Post-LS3 Experimental Options in ECN3

The Experimental Cavern North 3 (ECN3) is an underground experimental cavern on the CERN Pr\'evessin site. ECN3 currently hosts the NA62 experiment, with a physics programme devoted to rare kaon decays and searches of hidden particles approved until Long Shutdown 3 (LS3). Several options are proposed on the longer term in order to make best use of the worldwide unique potential of the high-intensity/high-energy proton beam extracted from the Super Proton Synchrotron (SPS) in ECN3. The current status of their study by the CERN Physics Beyond Colliders (PBC) Study Group is presented, including considerations on beam requirements and upgrades, detector R&D and construction, schedules and cost, as well as physics potential within the CERN and worldwide landscape.Comment: 113 pages, 39 figure

Optimizing Nanopore-based microbiome sequencing for characterizing biotechnologically-relevant ecosystems

La secuenciación de siguiente generación (NGS) ha cambiado completamente la manera en la que estudiamos el mundo microbiano. A lo largo de los últimos años, las plataformas Illumina han sido las más usadas para secuenciar ADN. No obstante, la secuenciación por nanoporos, un nuevo método impulsado por Oxford Nanopore Technologies (ONT), está incrementando gradualmente su popularidad dadas sus múltiples ventajas sobre las plataformas NGS. Estas ventajas incluyen la portabilidad y la capacidad para generar secuencias largas en tiempo real. A pesar de estos avances, la adopción de la secuenciación ONT se ha visto limitada por la elevada tasa de error asociada a esta tecnología en comparación con Illumina. El objetivo de la presente tesis fue optimizar la secuenciación por nanoporos para analizar comunidades microbianas de interés industrial y/o biotecnológico. Específicamente, se evaluó el uso de esta técnica de secuenciación para tres aplicaciones diferentes: la monitorización industrial, la bioprospección microbiana y el ensamblaje metagenómico. En el primer caso, se estudiaron las comunidades microbianas implicadas en la producción de biogás, un combustible obtenido por digestión anaerobia (DA), mediante secuenciación metataxonómica. Este trabajo permitió medir el efecto de diferentes parámetros sobre los microbiomas responsables de la DA y detectar marcadores microbianos relacionados con una producción optimizada de biogás. En segundo lugar, se usó la secuenciación ONT in situ para caracterizar distintas muestras durante una expedición de bioprospección al Desierto de Tabernas (Almería, España). Los análisis bioinformáticos se centraron en detectar bacterias resistentes a la radiación y a la desecación, que eran el objetivo de la bioprospección. Los datos obtenidos mediante secuenciación fueron consistentes con los resultados de las técnicas de cultivo, demostrando que la secuenciación portátil puede ser usada para predecir el potencial biotecnológico de las muestras in situ. Finalmente, se investigó el impacto de la secuenciación ONT en el ensamblaje metagenómico usando datos generados a partir de comunidades microbianas artificiales. Después de evaluar varias herramientas de ensamblaje, metaFlye mostró el mejor rendimiento, permitiendo recuperar varios genomas extremadamente contiguos directamente desde el metagenoma. En términos generales, esta tesis evidencia que las plataformas ONT pueden ser aplicadas con éxito para caracterizar microbiomas de relevancia biotecnológica y analiza críticamente las direcciones futuras de la secuenciación por nanoporos, teniendo en cuenta las limitaciones y ventajas de esta tecnología.Next-generation sequencing (NGS) has completely changed the way the microbial world is studied. Illumina platforms have been the most widely used DNA sequencers during the last 8-10 years. However, Nanopore sequencing, a third-generation sequencing method powered by Oxford Nanopore Technologies (ONT), is becoming increasingly popular due to its multiple advantages over NGS, including portability and the ability to read long DNA molecules in real time. Despite these improvements, the adoption of ONT platforms has been hampered by the higher error rate of this technology compared to Illumina. This thesis aimed at optimizing Nanopore sequencing to analyze microbial communities of industrial and/or biotechnological interest. Specifically, this sequencing technique was evaluated for its use in three different applications: industrial monitoring, microbial bioprospecting and metagenome assembly. In the first case, metataxonomic sequencing was applied to characterize the archaeal and bacterial communities involved in the production of biogas, which is an industrially relevant biofuel obtained by anaerobic digestion (AD). This work enabled to measure the effect of different operating parameters on the AD microbiome and to detect microbial markers associated with an improved production of biogas. Secondly, in situ Nanopore sequencing was used to study several samples during a bioprospecting expedition to the Tabernas Desert (Almeria, Spain). Microbiome analyses were focused on the detection of radiation- and desiccation-resistant bacteria, which were the target of the bioprospecting activities. Nanopore data was consistent with the results obtained by culture methods, thus demonstrating that portable sequencing can be used to predict the biotechnological potential of the samples in situ. Finally, the impact of Nanopore sequencing on metagenome assembly was investigated by using data generated from mock communities with different levels of complexity. After benchmarking various assembly tools, metaFlye showed the best overall performance, retrieving highly contiguous genomes directly from metagenomic data. Altogether, this thesis proves that ONT platforms can be efficiently applied to characterize biotechnologically-relevant ecosystems. Moreover, future directions of Nanopore sequencing are critically discussed considering all the advantages and limitations of this technology

