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Die Untersuchung von Proteininteraktionen ist unerlässlich um die komplexen Mechanismen der epigenetischen Kontrolle von Zugänglichkeit zum Chromatin und dessen Struktur zu verstehen. Zellspezifizierung während der Entwicklung von Organismen kann nur durch strikte Regulation von Chromatin gewährleistet werden, was auch für den Schutz von Zellenidentitäten im späteren Lebensverlauf wichtig ist. Die Modifizierung von Histon-Proteinen, welche integrale Komponenten des Chromatins sind, fördert entweder positive oder negative Genregulation. Eine Vielzahl von Chromatin regulierenden Proteinen hat jedoch keine enzymatische Aktivität für Histon- Modifikationen, so dass sie nur such Interaktionen mit anderen Proteinen regulatorisch einwirken können. Der Nematode Caenorhabditis elegans eignet sich als ein in vivo System, um die Schutzmechanismen der Zellen basierend auf Chromatinfaktoren zu untersuchen, indem systematisch Protein-Interaktionsnetzwerke bestimmt werden. Diese Dissertation beschriebt zunächst die Etablierung eines optimierten Verfahrens für die quantitative Analyse ohne Markierung von Proteinen in C. elegans, die mittels CRISPR mit einem Epitop fusioniert wurden. Mit Hilfe dieses Verfahrens wurden fünf Chromatin regulierende Proteine, die eine wichtige Rolle beim Schutz von Zellidentitäten spielen, charakterisiert. Es wurden in vivo Proteininteraktions-Netzwerke erstellt und dabei neue funktionsrelevante Interaktionspartner identifiziert. Darüber hinaus wurde eine vertiefende Analyse der Interaktionen des Chromatinfaktors MRG- 1 durchgeführt, das homolog zum humanen MRG15 ist. MRG-1 besitzt eine sogenannte Chromodomäne, um an methylierte Histone zu binden. Diese Studie zeigt, dass die Untersuchung der Proteininteraktionen von epigenetischen Faktoren in einem in vivo System ein bedeutendes Verfahren ist, um wichtige biologische Mechanismen der Schutzfunktion von Zellen zu entschlüsseln. / Elucidating protein-protein interactions has been instrumental to understand the complex mechanisms underlying epigenetic regulations to control chromatin accessibility and structure. Proper development and cell fate specification are established under strict chromatin regulation to safeguard cellular identities throughout an organism's life. Modifications of histone proteins as an integral component of chromatin can promote either positive or negative gene regulation. However, many chromatin-regulation proteins lack enzymatic activity and depend on protein-protein interaction to cooperate with other factors to regulate chromatin through histone modifications. The nematode Caenorhabditis elegans can be used as an in vivo system to study chromatin regulators that safeguard cell identity and offers an attractive model system for mapping in vivo protein interactions. The presented thesis includes establishment of an optimized protocol for a quantitative approach based on label-free interaction proteomics to accurately identify interactions of chromatin-regulating proteins, which were epitope-tagged using CRISPR in C. elegans. This protocol was utilized to reveal the interaction partners of five bait proteins involved in essential chromatin regulation mechanisms during cell fate maintenance. The present study generated an in vivo protein interaction network identifying new interactions of high functional relevance. Moreover, in-depth protein-protein interaction analysis of the chromodomain protein MRG-1, homolog of human MRG15, detected a strong association with the Small Ubiquitin-like Modifier (SUMO), besides previously described and novel interactions with other proteins. In summary, in vivo interactome mapping of epigenetic regulators is a powerful approach that can reveal crucial biological insights into how cell fate decisions are regulated.

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Epigallocatechin gallate (EGCG), also known as epigallocatechin-3-gallate, is a polyphenol that is most abundant in tea. It has been shown from many studies that consumption of EGCG can contribute to weight loss, however, the underlying mechanism is not fully understood. To determine how EGCG acts to reduce fat, an organism model Caenorhabditis elegans (C. elegans) is introduced, which is a useful animal system in exploring crucial biological mechanisms that are readily applicable to humans. In this study, different strains were raised for two days on a diet with or without 100µM and 200µM EGCG treatment: N2 (i.e., wild type) and mutants (i.e., knockdown of fat metabolism related genes). EGCG’s effect on fat reduction was characterized by triglyceride content, food consumption and physiological behaviors. Our results showed that 100 and 200 µM EGCG significantly reduced the triglyceride content of wild type worms by 10% and 20%, respectively, without affecting its food intake and physiological behaviors. Additionally, EGCG could effectively reduce fat accumulation in C. elegans dependent on acs-2 and atgl-1.

