http://hdl.handle.net/10366/4028 2026-09-16T09:36:44Z http://hdl.handle.net/10366/172697 [EN]Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures1-3. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time4. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms. 2024-04-24T00:00:00Z http://hdl.handle.net/10366/172678 [EN]Tumor initiation remains one of the least understood events in cancer biology, largely due to the challenge of dissecting the intricacy of the tumorigenic process in laboratory settings. The insufficient biological complexity of conventional in vitro systems makes animal models the primary experimental approach to study tumorigenesis. Despite providing valuable insights, these in vivo models function as experimental black boxes with limited spatiotemporal resolution of cellular dynamics during oncogenesis. In addition, their use raises ethical concerns, further underscoring the need for alternative ex vivo systems. Here we provide a detailed protocol to integrate state-of-the-art microfabrication, tissue engineering and optogenetic approaches to generate topobiologically complex miniature colons ('mini-colons') capable of undergoing tumorigenesis in vitro. We describe the key methodology for the generation of blue light-inducible oncogenic cells, the establishment of hydrogel-based mini-colon scaffolds within microfluidic devices, the development of mini-colons and the induction of spatiotemporally controlled tumorigenesis. This protocol enables the formation and long-term culture of complex cancerous tissues that capture in vivo-like tumoral biology while offering real-time and single-cell resolution analyses. It can be implemented in 4-6 weeks by researchers with prior experience in 3D cell culture techniques. We anticipate that these methodological guidelines will have a broad impact on the cancer research community by opening new avenues for tumorigenesis studies. 2026-01-01T00:00:00Z http://hdl.handle.net/10366/172674 [EN]Both the number and regenerative activity of hair follicle stem cells (HFSCs) are regulated by Vav2, a GDP/GTP exchange factor involved in the catalytic stimulation of the GTPases Rac1 and RhoA. However, whether Vav2 signaling changes in HFSCs over the mouse lifespan is not yet known. Using a mouse knock-in mouse model, we now show that the expression of a catalytically active version of Vav2 (Vav2Onc) promotes an extensive rewiring of the overall transcriptome of HFSCs, the generation of new transcription factor hubs, and the synchronization of many transcriptional programs associated with specific HFSC states and well-defined signaling pathways. Interestingly, this transcriptome rewiring is not fixed in time, as it involves the induction of 15 gene expression waves with diverse distribution patterns during the life of the animals. These expression waves are consistent with the promotion by Vav2Onc of several functional HFSC states that differ from those normally observed in wild-type HFSCs. These results further underscore the role of Vav2 in the regulation of the functional state of HFSCs. They also indicate that, unlike other Vav2-dependent biological processes, the signaling output of this exchange factor is highly contingent on age-dependent intrinsic and/or extrinsic HFSC factors that shape the final biological readouts triggered in this cell type. 2023-09-26T00:00:00Z http://hdl.handle.net/10366/172664 [ES]El establecimiento del sitio de división en Schizosaccharomyces pombe depende de la formación de un anillo medio contráctil y de la acumulación de los parches de F-actina a su alrededor. Al final de la anafase comienza la contracción del anillo y la síntesis centrípeta del septo de división, el cual está compuesto por un disco central de beta(1-3)-D-glucano lineal, denominado septo primario, y por una estructura similar a la de la pared celular a ambos lados del mismo, denominada septo secundario, y cuya síntesis es esencial para la integridad celular. Últimamente ha aumentado considerablemente la información sobre la formación y la activación de la contracción del anillo medio. Sin embargo, aún no se conoce cómo se produce la síntesis del septo. S. pombe posee cuatro posibles subunidades catalíticas del complejo beta(1-3)-D-glucán sintasa (BGS), denominadas Bgs1p-4p. En esta tesis hemos estudiado la función de Bgs1p y Bgs4p en la síntesis del beta-glucano y su importancia para la formación del septo durante la citocinesis y el mantenimiento de la integridad celular. La proteína Bgs1p/Cps1p ha sido identificada como una hipotética subunidad catalítica de la (BGS), con una posible función durante la citocinesis y el crecimiento polarizado. Para estudiar esta posibilidad, se crearon mutantes dobles de cps1-12 y diferentes mutantes cdc de septación. Los mutantes dobles obtenidos presentaron distintos