5,389 research outputs found
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Ensuring Access to Safe and Nutritious Food for All Through the Transformation of Food Systems
The development of ALICE-tRNA-sequencing and its use in exploring the role of tRNAs in translational control
Sustaining proliferative signalling and loss of translational control is arguably the most fundamental trait of cancer cells, enabling tumour growth and metastatic dissemination. Transfer RNAs (tRNAs) have long been considered abundant “housekeeping” RNAs, functioning to decipher the universal genetic code. However, exhaustive analyses have implicated tRNA participation in a host of regulatory networks including the cellular stress response and protein synthesis. Recent findings suggest that the expression of tRNAs for synonymous codon usage is dependent on the differentiation/proliferation status of the cell and are coordinated with changes in translation. Although the molecular mechanisms that govern these changes are yet to be elucidated, cellular tRNA composition potentially introduces an additional layer of translational control. tRNAs are the most post-transcriptionally modified RNA species, with well over 50 unique modifications identified in eukaryotes. Consequently, isoacceptor identification and the measuring of the tRNA pool using next generation sequencing has long been an area of interest, with many attempts being made in literature. Using the Escherichia coli dealkylating enzyme AlkB and the novel tRNA high throughput sequencing methodology ALICE-tRNA-seq, we have developed a methodology that can accurately measure relative tRNA pools in vitro and in vivo. We show how other published tRNA sequencing protocols show bias towards tRNA sub populations, with our method showing a more realistic distribution across all tRNAs. We also show relative distribution changes in cellular and genetically modified mouse models of cancer, opening up a high resolution approach to establish the role of tRNAs in translational control and cell fate decisions
Development of in-vitro in-silico technologies for modelling and analysis of haematological malignancies
Worldwide, haematological malignancies are responsible for roughly 6% of all the cancer-related deaths. Leukaemias are one of the most severe types of cancer, as only about 40% of the patients have an overall survival of 10 years or more. Myelodysplastic Syndrome (MDS), a pre-leukaemic condition, is a blood disorder characterized by the presence of dysplastic, irregular, immature cells, or blasts, in the peripheral blood (PB) and in the bone marrow (BM), as well as multi-lineage cytopenias.
We have created a detailed, lineage-specific, high-fidelity in-silico erythroid model that incorporates known biological stimuli (cytokines and hormones) and a competing diseased haematopoietic population, correctly capturing crucial biological checkpoints (EPO-dependent CFU-E differentiation) and replicating the in-vivo erythroid differentiation dynamics. In parallel, we have also proposed a long-term, cytokine-free 3D cell culture system for primary MDS cells, which was firstly optimized using easily-accessible healthy controls. This system enabled long-term (24-day) maintenance in culture with high (>75%) cell viability, promoting spontaneous expansion of erythroid phenotypes (CD71+/CD235a+) without the addition of any exogenous cytokines. Lastly, we have proposed a novel in-vitro in-silico framework using GC-MS metabolomics for the metabolic profiling of BM and PB plasma, aiming not only to discretize between haematological conditions but also to sub-classify MDS patients, potentially based on candidate biomarkers. Unsupervised multivariate statistical analysis showed clear intra- and inter-disease separation of samples of 5 distinct haematological malignancies, demonstrating the potential of this approach for disease characterization.
The work herein presented paves the way for the development of in-vitro in-silico technologies to better, characterize, diagnose, model and target haematological malignancies such as MDS and AML.Open Acces
Hunting Wildlife in the Tropics and Subtropics
The hunting of wild animals for their meat has been a crucial activity in the evolution of humans. It continues to be an essential source of food and a generator of income for millions of Indigenous and rural communities worldwide. Conservationists rightly fear that excessive hunting of many animal species will cause their demise, as has already happened throughout the Anthropocene. Many species of large mammals and birds have been decimated or annihilated due to overhunting by humans. If such pressures continue, many other species will meet the same fate. Equally, if the use of wildlife resources is to continue by those who depend on it, sustainable practices must be implemented. These communities need to remain or become custodians of the wildlife resources within their lands, for their own well-being as well as for biodiversity in general. This title is also available via Open Access on Cambridge Core
Investigating PAX6 and SOX2 dynamic interactions at the single molecule level in live cells
The abundance of transcription factor (TF) molecules in the nuclei of
eukaryotic cells are in the range of thousands. However, the functional binding
sites of most TFs lie in the range of hundreds. This suggests that there is a
surplus of the number of molecules for many TFs, relative to their binding sites
at any given time. Nevertheless, precise TF levels are instrumental for normal
development and maintenance, with haploinsufficiency (namely lowering the
dosage of a TF by half) being a hallmark of many TF-related human
developmental disorders. Qualitative methods assessing TF binding such as
chromatin immunoprecipitation, provide static information, from fixed cell
populations and so fail to provide insight into TF dynamic behaviour. Live-cell
imaging methodologies such as Fluorescence Correlation Spectroscopy
(FCS) offer the ability to measure kinetics of binding to chromatin, protein-protein interactions, absolute concentrations of molecules and the underlying
cell-to-cell variability.