<p>Microinjection is an established and reliable method to deliver transgenic constructs and other reagents to specific locations in the animal. Specifically, microinjection of a desired DNA construct into the distal gonad is the most widely used method to generate germ-line transformation of <em>C. elegans</em>. Although, current <em>C. elegans</em> microinjection method is an effective manner for creating transgenic worms, it requirements such as expensive multi DOF micromanipulator, detailed injection alignment procedure and skilled operator which makes the microinjection process slow and not suitable for scale to high throughput. Although many microfabricated microinjectors exist, none of them are capable of immobilizing a freely mobile animal such as <em>C.elegans</em> worm. In this research, a microfluidic microinjector was developed to simultaneously immobilize a freely mobile animal such as <em>C.elegans</em> and perform microinjection by using a simple and fast mechanism for needle actuation. The entire process of the microinjection takes ~30 seconds which includes 10s for worm loading and aligning, 5s needle penetration, 5s reagent injection and 5s worm unloading. The capability of the microinjector chip for creating transgenic <em>C. elegans</em> was illustrated (with success rate between 4% to 20%)</p> / Master of Science (MSc)

Tesis por compendio / [EN] Agri-food industry generates, because of its production processes, high amount of by-products, which cause a negative impact both economically and environmentally. For this reason, nowadays, their revalorization is one of the main aims of European Union in support of sustainable development. This doctoral thesis is focused on the revalorization of by-products from the horticultural industry as natural antimicrobials, by themselves or combined with no-thermal technologies for food preservation, like Pulsed Electric Fields (PEF) or High Hydrostatic Pressure (HHP) against the main foodborne pathogens. Also, it pretends to evaluate the microbial resistance development against the subletal antimicrobial treatments under study and the possible virulence changes using C. elegans as a model organism. The agri-food by-products studied have shown an important antimicrobial effect against the main foodborne pathogens, also the ASE extracts and the infusions obtained therefrom, being S. Typhimurium the most sensitive microorganism. In addition, the combination of subletal treatments of PEF and HHP with by-product infusions has resulted in the emergence of synergies, which permit us to achieve the desirable levels of microbial inactivation (5 log cycles) in a minor period of time. The application of subletal antimicrobial treatments under study consecutively, has shown that it causes microbial resistance development in S. Typhimurium. However, C. elegans studies show that the development of microbial resistance not imply the increase of its virulence against a host organism. For all these reasons, we can conclude that the revalorization of agri-food by-products as natural antimicrobials is a viable alternative as an additional control measure to ensure the microbial food safety by themselves or combined with PEF or HHP treatments, due to their synergistic effect. / [ES] La industria agroalimentaria genera, como resultado de sus procesos de producción, grandes cantidades de subproductos que suponen un impacto negativo a nivel económico y medioambiental. Es por ello que, en la actualidad, su revalorización es uno de los objetivos principales de la Unión Europea en apoyo al desarrollo sostenible. La presente tesis doctoral se centra en la revalorización de subproductos de la industria hortofructícola como antimicrobianos naturales en sí mismos o combinados con tecnologías no-térmicas de conservación de alimentos como los Pulsos Eléctricos de Alta Intensidad (PEF) o las Altas Presiones Hidrostáticas (HHP) frente a los microorganismos patógenos transmitidos por alimentos más importantes. Además, trata de evaluar el desarrollo de resistencias en los microorganismos a los tratamientos antimicrobianos subletales estudiados y sus posibles cambios de virulencia usando C. elegans como organismo modelo. Los subproductos hortofructícolas estudiados han demostrado un importante efecto antimicrobiano frente a los principales patógenos alimentarios, así como los extractos ASE y las infusiones obtenidas a partir de los mismos, siendo el microorganismo más sensible S. Typhimurium. Además, la aplicación de forma combinada de tratamientos subletales de PEF y HHP con infusiones de subproductos ha dado lugar a la aparición de sinergias que permiten alcanzar los niveles deseados de inactivación microbiana (5 ciclos logarítmicos) en un menor periodo de tiempo. La aplicación de los tratamientos antimicrobianos subletales estudiados de forma consecutiva se ha demostrado que da lugar a la generación de resistencia microbiana en S. Typhimurium. Sin embargo, los estudios con C. elegans ponen de manifiesto que el desarrollo de esta resistencia antimicrobiana no lleva consigo el aumento de su virulencia al infectar a un organismo hospedador. En base a todo lo anterior, podemos concluir que la revalorización de los subproductos de la industria hortofructícola como antimicrobianos naturales es una alternativa viable para su utilización como medida de control adicional de la seguridad microbiológica de productos alimenticios por sí mismos o en combinación con tratamientos de PEF o HHP, dado su efecto sinérgico. / [CA] La industria agroalimentària genera, com a resultat dels seus processos de producció, grans quantitats de subproductes que suposen un impacte negatiu a nivell econòmic y mediambiental. És per això que, en la actualitat, la seua revalorització és un dels objectius principals de la Unió Europea en recolzament al desenvolupament sostenible. La present tesi doctoral es centra en la revalorització de subproductes de la industria hortofructícola com a antimicrobians naturals per sí mateixa o combinats amb tecnologies no-tèrmiques de conservació d'aliments com els Polsos Elèctrics d'Alta Intensitat (PEF) o les Altes Pressions Hidrostàtiques (HHP) front als microorganismes patògens transmesos per aliments més importants. A més, tracta d'avaluar el desenvolupament de resistències en els microorganismes als tractaments antimicrobians subletals estudiats i els seus possibles canvis de virulència utilitzant C. elegans com a organisme model. Els subproductes hortofructícoles estudiats han demostrat un important efecte antimicrobià front als principals patògens alimentaris, així com els extractes ASE i les infusions obtingudes a partir dels mateixos, sent el microorganisme més sensible S. Typhimurium. A més, l'aplicació de forma combinada de tractaments subletals de PEF i HHP amb infusions de subproductes ha donat lloc a l'aparició de sinèrgies que permeten aplegar als nivells desitjats d'inactivació microbiana (5 cicles logarítmics) en un menor període de temps. L'aplicació dels tractaments antimicrobians subletals estudiats de forma consecutiva s'ha demostrat que dona lloc a la generació de resistència microbiana en S. Typhimurium. No obstant això, els estudis amb C. elegans posen de manifest que el desenvolupament d'esta resistència antimicrobiana no du implícit l'augment de la seua virulència al infectar a un organisme hospedador. En base a tot lo anterior, podem concloure que la revalorització dels subproductes de la industria hortofructícola com a antimicrobians naturals és una alternativa viable per a la seua utilització com a mesura de control addicional de la seguretat microbiològica de productes alimentaris per sí mateixa o en combinació amb tractaments de PEF o HHP, donat el seu efecte sinèrgic. / Sanz Puig, M. (2017). Valorización de subproductos de la industria agroalimentaria como antimicrobianos naturales frente a microorganismos patógenos mediante tecnologías no térmicas de conservación [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90508 / Compendio