fenotipos de hipersensibilidad y alteraciones en la distribución de la actina. El análisis de las relaciones epistáticas de los mutantes dobles mostró que las mutaciones de los procesos anteriores a la síntesis del septo eran dominantes sobre cps1-12, mientras que cps1-12 era dominante sobre el mutante de finalización de la septación cdc16-116, lo que sugiere que Bgs1p está implicada en la síntesis del beta(1-3)-D-glucano de la pared celular del septo durante la citocinesis. Hemos estudiado la localización fisiológica in vivo de Bgs1p en una estirpe bgs1D que contiene un gen GFP-bgs1+ funcional (una única copia integrada del gen y expresada bajo el control de su propio promotor). Durante el crecimiento vegetativo, Bgs1p siempre se localiza en las zonas de crecimiento: en uno ó ambos polos durante el crecimiento celular, y en el anillo medio y septo durante la citocinesis. La localización de Bgs1p en los mutantes cdc de septación indica que Bgs1p necesita las proteínas del anillo medio y de la ruta de activación de la septación (SIN) para localizarse correctamente con el resto de los componentes de la septación. La localización de Bgs1p en el mutante de actina cps8-188 muestra que la localización de Bgs1p depende de la localización de la actina. Además, Bgs1p permanece polarizada en los polos y septos deslocalizados de los mutantes tea1-1 y tea2-1. Durante el proceso meiótico del ciclo de vida, Bgs1p se localiza en las proyecciones de conjugación, en la zona de contacto entre las células durante la fusión celular y en el área del cuello durante la formación del cigoto. También, la localización de Bgs1p sugiere que colabora en la síntesis de la pared de la proespora y de la espora. Durante la germinación de la espora, Bgs1p se localiza primero alrededor de la espora durante el crecimiento isotrópico, a continuación, en la zona de crecimiento polarizado y finalmente, en el anillo medio y septo. Al final de la división entre la espora y la célula, el desplazamiento de Bgs1p hacía el polo viejo ocurre únicamente en la célula. Todos estos datos muestran que Bgs1p se localiza en las áreas de crecimiento polarizado de la pared celular, y por lo tanto, proponemos que Bgs1p podría estar implicada en la síntesis del beta(1-3)-D-glucano lineal del septo primario, así como de un beta(1-3)-D-glucano linear similar, que podría ser necesario en otros procesos de crecimiento ó de reparación de la pared celular. También hemos estudiado la función de Bgs1p y demostramos que es la subunidad de una actividad BGS, responsable de la síntesis del beta(1-3)-D-glucano lineal y del septo primario que origina. Hemos analizado el efecto letal que produce la represión de la expresión de bgs1+ en un fondo genético bgs1D. Para ello, hemos construido una serie de vectores que expresan bgs1+ bajo el control del promotor reprimible por tiamina nmt1+-81X, y que abarcan un rango de represión de aproximadamente 500 veces. Al reprimir la expresión de bgs1+ las células siguen presentando un fenotipo silvestre incluso cuando los niveles de Bgs1p son ya indetectables indicando el nivel tan bajo de Bgs1p que necesitan las células. La represión de bgs1+ sólo muestra un fenotipo letal con los tres vectores con mayor nivel de represión, originando células ramificadas y con múltiples septos, lo que demuestra que Bgs1p tiene un papel esencial durante la septación. La represión ó la sobreexpresión de bgs1+ no muestra ninguna alteración del beta-glucano, pero sí un aumento considerable del beta-glucano de la pared celular. Sin embargo, la actividad BGS disminuye en ambos casos. Por otro lado, las células con niveles reducidos de Bgs1p muestran un incremento notable de Bgs3p. Estos resultados sugieren que Bgs1p forma parte de un complejo multiproteico de la actividad BGS, y que las alteraciones producidas por las variaciones de Bgs1p inducen procesos compensatorios de otras subunidades Bgs y de la alfa(1-3)-D-glucán sintasa (AGS). El análisis de la represión de bgs1+ mediante tinción con Calcofluor white, un compuesto fluorescente que marca específicamente las zonas de crecimiento de la pared celular (polos y septo), y mediante marcaje con anticuerpos monoclonales específicos contra el beta(1-3)-D-glucano lineal, muestra en ambos casos, una reducción de la tinción de los septos. Los estudios mediante microscopía electrónica de transmisión y mediante inmunomicroscopía electrónica confirman que los septos presentan grandes alteraciones en su estructura, debidas principalmente a los defectos ó a la ausencia del septo primario y del beta(1-3)-D-glucano lineal del mismo. La deleción de bgs1+ es letal, pero las esporas bgs1D son capaces de germinar y originar células que pueden crecer durante varios ciclos mitóticos hasta morirse, dando lugar a células gigantes, multiseptadas y ramificadas. Estas células