Screening methodologies for the determination of water contaminant residues by compact disk technology

[EN] The contamination of water resources with many industrial, agricultural and other chemicals is one of the key environmental problems that humanity is facing nowadays. Despite the fact that they are usually present at very low concentration, they possess a significant risk to aquatic and human life. To address this issue many national and international institutions set different regulations to monitor and control the water quality. Currently, the monitoring of compounds included in official watch lists is conducted by chromatographic and mass spectroscopic methods. These techniques are approved as "gold standards" for analytical quantitation of organic residues in water. Although they are sensitive and reproducible, cannot be used on-site. The need of sampling and centralized laboratory measurements makes not only the overall cost high but lowering the efficiency of the analysis. Therefore, there is an urgent need to develop suitable field methods to facilitate the in situ measurements at a low cost. Biosensors are therefore an alternative technology that can provide sensitive results in a fast and affordable way. This thesis has focused on the development of a biosensor based on immunoassays and compact disk technology, for the multiplex detection of priority water contaminants. As the methods based on the immunorecognition events are challenging in terms of the selectivity and sensitivity, the major part of the thesis was the selection of the immunoreagents, assay form and procedure. For the detection part, gold nanostructures were selected as sensitive tags for signal enhancement. Therefore, different nanoparticles were studied in order to select the optimal size in terms of the signal enhancement, sensitivity and antibody amount used. Also, the assays performances with signal enhancement and without any amplification were evaluated. The best immunoassay was selected for developing the multiplexed assay. Furthermore, an approach to improve the readout sensitivity of microimmunoassays based on used of gold nanoparticles as both capture and detection species was demonstrated. The method is based on the performance of the immunorecognition event in a homogeneous mode and detection part in the heterogeneous format. Finally, representative water samples were analysed to confirm the applicability of the multi-residue assay. The analytical properties have been established for each methodology and the obtained results have been validated by comparison with reference techniques. The investigations carried out in this work, have resulted in new insights in immunoassay technique that could be useful for screening applications.[ES] La contaminación de aguas superficiales causada por plaguicidas y productos industriales es actualmente, uno de los grandes problemas medioambientales. Aunque estas sustancias están presentes a niveles muy bajos, tienen efectos perjudiciales en el medio en general y especialmente en humanos. Por este motivo, diferentes instituciones han regulado los niveles de contaminantes en áreas de controlar de la calidad de las aguas, creando listas prioritarias de sustancias peligrosas y tóxicas para el medio ambiente. Actualmente, la monitorización de los contaminantes incluidos en las listas oficiales se realiza mediante técnicas cromatográficas y espectrometría. Estos métodos analíticos están aprobados como técnicas de referencia para la determinación de residuos orgánicos presentes en aguas naturales. A pesar de ser técnicas fiables, reproducibles y sensibles, los métodos cromatográficos no están exentos de inconvenientes. Este tipo de metodologías requiere una instrumentación costosa y una laboriosa preparación de muestra, que hacen que el análisis sea en general complejo. Por ello, el desarrollo de métodos analíticos alternativos que faciliten hacer medidas in-situ a bajo coste y con gran capacidad de trabajo es de gran utilidad. En este sentido, las técnicas inmunoquímicas tienen un gran potencial analítico ya que son en general sensibles y selectivas, se pueden utilizar en el lugar de la toma de muestra y tienen capacidad multianalito. Esta tesis se ha centrado en el desarrollo de un sistema biosensor, basado en la tecnología de disco compacto, para la detección multianalito de diversos contaminantes prioritarios en aguas naturales. Las limitaciones más críticas para el desarrollo de un biosensor multianalito mediante métodos inmunoquímicos son los relacionados con su sensibilidad y selectividad. Por lo tanto, una parte importante de la tesis se ha centrado en la selección de inmunoreactivos, formato y optimización de diferentes parámetros claves del ensayo. Una estrategia utilizada para aumentar la sensibilidad de los ensayos ha consistido