SOX2 and PAX6 TFs exhibit haploinsufficiency in humans. Heterozygous point
mutations, deletions or insertions in these genes can lead to a plethora of
abnormal ocular developmental disorders (e.g. coloboma, aniridia,
microphthalmia, anopthalmia). SOX2 encodes a high-mobility group (HMG)
domain-containing TF, essential for maintaining self-renewal of embryonic
stem cells and is expressed in proliferating central nervous system (CNS)
progenitors. PAX6 contains two DNA binding domains; a PAIRED domain (PD)
and a homeodomain (HD). Both DNA binding domains present in PAX6 (PD
and HD) can function either jointly, or separately, to regulate a plethora of
genes implicated in the development and maintenance of the CNS, the eye
and the pancreas. Despite existing genetic and phenotypic evidence, it
remains unclear how PAX6 and SOX2 influence each other at the molecular
level and how sensitive their stoichiometry is during ocular development.
In this thesis I investigated the dynamic interplay between PAX6/SOX2 and
chromatin in live cells, at the molecular level. I compared wild-type protein
function with pathogenic missense variants using advanced fluorescence
microscopy techniques and assessed how these mutations quantitatively and
qualitatively affected molecular behaviour. My results showed that both SOX2
and PAX6 pathogenic missense mutants display differential subnuclear
localisation, as well as altered protein-protein and protein-chromatin
interactions, linking molecular diffusion to pathogenic phenotype in humans.
More importantly, I identified a novel role of SOX2 in stabilising PAX6-
chromatin complexes in live cells, providing further insight into the complex
and dynamic relation of PAX6 and SOX2 in ocular tissue specification,
maintenance and development
Novel Cardiac Mapping Approaches and Multimodal Techniques to Unravel Multidomain Dynamics of Complex Arrhythmias Towards a Framework for Translational Mechanistic-Based Therapeutic Strategies
[ES] Las arritmias cardíacas son un problema importante para los sistemas de salud en el mundo desarrollado debido a su alta incidencia y prevalencia a medida que la población envejece. La fibrilación auricular (FA) y la fibrilación ventricular (FV) se encuentran entre las arritmias más complejas observadas en la práctica clínica. Las consecuencias clínicas de tales alteraciones arrítmicas incluyen el desarrollo de eventos cardioembólicos complejos en la FA, y repercusiones dramáticas debido a procesos fibrilatorios sostenidos que amenazan la vida infringiendo daño neurológico tras paro cardíaco por FV, y que pueden provocar la muerte súbita cardíaca (MSC). Sin embargo, a pesar de los avances tecnológicos de las últimas décadas, sus mecanismos intrínsecos se comprenden de forma incompleta y, hasta la fecha, las estrategias terapéuticas carecen de una base mecanicista suficiente y poseen bajas tasas de éxito.
Entre los mecanismos implicados en la inducción y perpetuación de arritmias cardíacas, como la FA, se cree que las dinámicas de las fuentes focales y reentrantes de alta frecuencia, en sus diferentes modalidades, son las fuentes primarias que mantienen la arritmia. Sin embargo, se sabe poco sobre los atractores, así como, de la dinámica espacio-temporal de tales fuentes fibrilatorias primarias, específicamente, las fuentes focales o rotacionales dominantes que mantienen la arritmia. Por ello, se ha desarrollado una plataforma computacional, para comprender los factores (activos, pasivos y estructurales) determinantes, y moduladores de dicha dinámica. Esto ha permitido establecer un marco para comprender la compleja dinámica de los rotores con énfasis en sus propiedades deterministas para desarrollar herramientas basadas en los mecanismos para ayuda diagnóstica y terapéutica.