son parecidas a las observadas durante la represión de bgs1+, pero carecen completamente del septo primario y del beta(1-3)-D-glucano lineal, y no se tiñen con el Calcofluor. Todos estos datos muestran que el septo primario no es necesario para la síntesis del septo, que Bgs1p es responsable de la síntesis del beta(1-3)-D-glucano lineal y que éste a su vez es responsable de la estructura del septo primario, y que el polisacárido de la pared celular que reconoce específicamente y con una alta afinidad el Calcofluor es el beta(1-3)-D-glucano lineal. Finalmente, en esta tesis hemos clonado bgs4+ y mostramos que Bgs4p es la única subunidad que se ha encontrado hasta la fecha que forma parte de la enzima BGS y que es esencial para mantener la integridad celular durante la citocinesis y el crecimiento polarizado. También mostramos que bgs4+, cwg1+ (cwg1-1 presenta una reducción del beta-glucano de la pared celular y de la actividad catalítica de la BGS) y orb11+ (orb11-59 está alterado en la morfogénesis celular) son el mismo gen. bgs4+ es esencial para la germinación de la espora. En células vegetativas la represión de la expresión de bgs4+ produce la lisis celular en los polos de crecimiento y principalmente en el septo, antes de la separación celular, lo que sugiere que Bgs4p es esencial para el crecimiento de la pared celular y para compensar un exceso de degradación de la pared celular durante la citocinesis. El análisis de la represión y de la sobreexpresión de bgs4+ sugiere que Bgs4p forma parte de un complejo catalítico multiproteico de la BGS, y que las alteraciones del beta-glucano de la pared celular producidas por las variaciones de Bgs4p inducen mecanismos compensatorios a través de las otras subunidades Bgs y de la AGS. Los estudios de localización en condiciones fisiológicas mostraron que Bgs4p se localiza en los polos de crecimiento, el anillo medio, el septo y en cada uno de los sitios donde se produce síntesis ó remodelación de la pared celular durante la diferenciación sexual: conjugación, formación del cigoto y de la espora, y germinación de la espora. Los requerimientos para una correcta localización de Bgs4p durante la citocinesis, así como el patrón de localización de Bgs4p durante la maduración de la espora, difieren de los de Bgs1p. Bgs4p aparece en el anillo contráctil después de que se detecte Bgs1p y un material del septo teñido por el Calcofluor white, sugiriendo que Bgs4p está implicada en un proceso posterior, de síntesis del septo secundario ó de todo el septo en general. A diferencia de Bgs1p, Bgs4p necesita el anillo medio pero no las proteínas de la ruta de activación de la septación (SIN) para localizarse con los otros componentes de la septación. Además, la localización de Bgs4p depende de las proteínas del establecimiento de la polaridad. Finalmente, Bgs4p depende de la F-actina para su deslocalización de las zonas de crecimiento y su relocalización en las mismas, pero no necesita la actina para mantenerse estable en los sitios de crecimiento, polos y septo. Todos estos resultados muestran, por primera vez, el papel esencial de una subunidad Bgs en la síntesis de un beta(1-3)-D-glucano, que es necesario para preservar la integridad de la célula durante la síntesis ó reparación de la pared celular. 2006-01-01T00:00:00Z http://hdl.handle.net/10366/172277 [ES] Debido a la crisis generada por las bacterias resistentes a antibióticos, es necesario identificar nuevas moléculas con potencial antibiótico que permitan tratar las infecciones bacterianas actuales. Uno de los abordajes para conseguir este objetivo es el estudio de la regulación de la producción de antibióticos por microorganismos productores. Entre ellos, las bacterias del género Streptomyces poseen un gran potencial, dada su gran cantidad de genes que permiten la síntesis de metabolitos secundarios. Estos genes están muy controlados por factores de regulación que integran señales externas e internas, de modo que caracterizar estas redes es crucial para comprender qué factores son necesarios para incrementar la producción de antibióticos y para despertar genes no activados en condiciones de laboratorio, que puedan ser claves para la producción de nuevas moléculas. En este trabajo, se ha profundizado en el estudio de las redes de regulación de S. coelicolor, organismo modelo del género Streptomyces. Los sistemas de dos componentes (TCS) en estas bacterias cumplen un papel esencial en el control del desarrollo y del metabolismo. Algunos TCS descritos en nuestro grupo de investigación intervienen en la producción de antibióticos en S. coelicolor, y todos ellos regulan el gen bfr, que codifica la bacterioferritina Bfr. Entre estos TCS destaca el regulador huérfano Aor1, que tiene gran influencia en la expresión del genoma de S. coelicolor y cuya histidina quinasa