The stem cell-like seam cells of the nematode, Caenorhabditis elegans, represent a tractable and powerful model for studying stem cell biology. rnt-1, the worm homologue of the mammalian RUNX family of transcription factors, together with the CBFβ homologue bro-1, is essential for the proliferation of the seam cells. RUNX genes and CBFβ are important regulators of stem cell development in mammals, and are associated with a variety of cancers. The worm seam cell model offers an opportunity to examine how these genes function in stem cell biology. The aim of this work was to shed light on the genetic network in which bro-1 and rnt-1 function, and to reveal the identity of regulators of these genes as well the downstream targets of the bro-1/rnt-1 pathway. Here, a number of genes that interact with bro-1 and rnt-1 have been identified. ELT-1, a GATA transcription factor, is shown to be a direct regulator of bro-1. Findings which show that the MEIS gene unc-62 acts upstream of bro-1/rnt-1 and regulates the symmetry of seam cell divisions are also presented. The seam cell marker, scm::gfp, is widely used in studies of the seam cells; here the results of an investigation into its identity and functional links are described. In addition, the mechanism underlying spatial regulation of rnt-1 was examined; this led to the discovery of distinct tissue-specific enhancer modules within an intron of this gene. Finally, interactions between pal-1 and bro-1/rnt-1 are reported and described. Together, these findings provide a framework for furthering our understanding of the mechanisms and genes associated with the functioning of bro-1 and rnt-1 in the worm.

Alcohol abuse and dependence are complex disorders that are influenced by many genetic and environmental factors. Acute behavioral responses to ethanol have predictive value for determining an individual’s long-term susceptibility to alcohol abuse and dependence. These behavioral responses are strongly influenced by genetics. Here, we have explored the role of genetic influences on acute behavioral responses to ethanol using the nematode worm, Caenorhabditis elegans. First, we explored the role of ethanol metabolism in acute behavior responses to ethanol. Natural variation in human ethanol metabolism machinery is one of the most reported and reproducible associations found to alter drinking behavior. Ethanol metabolism is conserved across phyla and alteration in this pathway alters acute behavioral responses to ethanol in humans, mice, rats, and flies. We have extended these findings to the worm and have shown that loss of either alcohol dehydrogenase or aldehyde dehydrogenase results in an increase in sensitivity to the acute effects of ethanol. Second, we explored the influence of differences in basal and ethanol-induced gene expression in ethanol responsive behaviors. We identified a set of candidate genes using the basal gene expression differences in npr-1(ky13) mutant animals to enrich for genes involved in AFT. This analysis revealed ethanol changes to the expression of genes involved in a variety of biological processes including lipid metabolism. We focused on a gene involved in the metabolism of fatty acids, acs-2. acs-2 encodes an acyl-CoA synthetase that activates fatty acids for mitochondrial beta-oxidation. Animals carrying mutant acs-2 have significantly reduced AFT and we explored the role of genes in the mitochondria beta-oxidation pathway for alterations in ethanol responsive behaviors. We have shown that knockdown of ech-6, an enoyl-CoA hydratase, enhances the development of AFT. This work has uncovered a role for fatty acid utilization pathways in acute ethanol responses and we suggest that natural variation in these pathways in humans may impact the acute alcohol responses to alcohol that in turn influence susceptibility to alcohol abuse and dependence.