Comprender los procesos fibrilatorios es clave para desarrollar marcadores y herramientas fisiológica- y clínicamente relevantes para la ayuda de diagnóstico temprano. Específicamente, las propiedades espectrales y de tiempo-frecuencia de los procesos fibrilatorios han demostrado resaltar el comportamiento determinista principal de los mecanismos intrínsecos subyacentes a las arritmias y el impacto de tales eventos arrítmicos. Esto es especialmente relevante para determinar el pronóstico temprano de los supervivientes comatosos después de un paro cardíaco debido a fibrilación ventricular (FV).
Las técnicas de mapeo electrofisiológico, el mapeo eléctrico y óptico cardíaco, han demostrado ser recursos muy valiosos para dar forma a nuevas hipótesis y desarrollar nuevos enfoques mecanicistas y estrategias terapéuticas mejoradas. Esta tecnología permite además el trabajo multidisciplinar entre clínicos y bioingenieros, para el desarrollo y validación de dispositivos y metodologías para identificar biomarcadores multi-dominio que permitan rastrear con precisión la dinámica de las arritmias identificando fuentes dominantes y atractores con alta precisión para ser dianas de estrategias terapeúticas innovadoras. Es por ello que uno de los objetivos fundamentales ha sido la implantación y validación de nuevos sistemas de mapeo en distintas configuraciones que sirvan de plataforma de desarrollo de nuevas estrategias terapeúticas. Aunque el mapeo panorámico es el método principal y más completo para rastrear simultáneamente biomarcadores electrofisiológicos, su adopción por la comunidad científica es limitada principalmente debido al coste elevado de la tecnología. Aprovechando los avances tecnológicos recientes, nos hemos enfocado en desarrollar, y validar, sistemas de mapeo óptico de alta resolución para registro panorámico cardíaco, utilizando modelos clínicamente relevantes para la investigación básica y la bioingeniería.[CA] Les arítmies cardíaques són un problema important per als sistemes de salut del món desenvolupat a causa de la seva alta incidència i prevalença a mesura que la població envelleix. La fibril·lació auricular (FA) i la fibril·lació ventricular (FV), es troben entre les arítmies més complexes observades a la pràctica clínica. Les conseqüències clíniques d'aquests trastorns arítmics inclouen el desenvolupament d'esdeveniments cardioembòlics complexos en FA i repercussions dramàtiques a causa de processos fibril·latoris sostinguts que posen en perill la vida amb danys neurològics posteriors a la FV, que condueixen a una aturada cardíaca i a la mort cardíaca sobtada (SCD). Tanmateix, malgrat els avanços tecnològics de les darreres dècades, els seus mecanismes intrínsecs s'entenen de forma incompleta i, fins a la data, les estratègies terapèutiques no tenen una base mecanicista suficient i tenen baixes taxes d'èxit.
La majoria dels avenços en el desenvolupament de biomarcadors òptims i noves estratègies terapèutiques en aquest camp provenen de tècniques valuoses en la investigació de mecanismes d'arítmia. Entre els mecanismes implicats en la inducció i perpetuació de les arítmies cardíaques, es creu que les fonts primàries subjacents a l'arítmia són les fonts focals reingressants d'alta freqüència dinàmica i AF, en les seves diferents modalitats. Tot i això, se sap poc sobre els atractors i la dinàmica espaciotemporal d'aquestes fonts primàries fibril·ladores, específicament les fonts rotacionals o focals dominants que mantenen l'arítmia. Per tant, s'ha desenvolupat una plataforma computacional per entendre determinants actius, passius, estructurals i moduladors d'aquestes dinàmiques. Això va permetre establir un marc per entendre la complexa dinàmica multidomini dels rotors amb ènfasi en les seves propietats deterministes per desenvolupar enfocaments mecanicistes per a l'ajuda i la teràpia diagnòstiques.