asociada, encargada de activarlo, se desconoce. En primer lugar, en esta tesis se describe el papel de la bacterioferritina codificada por el gen bfr en el desarrollo de S. coelicolor mediante el estudio del mutante de deleción Δbfr. Bfr es un elemento necesario para una correcta diferenciación y producción de antibióticos (actinorrodina, prodigiosinas y coelimicinas) en medios de cultivo complejos, mientras que en medio mínimo su efecto depende de la concentración de hierro y del estado (sólido o líquido) del medio. Bfr está implicada en la homeostasis del hierro, pero esta no es su función principal al no alterarse los niveles de hierro en el mutante Δbfr. La ausencia de bfr causa cambios a nivel proteómico: aumenta la abundancia de proteínas de estrés oxidativo (y los niveles de especies reactivas de oxígeno en el mutante), del antibiótico dependiente de calcio y de proteínas de respiración, mientras que disminuye la abundancia de proteínas de desarrollo, de varios clústeres para biosíntesis de metabolitos y de traducción. Además, varias proteínas de señalización alteran sus niveles en el mutante Δbfr. Con los datos de esta tesis, se propone un modelo en el que Bfr es necesaria para donar hierro a proteínas de regulación importantes para la síntesis de metabolitos secundarios. En cuanto al regulador huérfano Aor1, mediante varias estrategias, se han identificado varias quinasas que podrían estar asociadas a este regulador: SCO3750 (quinasa huérfana), SCO2215 (asociada al regulador SCO2216) y SCO2359 (asociada al regulador SCO2358). La asociación entre estas proteínas y Aor1 se ha estudiado mediante la generación de mutantes de deleción y CRISPR de interferencia, y se ha validado mediante ensayos de dos híbridos. También se ha estudiado brevemente la relación entre el sistema AbrC, previamente caracterizado en el laboratorio, con Aor1. Aún quedan por caracterizar las señales a las que responden estas proteínas y su mecanismo de regulación. Finalmente, se realizó un estudio metabolómico de varias cepas de interés relacionadas con los TCS estudiados en este trabajo: la cepa silvestre y los mutantes ΔabrA, Δbfr, Δaor1, ΔSCO3750, ΔSCO2359 y ΔSCO2359/2358. Muchos de los metabolitos anotados al comparar los metabolomas de cada mutante con el silvestre no se pudieron identificar con exactitud, lo cual refleja el conocimiento que falta en el metabolismo de S. coelicolor. La identificación de estos metabolitos sería clave para profundizar en los mecanismos de regulación de estos sistemas. En conclusión, esta tesis caracteriza en gran medida el papel de bfr en la red regulatoria de la producción de antibióticos de S. coelicolor, y profundiza en la red molecular del regulador huérfano Aor1 mediante su relación con nuevos elementos no descritos anteriormente (la quinasa huérfana SCO3750 y los TCS SCO2215/2216 y SCO2359/2358) y con el sistema AbrC. Además, este trabajo inicia el estudio metabolómico de estos sistemas involucrados en la síntesis de antibióticos y propone su estudio más detallado para comprender las redes de regulación de S. coelicolor y mejorar la producción y descubrimiento de metabolitos secundarios.; [EN] Due to the crisis caused by antibiotic-resistant bacteria, it is necessary to identify new molecules with antibiotic potential that can treat current bacterial infections. One approach to achieve this goal is to study the regulation of antibiotic production by antibiotic-producing microorganisms. Among these, bacteria from the genus Streptomyces have great potential, given their large number of genes that enable the synthesis of secondary metabolites. These genes are tightly controlled by regulatory factors that integrate external and internal signals, so characterizing these networks is crucial to understand which factors are necessary to increase antibiotic production and to turn on genes that are not activated under laboratory conditions, which may be key to the production of new molecules. In this work, we have delved deeper into the study of the regulatory networks of S. coelicolor, a model organism of the Streptomyces genus. Two-component systems (TCS) in these bacteria play an essential role in controlling development and metabolism. Some TCS described in our research group are involved in the production of antibiotics in S. coelicolor, and all of them regulate the bfr gene, which encodes the bacterioferritin Bfr. Among these TCS, the orphan regulator Aor1 stands out, which has a major influence on the expression of the genome of S. coelicolor and whose associated histidine kinase, responsible for activating it, is unknown. Firstly, this thesis describes the role of the bacterioferritin codified by the bfr gene in the development of S. coelicolor by studying the Δbfr deletion mutant. Bfr is a necessary element for the correct