Redox regulation of photosynthetic metabolism in chloroplasts of Arabidopsis thaliana

En la tesis existe un contrato de cesión de derechos que afecta a los capítulos 1, 2, 3, 4 y 5La regulación redox basada en intercambio disulfuro-ditiol es un mecanismo regulatorio universal que afecta a la conformación y actividad de las proteínas. En organismos heterótrofos, la regulación redox se lleva a cabo mediante un sistema de dos componentes, tioredoxina (Trx) y NADPH tioredoxina reductasa (NTR), el denominado sistema NTR/Trx. Por el contrario, la regulación redox en los cloroplastos de plantas muestra una mayor complejidad. Además de un número elevado de tioredoxinas, alrededor de 20 en el cloroplasto de Arabidopsis thaliana, este orgánulo posee dos sistemas de regulación redox: la vía clásica, donde la ferredoxina (Fd), reducida fotosintéticamente, reduce el set de Trxs del cloroplasto por medio de la actividad de una Fd Trx reductasa (FTR), conocido como sistema FTR/Trx; y el sistema NTRC, enzima que presenta un dominio NTR y un dominio Trx en el mismo polipéptido, y que utiliza el poder reductor del NADPH. En esta tesis doctoral se ha llevado a cabo una serie de aproximaciones genéticas, fisiológicas y bioquímicas, para abordar el estudio de la relación funcional que existe entre ambos sistemas redox, así como su implicación en la regulación de enzimas del cloroplasto, y el impacto de estos sistemas en el desarrollo de la planta. A lo largo de este trabajo, se ha descrito que NTRC es esencial para la actividad de Trxs con funciones diferentes, como la Trx x o la Trx f. También hemos demostrado el papel clave de NTRC en la regulación redox del cloroplasto, siendo crítico durante los primeros estadíos del desarrollo de la planta. Previamente ha sido descrito que NTRC es la principal enzima reguladora de la 2-cisteina peroxiredoxina (2-Cys Prx), enzima capaz de reducir el peróxido de hidrógeno a agua. En esta tesis se ha determinado el papel clave que juegan las 2-Cys Prxs en la homeostasis redox del cloroplasto, integrando las actividades de los sistemas FTR/Trx y NTRC. Aunque se conocen bien los mecanismos moleculares del proceso de reducción de las enzimas del cloroplasto durante el día, se desconoce cómo se produce la oxidación de estas enzimas durante la oscuridad. En esta tesis se ha elucidado el papel clave que las 2-Cys Prxs juegan en este proceso, siendo el peróxido de hidrógeno el aceptor final de electrones.Premio Extraordinario de Doctorado U

Metabolic implications and characterization of hepatic extracellular vesicles in drug-induced liver injury

170 p.El daño hepático inducido por drogas (DILI) es un grave problema en las sociedadesdesarrolladas, siendo el responsable de más del 50% de los casos de fallo hepático agudo. Unárea de gran interés, tanto en el diagnóstico clínico como en la industria farmacéutica, es labúsqueda de marcadores no invasivos, tanto para el diagnóstico de esta patología, como paradilucidar el mecanismo molecular que se encuentra tras ella. En este sentido, eldescubrimiento de vesículas extracelulares (EVs) secretadas por células, ha proporcionado unnuevo componente celular con la posibilidad de ser un agente activo en diferentes procesostanto fisiológicos como patológicos. Estas vesículas han sido detectadas tanto en muestras desangre como de orina, proporcionando una nueva plataforma para la identificación demarcadores no invasivos de enfermedades de diversa índole. Estas vesículas son secretadaspor la mayoría de tipos celulares, incluyendo el hepatocito. Sin embargo, a pesar de la intensainvestigación llevada a cabo en estas vesículas, actualmente poco se conoce acerca de supapel fisiológico y de su implicación en el desarrollo de enfermedades.Los hepatocitos secretan EVs que contienen enzimas implicadas en el metabolismo de drogas,sugiriendo una posible implicación de estas vesículas en el desarrollo de DILI. En estesentido, en esta tesis doctoral se ha caracterizado la secreción de EVs hepáticas en diferentesmodelos de DILI in vitro mediante el uso de técnicas tanto bioquímicas como moleculares.Los resultados obtenidos muestran que el contenido de las EVs hepáticas es sensible a losdiferentes modelos de DILI empleados, lo que podría ser de utilidad para fines diagnósticos.Además, las EVs hepáticas contienen diversas enzimas activas relacionadas con DILIincluyendo: la carboxylesterasa (CES), methionine adenosyl transferasa (MAT) y catechol Omethyltransferasa(COMT). En base a la caracterización mediante UPLC-MS del metabolomaasociado a EVs hepáticas, se han detectado diversas rutas celulares así como funcionessusceptibles de ser modificadas mediante estas vesículas. Un ejemplo de ello son el ciclo de lametionina, el metabolismo enérgético, la síntesis de purinas y ADN o el control de la presiónsanguínea local.En conclusión, los resultados obtenidos apoyan que las EVs hepáticas podrían estarimplicadas tanto en DILI como en la respuesta a otro tipo de daño hepático. Asimismo,también se presentan varias enzimas y moléculas como candidatos a marcadores de bajainvasividad para la detección de DIL