La comprensió dels processos fibril·latoris és clau per desenvolupar puntuacions i eines rellevants fisiològicament i clínicament per ajudar al diagnòstic precoç. Concretament, les propietats espectrals i de temps-freqüència dels processos fibril·latoris han demostrat destacar un comportament determinista important dels mecanismes intrínsecs subjacents a les arítmies i l'impacte d'aquests esdeveniments arítmics. Mitjançant coneixements previs, processament de senyals, tècniques d'aprenentatge automàtic i anàlisi de dades, es va desenvolupar una puntuació de risc mecanicista a la aturada cardíaca per FV.
Les tècniques de cartografia òptica cardíaca i electrofisiològica han demostrat ser recursos inestimables per donar forma a noves hipòtesis i desenvolupar nous enfocaments mecanicistes i estratègies terapèutiques. Aquesta tecnologia ha permès durant molts anys provar noves estratègies terapèutiques farmacològiques o ablatives i desenvolupar mètodes multidominis per fer un seguiment precís de la dinàmica d'arrímies que identifica fonts i atractors dominants. Tot i que el mapatge panoràmic és el mètode principal per al seguiment simultani de paràmetres electrofisiològics, la seva adopció per part de la comunitat multidisciplinària d'investigació cardiovascular està limitada principalment pel cost de la tecnologia. Aprofitant els avenços tecnològics recents, ens centrem en el desenvolupament i la validació de sistemes de mapes òptics de baix cost per a imatges panoràmiques mitjançant models clínicament rellevants per a la investigació bàsica i la bioenginyeria.[EN] Cardiac arrhythmias are a major problem for health systems in the developed world due to their high incidence and prevalence as the population ages. Atrial fibrillation (AF) and ventricular fibrillation (VF), are amongst the most complex arrhythmias seen in the clinical practice. Clinical consequences of such arrhythmic disturbances include developing complex cardio-embolic events in AF, and dramatic repercussions due to sustained life-threatening fibrillatory processes with subsequent neurological damage under VF, leading to cardiac arrest and sudden cardiac death (SCD). However, despite the technological advances in the last decades, their intrinsic mechanisms are incompletely understood, and, to date, therapeutic strategies lack of sufficient mechanistic basis and have low success rates.
Most of the progress for developing optimal biomarkers and novel therapeutic strategies in this field has come from valuable techniques in the research of arrhythmia mechanisms. Amongst the mechanisms involved in the induction and perpetuation of cardiac arrhythmias such AF, dynamic high-frequency re-entrant and focal sources, in its different modalities, are thought to be the primary sources underlying the arrhythmia. However, little is known about the attractors and spatiotemporal dynamics of such fibrillatory primary sources, specifically dominant rotational or focal sources maintaining the arrhythmia. Therefore, a computational platform for understanding active, passive and structural determinants, and modulators of such dynamics was developed. This allowed stablishing a framework for understanding the complex multidomain dynamics of rotors with enphasis in their deterministic properties to develop mechanistic approaches for diagnostic aid and therapy.
Understanding fibrillatory processes is key to develop physiologically and clinically relevant scores and tools for early diagnostic aid. Specifically, spectral and time-frequency properties of fibrillatory processes have shown to highlight major deterministic behaviour of intrinsic mechanisms underlying the arrhythmias and the impact of such arrhythmic events. Using prior knowledge, signal processing, machine learning techniques and data analytics, we aimed at developing a reliable mechanistic risk-score for comatose survivors of cardiac arrest due to VF.
Cardiac optical mapping and electrophysiological mapping techniques have shown to be unvaluable resources to shape new hypotheses and develop novel mechanistic approaches and therapeutic strategies. This technology has allowed for many years testing new pharmacological or ablative therapeutic strategies, and developing multidomain methods to accurately track arrhymia dynamics identigying dominant sources and attractors. Even though, panoramic mapping is the primary method for simultaneously tracking electrophysiological parameters, its adoption by the multidisciplinary cardiovascular research community is limited mainly due to the cost of the technology. Taking advantage of recent technological advances, we focus on developing and validating low-cost optical mapping systems for panoramic imaging using clinically relevant models for basic research and bioengineering.Calvo Saiz, CJ. (2022). Novel Cardiac Mapping Approaches and Multimodal Techniques to Unravel Multidomain Dynamics of Complex Arrhythmias Towards a Framework for Translational Mechanistic-Based Therapeutic Strategies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182329TESI
Learning from biology to design stimuli-responsive capsules
As well as being model cell membranes, lipid vesicles are widely used as an encapsulation technology due to their impermeable membrane. Hydrogels are similarly useful due to their biocompatibility, mechanical strength, and potential for stimulus-responsive behaviour.