differentiation and production of antibiotics (actinorhodin, prodigiosins and coelimycins) in complex culture media, while in minimal media its effect depends on the iron concentration and the state (solid or liquid) of the medium. Bfr is involved in iron homeostasis, but this is not its main function, as iron levels are not altered in the Δbfr mutant. The lack of bfr produces changes at the proteomic level: the abundance of proteins linked to oxidative stress (and levels of reactive oxygen species in the mutant), calcium-dependent antibiotic and respiration proteins increases, while the abundance of developmental proteins, various clusters for metabolites biosynthesis and translation proteins decreases. In addition, several signaling proteins alter their levels in the Δbfr mutant. Based on the data from this thesis, a model is proposed in which Bfr is necessary to donate iron to regulatory proteins important for the synthesis of secondary metabolites. As for the orphan regulator Aor1, several strategies have been used to identify several kinases that could be associated with this regulator: SCO3750 (orphan kinase), SCO2215 (associated with the regulator SCO2216) and SCO2359 (associated with the regulator SCO2358). The association between these proteins and Aor1 has been studied by generating deletion mutants and CRISPR interference and has been validated by two-hybrid assays. The relationship between the AbrC system, previously characterized in the laboratory, and Aor1 has also been briefly studied. The signals these proteins respond to, and their regulatory mechanism, remain to be characterized. Finally, a metabolomic study was performed on several strains of interest related to the TCS studied in this work: the wild-type strain and the mutants ΔabrA, Δbfr, Δaor1, ΔSCO3750, ΔSCO2359 and ΔSCO2359/2358. Many of the metabolites noted when comparing the metabolomes of each mutant with the wild type could not be identified with certainty, reflecting the lack of knowledge about the metabolism of S. coelicolor. The identification of these metabolites would be key to further understanding the regulatory mechanisms of these systems. In conclusion, this thesis largely characterizes the role of bfr in the regulatory network of antibiotic production in S. coelicolor and delves into the molecular network of the orphan regulator Aor1 through its relationship with new, previously undescribed elements (the orphan kinase SCO3750 and the TCS SCO2215/2216 and SCO2359/2358) and with the AbrC system. Furthermore, this work initiates the metabolomic study of these systems involved in antibiotic synthesis and proposes more detailed studies to understand the regulatory networks of S. coelicolor and improve the production and discovery of secondary metabolites. 2026-01-01T00:00:00Z http://hdl.handle.net/10366/172269 [EN]It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells. 2022-06-01T00:00:00Z http://hdl.handle.net/10366/171615 [EN]To enhance the framework of the mechanisms of action used by Trichoderma gamsiiT6085 for the control of Fusarium head blight (FHB), this work investigated its abil-ity to modulate the expression of defence-related genes of wheat (Triticum aestivum‘Apogee’) in response to endophytic colonization of plant tissues. Changes in relativeexpression of pal1, pr1, pgip2 and lox1 genes were assessed over time in wheat roots,in spikes colonized by T6085 alone and both T6085 and Fusarium graminearum, and inleaves from wheat seedlings root-inoculated with T6085. Results indicate the ability ofT6085 to induce local and systemic defence responses in wheat plants in the presenceof one of the causal agents of FHB. There was a general significant up-regulation ofthe plant defence-related genes analysed, especially in the first days after the applica-tion of T6085. According to these results, modulation of plant defence genes could beincluded within the arsenal of mechanisms used by T6085 when applied to wheat, anadditional feature of interest in the management of FHB. To evaluate the effect of theplant genotype on the ability of T6085 to endophytically colonize roots, root coloniza-tion was assessed on four cultivars of T. aestivum and two cultivars of T. durum. Datashowed that roots of only two T. aestivum cultivars were endophytically colonized byT6085, similar to cv. Apogee used here as control, thus demonstrating an effect of thehost genotype on the endophytic ability of T6085. 