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite ( SeO3 2- ) into the less bioavailable elemental selenium (Se0 ) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of SeO3 2- exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with SeO3 2- . The results show substantial biochemical changes in SeITE01 cells when exposed to SeO3 2- . The initial uptake of SeO3 2- by SeITE01 cells (3 h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against SeO3 2- are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of SeO3 2- to Se0 . In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from SeO3 2- toxicity while it is converting into SeNPs

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite ((Formula presented.)) into the less bioavailable elemental selenium (Se0) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of (Formula presented.) exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with (Formula presented.). The results show substantial biochemical changes in SeITE01 cells when exposed to (Formula presented.). The initial uptake of (Formula presented.) by SeITE01 cells (3h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against (Formula presented.) are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of (Formula presented.) to Se0. In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from (Formula presented.) toxicity while it is converting into SeNPs

Antisense Therapy

Antisense Therapy offers a comprehensive, state-of-the art perspective on the role of antisense therapy in the treatment of human disease, with a special focus on cancer. Use of antisense oligonucleotides is a growing field of pharmaceutical and biotech companies and research programs for treatment of several diseases. This book summarizes and presents the best updates, therapeutic principles, methods, and applications in the field and offers meaningful information to move treatment discovery forward

Bryophytes: how to conquer an alien planet and live happily (ever after)

There are many push and pull factors that commonly drive individuals to leave their homeland. For example, escaping competition and occupying a novel habitat undoubtedly offer the advantage of new opportunities to pilgrims, but the absence of unfavorable biotic interactions can be counterbalanced by other antagonistic abiotic forces. After all, conquering an alien planet is not now nor ever was an easy task. We cannot know how many attempts and failures have punctuated the journey that led ancestral, photosynthetic organisms to leave the aquatic world and successfully establish on dry land. However, some traits developed by the ancestors of modern bryophytes that allowed them to adapt their life cycle to such a different habitat and persist there, have been undoubtedly identified

Eukaryotic Ribosome assembly and Nucleocytoplasmic Transport

The process of eukaryotic ribosome assembly stretches across the nucleolus, the nucleoplasm and the cytoplasm, and therefore relies on efficient nucleocytoplasmic transport. In yeast, the import machinery delivers ~140,000 ribosomal proteins every minute to the nucleus for ribosome assembly. At the same time, the export machinery facilitates translocation of ~2000 pre-ribosomal particles every minute through ~200 nuclear pore complexes (NPC) into the cytoplasm. Eukaryotic ribosome assembly also requires >200 conserved assembly factors, which transiently associate with pre-ribosomal particles. Their site(s) of action on maturing pre-ribosomes are beginning to be elucidated. In this chapter, we outline protocols that enable rapid biochemical isolation of pre-ribosomal particles for single particle cryo-electron microscopy (cryo-EM) and in vitro reconstitution of nuclear transport processes. We discuss cell-biological and genetic approaches to investigate how the ribosome assembly and the nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. Keywords: Budding Yeast; Nuclear Export; Nuclear Import; Ribosome Assembly; preribosome structure

Ribosome Biogenesis

This Open Access volume provides comprehensive reviews and describes the latest techniques to study eukaryotic ribosome biogenesis. For more than 50 years ribosomes are a major research topic. Our knowledge about ribosome biogenesis and function such as transcription, mRNA modification, and translation was the sine qua non for developing the powerful RNA-based vaccines against RNA-viruses causing the world-threatening Covid-19 pandemia. The chapters in this book are organized into six parts. Part One discusses a comparative survey about the unity and diversity of ribosome biogenesis in pro- and eukaryotic cells. Part Two deals with the genomic organization of eukaryotic rDNA and the role of RNA polymerase I in ribosomal RNA transcription. Part Three explores in vitro methods to study RNA polymerase I structure and its function, and Part Four analyzes the nucleo-cytoplasmic transport of assembled ribosomes and RNP complexes. Part Five covers modifications that increase the complexity of rRNAs, and Part Six provides readers with a review of eukaryotic translation and - for the first time - describes a new method to analyze translation in vitro. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Ribosome Biogenesis: Methods and Protocols is a valuable resource for scientists and researchers interested in learning more about the increasing importance of in vitro RNA-technologies

Challenges and Opportunities in the Present Era of Marine Algal Applications

Marine algae are of high importance in their natural habitats and even more now in the world of green technology. The sprouting interest of the scientific community and industries in these organisms is driven by the fast-growing world of modern biotechnology. Genomics, transcriptomics, proteomics, metabolomics and their integration collectively termed here as ‘marine algal-omics’ have broadened the research horizon in view of enhancing human’s life by addressing environmental problems and encouraging novelty in the field of pharmaceuticals among so many more. Their use in the human society dates back to 500 B. C. in China and later across the globe; they are still being used for similar purposes and more today. There is a hiking interest in marine algae and their derivatives—from phycoremediation, food supplements, pharmaceuticals to dyes. Marine algae are currently considered as an emerging panacea for the society. They are being studied in a multitude of arenas. The multi-use of marine algae is enticing and promises to be a boon for industrial applications. Yet, most marine algae face challenges that might variably constrain their commercialisation. This chapter gives an overview of marine algae including all the ‘omics’ technologies involved in studying marine algae and it explores their multitude applications. It also draws the various successful industries budded around them and presents some of the challenges and opportunities along with future directions