By combining these two structures, the benefits of both can be reaped, giving a structure with both mechanical strength and the possibility to encapsulate actives within an impermeable membrane.
However, the interactions between the gel and membrane, and their implications, are not well understood. This thesis considers two different composite structures of lipid vesicles and hydrogels as potential systems for encapsulation and controlled release. These structures are hydrogel-embedded vesicles, and Gel-Filled Vesicles (GFVs). The hydrogel-embedded vesicles are subjected to different types of mechanical stresses. Osmotic shocks are used to apply a uniform pressure on the lipid bilayer, and compression of the hydrogel by a micromanipulator is used to cause a uni-directional force.
Agarose-embedded vesicles are shown to experience an adhesive interaction between the membrane and the gel, causing vesicle behaviours to be altered in comparison to free-floating vesicles. Of particular note is the formation of a buckled morphology for embedded vesicles subjected to hyperosmotic shocks.
Additionally, the formation of GFVs demonstrating the poration mechanism of controlled release is attempted. A suitable gel core of poly(acrylamide-co-acrylic acid) is synthesised and characterised for Upper Critical Solution Temperature behaviour.
In summary, this thesis demonstrates that interactions between the lipid bilayer and a hydrogel can strongly affect membrane behaviours, and therefore their uses for either encapsulation systems or for biophysical models
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Decellularised Normal and Tumour Scaffolds for Cancer Organoid Cultures as a Model of Colorectal Peritoneal Metastases
Peritoneal metastasis (PM) is one of the most common routes of dissemination for colorectal cancer and remains a lethal disease. PM development is caused by a cross-talk between invading cancer cells and the rearrangement of the extracellular matrix (ECM). This interplay is governed by biochemical and biomechanical events that allow the development of a specific microenvironment: the so-called metastatic niche. ECM remodeling may be critical for PM spread. In fact, it has been demonstrated that ECMs are not only able to provide structural support to the exfoliated neoplastic cells, but also to trigger specific molecular pathways, paving the path for the seed of cancer cells, directly to their "pre-educated" soil. The mechanisms that determine the interactions within cancer cells and the ECM are still obscure and could be elucidated by an in vitro 3D-culture system that integrates all the elements involved in PM development. Cancer organoids have shown a profound impact in the field of oncology since they better reflect the main characteristics of the native organs compared to the traditional cell culture models. However, they still fail to represent the heterogeneity of the microenvironment. Methodologies have been recently established to remove cells from tissues and obtain matrices in which ECM and tissue architecture are maintained (dECM models), that could be used as the most representative scaffold on which implant 3D cultures.
I aimed to obtain a 3D-model that closely recapitulates the microenvironment where the PM develops and includes d-ECM repopulated with PM-derived organoids (3D-dECM model). I removed the cellular component of ECMs derived from peritoneal cavity obtained from both PM samples and r matched normal peritoneum using detergents and enzymatic methods. dECMs analyses demonstrated that the procedure maintained the specific characteristics of their tissue of origin also in terms of distribution, localization, and architectural organization of ECM-related proteins. The obtained dECMs showed a different spatial rearrangement between normal and PM-derived peritoneum, suggesting that dECM scaffolds closely recapitulate the native PM microenvironment. Moreover, when I repopulated dECMs with PM-derived organoids I found that PM- and normal peritoneum-derived dECMs differentially regulated the localization and organization of the seeded organoids, which was the same as in the original tissue. The two 3D-ECM models presented different ability in supporting cell proliferation, where PM-derived 3D-dECMs showed a higher proliferation index and a major ability to maintain the stemness phenotype. PM- and normal peritoneum-derived 3D-dECMs differently modulated cell homeostasis and proliferation ratio.
A gene expression analysis of organoids, grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. The impact of the ECM on the response to standard chemotherapy treatment for PM was also observed.
This demonstrated the value of ex vivo 3D models obtained by combining patient-derived extracellular matrices depleted of cellular components and organoids to mimic the metastatic niche, which could provide tools to develop new therapeutic strategies in a biologically relevant context, to personalize treatments and increase their efficacy
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