2023-01-01T00:00:00Z http://hdl.handle.net/10366/171614 [EN]We explore the combined use of two beneficial fungal isolates, Trichoderma gamsii T6085 (Tg) and Clonostachys rosea IK726 (Cr), to enhance Fusarium head blight (FHB) management by biological control. We found no evidence for mycoparasitism or inhibition via diffusible metabolites, but Tg volatiles inhibited Cr growth slightly. Although Cr reduced Tg spore germination and mycelial growth in liquid culture, this effect seemed absent in planta. The BCAs differently modulated defence-related (DR) genes when colonizing roots or spikes. At seven days post-inoculation (dpi), root-applied Cr, alone and co-inoculated, induced a minor upregulation of PR1. In leaves, a systemic signalling response by root inoculation was detected. In spikes, Pal1, PR1, and Lox1 were upregulated by Cr alone and co-inoculated at 96 hours post-inoculation (hpi). However, Lox1 activation was enhanced by co-inoculation. On spikes inoculated with Fg, the BCAs revealed different patterns of DR gene modulation indicating involvement of different biocontrol mechanisms. In detail, Pgip2 was primarily upregulated at 24 hpi in co-inoculated spikes whereas at 72 hpi activation of DR genes was observed only with Tg. Notably, the disease incidence was reduced by 93 % by co-inoculation. In addition, the inoculum potential of F. graminearum on straw was reduced by all BCAs treatments, with ≥ 96 % reduction of perithecia after six months incubation. Our results show the potential of combining Tg and Cr as a more effective and stable FHB management strategy, than by treatments with the individual strains. 2025-07-01T00:00:00Z http://hdl.handle.net/10366/171613 [EN]Fusarium graminearum (Fg) is one of the most virulent causal agents of Fusarium head blight (FHB) in Central Europe. The disease is also caused by other Fusarium species within the FHB Species Complex (FHBSC). Some Fusarium species secrete killer proteins (KPs) during host plant infection. Fg produces KP4-L killer toxins (FgKP4L), of which the clustered Fgkp4l-1, -2, -3 genes and Fgkp4l-4 (encoding for the heterodimeric KP4L-4 protein) are expressed in competitive interactions against the biocontrol agent Trichoderma gamsii T6085. We investigated the involvement of the four Fgkp4l genes in the competition with other plant-pathogenic Fusarium species either lacking KP4L proteins or carrying different combinations of them. Fusarium sporotrichioides (Fs) and Fusarium langsethiae (Fl) belonging to the FHBSC, and the outgroup Fusarium verticillioides (Fv), were used for both in vitro and in vivo tests. To monitor mycotoxin production, relative expression of Tri4 gene included within the trichothecene biosynthetic pathway was also evaluated. In dual cultures, modulation of KP4L-encoding genes was tailored according to the facing species and the distance between fungi. Against Fs, the three clustered Fgkp4l genes were up-regulated but no changes on gene expression occurred with Fl. Fgkp4l-2 was up-regulated facing Fv during the two contact independent stages (Early sensing and Sensing) of the interaction. The Tri4 gene was expressed only during the in vitro interaction with Fv, but not on spikes. Fgkp4l-2 and -3 were up-regulated at 3 days post-inoculation on wheat heads inoculated with a mix of the four Fusarium species. Competition against the other Fusarium did not influence Fg growth rate on spikes. 2025-01-01T00:00:00Z http://hdl.handle.net/10366/171612 [EN]Citrus fruits, widely consumed around the world, can be negatively affected by pests and fungal infections during their cultivation, handling, transportation, and storage, thus resulting in substantial yield losses and food waste. The use of natural preservatives like chitosan (CHT) and essential oils (EOs) is a promising approach for reducing chemical inputs to preserve food products. This study investigated the effects of CHT, extracted from crab shells and the fungus Pleorotus ostreatus, alone and in combination with mandarin (Citrus × reticulata Blanco, Rutaceae) essential oil (MEO), as an oviposition deterrent towards Ceratitis capitata (Diptera: Tephritidae), the Mediterranean fruit fly, and growth inhibitor of Penicillium (P. expansum, P. digitatum, and P. italicum) spp. fungi, the causal agents of apple and citrus rot. A solution of 1.0% CHT of both origins (from crab shells and P. ostreatus) added with MEO resulted as the best combination to significantly reduce the oviposition percentage of C. capitata as well as mycelial growth and spore germination of Penicillium isolates and their pathogenic activity on Citrus japonica Thunb. (kumquats) fruits. According to results here collected, CHT added with MEO represents a valid combination to be used as an edible film and coating as part of an integrated control strategy to improve the shelf-life of fresh citrus fruits. Furthermore, fungal CHT, here used for the first time in combination with MEO, can be an excellent alternative to reply to the eating habits and necessities of the final consumers. 