature review, some studies were selected as a reference basis for the study. In this, some wavelength management techniques were analyzed aiming a possible technological migration of the network access equipment at the user side, the optical network units (ONU), whereas it is possible to use efficiently the ONUs distribution in each pair of wavelength. As a result, it becomes possible to switch off some management interfaces thus assuring a degree of power consumption save. In parallel, methods for providing an \"adaptive record\" of ONUs have been aggregated to improve the performance of those ONUs that are migrating and/or entering the network, avoiding collisions between their signals and increasing the chance of their registration in the optical line terminal (OLT). Furthermore, this work deals with the fulfillment of the service agreement (SLA) signed by telecommunications operators and proposes ways to design a passive optical network (PON) in order to offer services with quality metrics assurance (QoS). Different scenarios were treated to vary the percentage of active users on the network, and it was noticed that when the PON operates with low rate of activity (average 10% of active ONUs), it was possible to save over 80% energy in the management interfaces. Moreover, by involving more realistic scenarios in optical networks (50%, in average, of active ONUs), the TWDM-PON saved almost 50% of energy at the interfaces to be configured with low migration period (5 minutes). Additionally, it reached an SLA of approximately 100%, a value considered above the usual when it comes to compliance services. As an emerging technology, TWDM-PON stands out in a scenario that should be designed to be compatible with deployed technologies, and beyond that, to overcome the legacy of PON networks in energy saving and quality of services offered.