en marcar la inmunoreacción con nanopartículas de oro. Para ello, se ha estudiado diferentes tipos (esféricas y cilíndricas) de distinto tamaño y se han comparado sus prestaciones analíticas (relación señal ruido, sensibilidad etc.) También, se han desarrollado inmunoensayos cuantitativos sin necesidad de amplificación de la señal. Por otro lado, se ha desarrollado una aproximación que hemos denominado "inmunocaptura" basada en el uso de nanopartículas de oro como especie de captura de analitos en disolución y que actúa como marcador de la inmunointeracción que tiene lugar en la fase sólida. Finalmente, se han analizado muestras de agua naturales dopadas con distintos niveles de los analitos objeto de estudio para evaluar la utilidad de las metodologías desarrolladas como herramienta de screening masivo en el área medioambiental. Los resultados obtenidos han sido comparados con los obtenidos mediante las técnicas de referencia. Las investigaciones realizadas han permitido desarrollar nuevos formatos de ensayo y conocimientos inmunoquímicos aplicados a la tecnología de disco compacto, aportando nuevas herramientas de screening que permiten la determinación simultánea de contaminantes en aguas naturales por debajo de las concentraciones establecidas en la normativa europea de calidad de agua.[CA] La contaminació d'aigües superficials causada principalment per plaguicides i altres productes industrials és un dels grans problemes mediambientals actuals. Malgrat que aquestes substàncies estan presents en nivells molt baixos, tenen efectes perjudicials en humans i animals. Per aquest motiu, diferents institucions estatals han regulat els nivells de contaminants en àrees de control de la qualitat de l'aigua, creant llistes prioritàries de substàncies perilloses i tòxiques per al medi ambient. Actualment, la monitorització dels contaminants inclosos en les llistes oficials es realitza mitjançant tècniques cromatogràfiques i d'espectroscòpia de masses. Aquests mètodes analítics estan aprovats com a tècniques de referència per a la determinació de residus orgànics presents en aigües naturals. Malgrat ser tècniques fiables, reproduïbles i sensibles, els mètodes cromatogràfics no estan exempts d'inconvenients. Aquest tipus de metodologies requereix una instrumentació costosa i una laboriosa preparació de mostres que fan que l'anàlisi sigui, en general, complex. Per això, el desenvolupament de mètodes analítics alternatius que facilitin la possibilitat de fer mesures in-situ a baix cost i amb gran capacitat analítica és de gran utilitat. En aquest sentit, les tècniques inmunoquímiques tenen un gran potencial analític ja que són, en general, sensibles i selectives, es poden utilitzar en el lloc de presa de la mostra i tenen capacitat multianalit. Aquesta tesi s'ha centrat en el desenvolupament d'un sistema biosensor, basat en la tecnologia de disc compacte, per a la detecció multianalit de diversos contaminants prioritaris en aigües naturals. Les limitacions més crítiques per al desenvolupament d'un biosensor multianalit mitjançant mètodes inmunoquímics són sensibilitat i selectivitat. Per tant, una part important de la tesi es va centrar en la selecció de inmunoreactius, format i optimització de diferents paràmetres clau de l'assaig. La detecció es va dur a terme mitjançant l'ús de nanopartícules d'or com a marcadors de la inmunointeracció i amplificació de la senyal analítica. S'han estudiat diferents estructures d'or (esferes i cilindres) de diferents tamanys, i s'han comparat les seves prestacions analítiques (relació senyal-soroll, sensibilitat, etc.). També s'han desenvolupat immunoassaigs quantitatius sense necessitat d'amplificació del senyal. Per altra banda, s'ha desenvolupat una aproximació que hem denominat "inmunocaptura", basada en l'ús de nanopartícules d'or com a espècie de captura d'analits en dissolució i que actua com a marcador de la inmunointeracció que té lloc en la fase sòlida. Finalment, s'han analitzat mostres d'aigües naturals dopades amb diferents nivells dels analits objecte d'estudi per avaluar la utilitat de les metodologies desenvolupades com a eina de "screening" massiu en l'àrea mediambiental. Els resultats obtinguts han sigut avaluats per comparació amb els obtinguts mitjançant tècniques de referència. Les investigacions realitzades han permès desenvolupar nous formats d'assaig i coneixements inmunoquímics aplicats a la tecnologia de disc compacte, aportant noves eines de "screening" que permetin la determinació de contaminants en aigües naturals per sota dels límits de concentració establerts per les normes internacionals de la qualitat de l'aigua.Dobosz, PD. (2017). Screening methodologies for the determination of water contaminant residues by compact disk technology [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79548TESI