2024-07-13T00:00:00Z http://hdl.handle.net/10366/171611 [EN]Plants constantly face the environment that surrounds them and fight for survival against biotic and abiotic stress factors. To deal with harmful conditions, plants have developed a multilayer defence system, making them capable of recognising threats and promptly recovering from them. This phenomenon, which takes advantage of the “memory effect”, is referred to as bio-priming and represents a new frontier in terms of crop protection. Here, we investigated the “indirect” protective mechanisms of a new yeast extract formulate in Vitis vinifera cv. Sangiovese plants at both the biochemical and genic levels. The formulate was applied once a week for three consecutive weeks, and grapevine leaves were sampled from the first to the fifth day after treatment (dat) at every week of the experiment. Increased levels of jasmonic acid (every week at 2 dat; +70% as average) and abscisic acid (at 1 dat of the first week, more than 1.7-fold higher than the control) and the underproduction of salicylic acid (from 2 dat; −18%) confirmed that these signalling molecules/”specialised compounds” are actively involved in the early activation of defence pathways in treated vines. In addition, pr2 and chit1b, two genes involved in regulating hormonal crosstalk, were significantly up-regulated (both in the first and second week of the trial) and were also found to underlie upstream molecular activation. The results obtained by this investigation confirm the use of this new product to prime and protect grapevines from a wide range of fungal and fungal-like plant pathogens through the induction of defence responses. 2024-06-01T00:00:00Z http://hdl.handle.net/10366/170354 [ES] Las RASopatías asociadas a alteraciones en los genes NF1 y SPRED1 presentan una marcada heterogeneidad clínica y molecular, donde el principal obstáculo tras el diagnóstico genético es la interpretación funcional de las variantes identificadas. Con el objetivo de superar este desafío, la presente tesis doctoral ha creado un flujo de trabajo experimental multidisciplinar que combina el análisis de variantes en NF1 y SPRED1 reportadas en bases de datos públicas y de pacientes de la DIERCyL, el modelado estructural y de estabilidad proteica in silico, la señalización celular, proliferación, y morfometría en las líneas celulares HEK293T y NIH3T3. Los análisis de las bases de datos genéticas identificaron hotspots estructurales críticos en la proteína neurofibromina (CSRD, GRD y Armadillo1), mientras que las variantes en SPRED1 mostraron una distribución homogénea, sugiriendo mecanismos patogénicos distintos. En el caso de las variantes en NF1 y SPRED1 de los pacientes, el análisis energético reveló un espectro continuo en neurofibromina que va desde haploinsuficiencia hasta dominancia negativa en truncantes tardías, mientras que en SPRED1 predominó la pérdida de función. Los ensayos celulares in vitro, mostraron dinámicas de señalización celular diferentes según el tipo de variante analizada en los genes NF1 y SPRED1. En particular, la hiperactivación de ERK emergió como el evento molecular más determinante para la patogenicidad de las variantes. La coactivación de p38 identificó variantes de mayor severidad clínica, mientras que la relación inversa p- ERK/p-AKT(Ser473) reveló un circuito de retroalimentación negativa entre las rutas de señalización MAPK y PI3K/AKT. Los ensayos de proliferación celular y el análisis cuantitativo del tamaño celular revelaron fenotipos consistentes con el grado de desregulación de MAPK, proporcionando biomarcadores fenotípicos adicionales para diferenciar entre variantes patogénicas y benignas en ambos genes. La relevancia tisular de HEK293T (contextos epiteliales/neuroectodérmicos) y NIH3T3 (mesenquimales) permitió reproducir la heterogeneidad clínica observada en pacientes. Los resultados de esta tesis doctoral han permitido reclasificar variantes de significado incierto en ambos genes e identificar la hiperactivación de ERK como diana terapéutica prioritaria, apoyando el uso de inhibidores de MEK en contextos compatibles con esta desregulación. En conjunto, este trabajo establece un marco integral para la interpretación funcional de variantes en RASopatías y para el desarrollo de futuras estrategias de medicina personalizada.; [EN] RASopathies associated with alterations in the NF1 and SPRED1 genes exhibit marked clinical and molecular heterogeneity, where the main challenge after genetic diagnosis lies in the functional interpretation of the identified variants. To address this issue, this doctoral thesis established a multidisciplinary experimental workflow that integrates the analysis of NF1 and SPRED1 variants reported in public databases and in patients from the DIERCyL cohort, together with in silico structural and protein-stability modeling, cellular signaling assays, proliferation studies, and morphometric analyses in HEK293T and NIH3T3 cell lines. Database analyses