Advances in Forensic Genetics

The book has 25 articles about the status and new directions in forensic genetics. Approximately half of the articles are invited reviews, and the remaining articles deal with new forensic genetic methods. The articles cover aspects such as sampling DNA evidence at the scene of a crime; DNA transfer when handling evidence material and how to avoid DNA contamination of items, laboratory, etc.; identification of body fluids and tissues with RNA; forensic microbiome analysis with molecular biology methods as a supplement to the examination of human DNA; forensic DNA phenotyping for predicting visible traits such as eye, hair, and skin colour; new ancestry informative DNA markers for estimating ethnic origin; new genetic genealogy methods for identifying distant relatives that cannot be identified with conventional forensic DNA typing; sensitive DNA methods, including single-cell DNA analysis and other highly specialised and sensitive methods to examine ancient DNA from unidentified victims of war; forensic animal genetics; genetics of visible traits in dogs; statistical tools for interpreting forensic DNA analyses, including the most used IT tools for forensic STR-typing and DNA sequencing; haploid markers (Y-chromosome and mitochondria DNA); inference of ethnic origin; a comprehensive logical framework for the interpretation of forensic genetic DNA data; and an overview of the ethical aspects of modern forensic genetics

Drug Eluting Stent Implantation for High Risk Patients and Novel Technologies in Percutaneous Coronary Intervention

Percutaneous coronary intervention is a major treatment strategy for patients with coronary artery disease, and currently coronary stents are widely used in the world.1 Although stent implantation itself has shown to reduce restenosis by preventing both early elastic recoil and late vascular remodeling compared to balloon angioplasty, in-stent restenosis (ISR) still occurs in 10-40% of patients and has been the ‘Achilles’ heel’ of coronary interventions, frequently resulting in repeated revascularization.2,3 Restenosis after coronary stenting occurs secondary to the accumulation of smooth muscle cells and extracellular matrix proteoglycans.4 Despite the sophistication of the new techniques and enormous advance in devices, ISR requiring repeat procedure has been considered as a main limitation of coronary stenting. The advent of drug eluting stents (DES), which consist of a drug (immunosuppressive or antiproliferative drug), a polymer and a metallic platform, has revolutionized the practice of interventional cardiology by significantly reducing the rates of restenosis and repeat revascularization as compared to bare metal stents.5 After the first approval of DES, a large number of patients with coronary artery disease have undergone percutaneous revascularization with DES. However, many trials conducted in the ‘real world’ showed that the problem of restenosis was not completely resolved and still persists. Effect of DES for patients at high risk for ISR, such as acute myocardial infarction, small coronary vessels, aorto-ostial lesions, or lesions of chronic total occlusion (Part 1 of this thesis), have not been fully investigated. In addition, certain potential safety concerns regarding the widespread use of DES have arisen. The most notable drawback of DES is that they could increase the risk of thrombotic complication, especially late stent thrombosis6, although its incidence is low.7 The increased risk of thrombosis with DES utilization may be associated with altered endothelial function8 and/or delayed vascular healing9 induced by cytotoxic and cytostatic drug use. Localized hypersensitivity reactions to the polymer coating of DES and drug itself may also contribute to stent thrombosis.10 To retain the positive clinical aspects of DES and overcome their drawbacks, new concept stents have been developed (Part 2 of this thesis)

The implementation of pharmacogenetics: evidence and preferences

Pharmacogenetics has huge potential to transform the field of medicine and deliver personalised treatments to patients. However, its wider use is limited by many factors, particularly a lack of suitable evidence of efficacy or safety for regulatory approval and clinical use. The evidence required can be difficult to ascertain, presenting three main problems. The first issue is that regulatory guidance for the evidence required is complex and varies greatly between different authorities and contexts. Guidance from the UK Medicines and Healthcare products Regulatory Authority (MHRA) and the US Food and Drug Administration (FDA) was reviewed along with criteria formulated by other industry and academic groups. It was found that there is a clear need for a unified set of standards for evidence gathering in pharmacogenetics. This was strengthened by an analysis of the evidence used by five different randomised controlled trials to justify the inclusion of their pharmacogenetic biomarker. Large variation in the quality and type of this evidence was found. These findings were used to make recommendations for future evidence gathering for trials, regulators, and journals. Additionally, the evidence required for clinical implementation has traditionally been the prospective randomised controlled trial. Gathering information from two novel systematic reviews and meta-analyses of carbamazepine-induced Stevens-Johnson syndrome, it was shown how these sources of observational evidence can produce effect estimates and measures of clinical validity of greater precision than that of a prospective trial. Finally, the level of evidence for a pharmacogenetic test that would be acceptable to the general public is not known. A discrete choice experiment (DCE) was designed to quantify these views. The first step was a systematic review of existing DCEs in this area, to extract useful information from these to inform the work. An extensive programme of qualitative work with healthcare professionals, patients, and the general public then further informed the design of this novel DCE. Participants were randomised to complete one of eight DCEs in different disease areas, with either a ‘high’ evidence scenario or a ‘low’ evidence scenario described. Launched in May 2021, over 2,000 responses were collected and the results were analysed in preference-weighted utility models. Although there was no difference in utility between ‘high’ and ‘low’ evidence tests, several important insights were generated (particularly in regard to data sharing and privacy) that will potentially have large impacts on policy in this area