On-a-chip biosensing based on all-dielectric nanoresonators

Nanophotonics has become a key enabling technology in biomedicine with great promises in early diagnosis and less invasive therapies. In this context, the unique capability of plasmonic noble metal nanoparticles to concentrate light on the nanometer scale has widely contributed to biosensing and enhanced spectroscopy. Recently, high-refractive index dielectric nanostructures featuring low loss resonances have been proposed as a promising alternative to nanoplasmonics, potentially offering better sensing performances along with full compatibility with the microelectronics industry. In this letter we report the first demonstration of biosensing with silicon nanoresonators integrated in state-of-the-art microfluidics. Our lab-on-a-chip platform enables detecting Prostate Specific Antigen (PSA) cancer marker in human serum with a sensitivity that meets clinical needs. These performances are directly compared with its plasmonic counterpart based on gold nanorods. Our work opens new opportunities in the development of future point-of-care devices towards a more personalized healthcare

Targeted therapy with histamine antagonists - New challenges to fight with breast cancer

Breast cancer (BC) is currently the most commonly diagnosed cancer in women. BC is most often derived from the epithelial tissue of the mammary gland and is a global problem due to the steady increase in morbidity and mortality in most countries. A particular problem today is the triple negative subtype (TNBC), which accounts for approximately 10-15% of breast cancer cases. BC occurs most frequently in young women and is characterised by various biological characteristics, an unfavourable clinical course and a poor prognosis. Recent studies to detect the effects of histamine receptors on breast cancer have shown that all H1R-H4R receptors are also hyperactive in the cancer microenvironment. Chronically maintaining a high level of histamine in the tumour-affected tissue contributes to increased angiogenesis at this site, induction of cancer cells proliferation and T lymphocyte dysfunction. The rising incidence of breast cancer is contributing to an increasing amount of research into targeted therapies. Studies on the effect of histamine antagonists through H1R-H4R receptors have proven their effectiveness in the treatment of breast cancer. Among those in the study, there was a reduction in tumour growth, cell proliferation and an increase in apoptosis. The use of histamine antagonists also contributed to a reduced risk of death from breast cancer and increased overall survival (OS). Therefore, targeted therapy is needed to improve the prognosis of patients with breast cancer.&nbsp

The impact of coffee on human health

Introduction: Coffee is one of the most popular beverages in the world. It contains lots of biologically active ingredients. These compounds not only have aromatic properties, but many of them also have antioxidant, hepatoprotective, anti-inflammatory, antimicrobial, antiviral and relaxing smooth muscle properties. The aim of the study: The purpose of the study is to collect and review scientific publications about the impact of coffee on health. Material and method: The paper uses standard criteria as the research method. Additionally, during the literature review on PubMed and Google Scholar platforms, keywords such as coffee, caffeine, health, impact were used. Description of the state of knowledge: Numerous studies suggest that coffee impact on the long-term functioning of the organism is negligible and is associated with the consumption of coffee for a longer period of time. Chlorogenic acid, caffeine and trigoneline are primarily responsible for the positive effect. They have a hypoglycemic, bactericidal and antioxidant effect. Diterpenes, such as kahweol and cafestol may have negative influence on health. Research also suggests that coffee can reduce the risk of Parkinson's disease and type 2 diabetes. Summary: The results show that coffee ingredients can have both positive and negative effects on health. However, before these observations can be used to create nutritional advice, further research is needed. They will allow a better understanding of the mechanisms of action of the compounds responsible for reduced risk of diabetes mellitus or Parkinson's disease. Keywords: coffee, caffeine, health, cardiovascula

Cryo-EM structures of the human Elongator complex at work

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action

Cryo-EM structure of the fully assembled elongator complex

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes

Security theory and practice: Germany towards security problems and international cooperation in the 21st century

Z wprowadzenia: "Pierwsze ćwierćwiecze XXI wieku obfituje w dyskusje na temat wagi i znaczenia wyzwań międzynarodowych. Również niemieccy politycy prezentują strategie i programy osiągania przez Niemcy celów i interesów na arenie międzynarodowej. Ocena realizacji niemieckiej polityki bezpieczeństwa oraz poziomu współpracy międzynarodowej wymaga uwzględnienia wielu czynników o charakterze podmiotowym i przedmiotowym. Minister obrony Ursula von der Leyen proponowała „przywództwo z centrum”, a prezydent Joachim Gauck postulował zwiększenie „odpowiedzialności międzynarodowej” Niemiec. Podczas gdy minister spraw zagranicznych Guido Westerwelle opowiedział się za trwaniem przy „kulturze wstrzemięźliwości”, socjaldemokraci Sigmar Gabriel i Frank-Walter Steinmeier optowali za polityką zaangażowania Niemiec. Kanclerz Angela Merkel odnosząc się do dyskusji dotyczących rozbrojenia podkreślała, że odstraszanie stanowi istotny element stabilizacji środowiska międzynarodowego i postulowała realistyczne podejście do problematyki zbrojeń."(...

The molecular basis of tRNA selectivity by human pseudouridine synthase 3

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Peer reviewe

Gold-nanoparticle based immunoassay on a Digital Versatile Disk for the determination of priority pollutants residues in water

The aim of this work is the development of an immunoassay adapted for compact disc technology to determine the priority pollutants residues in natural water. For that, specific antibody will be labeled with gold nanoparticles and the immunoreaction would be detected in one reaction step. As a proof of concept, the immunoassay for the determination of chlorpyrifos will be developed. Different aspects of the immunoassay protocol have to be considered. First, gold nanoparticles of different sizes should be synthesized. Secondly, as particular aims, the following points should be evaluated: - the influence of nanoparticles size on the conjugation efficiency and assay sensitivity - the influence of sample volume on the reproducibility of the signal intensity - the influence of immunoreaction time on the sensitivity and reproducibility of the assay. Finally, the performance of the DVD microimmunoassay method should to be evaluated through the determination of organic pollutants residues in water samples. The immunoassay should achieve the detection limit established in E.U Water Framework Directive.Dobosz, P. (2013). Gold-nanoparticle based immunoassay on a Digital Versatile Disk for the determination of priority pollutants residues in water. Universitat Politècnica de València. http://hdl.handle.net/10251/63122Archivo delegad