identified critical structural hotspots in neurofibromin (CSRD, GRD, and Armadillo1), whereas SPRED1 variants showed a more homogeneous distribution, suggesting distinct pathogenic mechanisms. In patient-derived NF1 and SPRED1 variants, energy-based analyses revealed a continuous spectrum in neurofibromin, ranging from haploinsufficiency to dominant-negative effects in late truncating variants, while SPRED1 alterations predominantly showed loss-of-function behavior. The in vitro cellular assays revealed distinct signaling dynamics depending on the type of variant analyzed in NF1 and SPRED1. Notably, ERK hyperactivation emerged as the most determinant molecular event for pathogenicity. Co-activation of p38 identified variants associated with more severe clinical outcomes, whereas the inverse p-ERK/p- AKT(Ser473) relationship revealed a negative feedback circuit between the MAPK and PI3K/AKT pathways. Cell-proliferation assays and quantitative cell-size analyses uncovered phenotypes consistent with the degree of MAPK deregulation, providing additional phenotypic biomarkers to discriminate between pathogenic and benign variants in both genes. The tissue relevance of HEK293T (epithelial/neuroectodermal contexts) and NIH3T3 (mesenchymal) cells allowed us to recapitulate the clinical heterogeneity observed in patients. The results of this doctoral thesis enabled the reclassification of variants of uncertain significance in both genes and identified ERK hyperactivation as a priority therapeutic target, supporting the use of MEK inhibitors in contexts consistent with this dysregulation. Taken together, this work establishes a comprehensive framework for the functional interpretation of variants in RASopathies and for the development of future personalizedmedicine strategies. 2025-01-01T00:00:00Z http://hdl.handle.net/10366/169986 [EN]Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing 2021-01-01T00:00:00Z http://hdl.handle.net/10366/169937 [EN]Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen 2021-12-01T00:00:00Z http://hdl.handle.net/10366/169930 [EN]KP4 killer toxins are secreted proteins that inhibit cell growth and induce cell death in target organisms. In Fusarium graminearum, KP4-like (KP4L) proteins contribute to fungal virulence in wheat seedling rot and are expressed during Fusarium head blight development. However, fungal KP4L proteins are also hypothesized to support fungal antagonism by permeabilizing cell walls of competing fungi to enable penetration of toxic compounds. Here, we report the differential expression patterns of F. graminearum KP4L genes (Fgkp4l-1, -2, -3 and -4) in a competitive interaction, using Trichoderma gamsii as the antagonist. The results from dual cultures indicate that Fgkp4l-3 and Fgkp4l-4 could participate in the recognition at the distance of the antagonist, while all Fgkp4l genes were highly activated in the pathogen during the physical interaction of both fungi. Only Fgkp4l-4 was up-regulated during the interaction with T. gamsii in wheat spikes. This suggests the KP4L proteins could participate in supporting F. graminearum interspecific interactions, even in living plant tissues. The distribution of KP4L orthologous within the genus Fusarium revealed they are more represented in species with broad host-plant range than in host-specific species. Phylogeny inferred provides evidence that KP4L genes evolved through gene duplications, gene loss and sequence diversification in the genus Fusarium 2022-09-16T00:00:00Z http://hdl.handle.net/10366/169920 [EN]Species of fungal genus Trichoderma are characterized by a versatile lifestyle, high adaptability to the changing environmental conditions and the ability to establish sophisticated interactions with other organisms. Due to their ability to antagonize plant pathogens and to elicit the plant defence responses against biotic/abiotic stresses, Trichoderma spp. are commonly used as commercially biopesticides and biofertilizers. The Trichoderma success in the rhizosphere is supported by a wide arsenal of specialised metabolites (SMs) providing morphological and physiological autoregulation, self-protection and facilitating fungal communication. This review aims to explore the roles of SMs in the biology of fungi, with special emphasis on the genus Trichoderma and on how divergence in the SMs genetic structure determine Trichoderma lifestyles. Trichoderma genomes are endowed with a high number of SMs biosynthetic genes, and understanding the genetic basis of their biosynthesis is crucial for determining the role of these metabolites in Trichoderma ecophysiology and for expanding their application in crop protection. Recent advances on the characterization of the Trichoderma SMs genetic inventory driven by computational biology are discussed 2022-01-01T00:00:00Z