Tesis Doctorals - Departament - Genètica, Microbiologia i Estadística

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Expresión génica y regulación transcripcional de los genes "nrd" en bacterias
(Universitat de Barcelona, 2025-02-21) Marchan del Pino, Domingo; Torrents Serra, Eduard; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[spa] A lo largo de la historia, el ser humano ha estado en contacto con el mundo microbiano, y un claro ejemplo de ello son las bacterias, causantes de diversas enfermedades infecciosas, aunque en otras ocasiones beneficiosas para nuestro organismo. El descubrimiento de los antibióticos marcó un hito en el tratamiento de estas infecciones, constituyendo la primera revolución en el tratamiento de las mismas. Sin embargo, en la actualidad, se hace cada vez más imperativo el desarrollo de nuevas metodologías para abordar y estudiar diferentes microorganismos patógenos, dado que estos pueden generar resistencia a los antibióticos disponibles. Aunque las nuevas generaciones de antibióticos son más sofisticadas y efectivas, están surgiendo bacterias multirresistentes que pueden evadir casi cualquier tipo de tratamiento antibiótico, lo que representa un gran desafío para la salud pública y la medicina moderna. En este contexto, se buscan nuevas alternativas que actúen en dianas antimicrobianas diferentes a los antibióticos convencionales. Por lo tanto, se requiere una comprensión a nivel molecular de los patógenos bacterianos para diseñar terapias dirigidas a objetivos moleculares previamente desconocidos o distintos de los abordados por los antibióticos actuales. Pseudomonas aeruginosa es una bacteria Gram-negativa multirresistente que codifica en su genoma las ribonucleótido reductasas (RNRs), enzimas encargadas de suministrar los desoxirribonucleótidos (dNTPs) necesarios para la síntesis y reparación del ADN. Estas enzimas se han considerado como dianas antimicrobianas en estudios recientes. Aunque en eucariotas sólo se encuentra codificada la RNR de clase Ia, eubacterias como P. aeruginosa, presentan las tres clases de RNR (clase I, II y III), lo que le confiere una gran versatilidad para adaptarse a diferentes ambientes, ya que cada clase tiene unas características que favorecen su actividad en diversas condiciones ambientales. En las últimas décadas, se han identificado varios factores transcripcionales que regulan la expresión génica de las RNR. Uno de estos factores es NrdR, capaz de reprimir todas las clases de RNR, aunque su regulación es incierta. Además, P. aeruginosa tiene capacidad de formar biopelículas o biofilms, donde suele haber un gradiente en la concentración de oxígeno, por lo que es interesante ver cómo puede cambiar la expresión génica de múltiples genes de forma simultánea. En este estudio, nos proponemos profundizar en el conocimiento de la expresión génica y la regulación transcripcional de los genes nrd en bacterias. Por un lado, buscamos los factores transcripcionales asociados a la regulación de la expresión génica del gen regulador de las RNR, nrdR. Hemos demostrado que FleQ regula el gen nrdR al unirse a la caja de unión FleQ Box bajo diferentes condiciones oxigénicas (aeróbicas y anaeróbicas) y durante la formación de biofilm bacteriano. También evaluamos el papel de FleQ durante la infección en el modelo in vivo de Galleria mellonella. Además, hemos desarrollado un sistema que combina el uso de un casete sintético llamado GLOW, que codifica para proteínas fluorescentes como genes reporteros, con plásmidos que pueden permanecer en el citoplasma de la bacteria o insertarse cromosómicamente en el genoma bacteriano. Probamos este sistema en diferentes condiciones comúnmente descritas en la expresión génica en bacterias, utilizando para ello los promotores génicos de las distintas RNRs de P. aeruginosa estudiados en nuestro laboratorio, obteniendo resultados similares a los previamente obtenidos y corroborando la utilidad de nuestro sistema. En conclusión, el entendimiento de la regulación transcripcional del gen nrdR es fundamental y se encuentra un poco más completo gracias a al papel que efectúa el factor transcripcional FleQ. Además, demostramos que el sistema de plásmidos que contiene el casete GLOW es una herramienta efectiva y fácil para estudiar simultáneamente varios genes bacterianos mediante proteínas fluorescentes bajo condiciones ambientales dinámicas.
Deciphering the role of the Arabidopsis thaliana METACASPASE I (AtMC1) as a stress granule protein in proteotoxic stress responses
(Universitat de Barcelona, 2025-03-20) Ruiz i Solaní, Nerea; Valls i Matheu, Marc; Sánchez Coll, Núria; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Rising temperatures pose a significant challenge for plant growth, metabolism, and productivity worldwide. One of the most detrimental consequences of heat stress is the accumulation of misfolded proteins into aggregates, which disrupt cellular homeostasis and can ultimately result in cell death. To counteract this phenomenon, cells activate sophisticated proteostasis mechanisms that aid in maintaining protein quality and eliminating aggregates. A crucial component of this response is the formation of stress granules (SGs), which are biomolecular condensates that assemble in response to diverse environmental stresses, such as heat, oxidative stress, and infections. Although SGs are highly conserved across eukaryotic organisms including mammals, plants, yeast, and protozoa, their precise functions in plants remain poorly understood. However, emerging evidence suggests that SGs play a central role in coordinating plant stress responses. This thesis aims to expand our understanding of SGs in plants, focusing on the Arabidopsis thaliana type I metacaspase 1 (MC1). In Chapter 1, we characterized the function and activity of MC1, demonstrating that it localizes to SGs during proteotoxic stress. Using predictive software, we identified two intrinsically disordered regions (IDRs) in the amino acid sequence of MC1 that regulate its recruitment to SGs. This discovery enabled us to generate and purify a recombinant version of MC1 (rMC1), overcoming previous limitations in isolating type I metacaspases in vitro. Functional assays revealed that rMC1 exhibits a strong and evolutionary conserved capacity to clear protein aggregates, including those formed by pathological protein forms that cause a diversity of life-threatening diseases in humans. We further studied the role of MC1 in proteostasis in aging tissues. Notably, we showed that plant overexpressing MC1 present a delay in leaf aging. Based on this data and previous evidences, we infer that MC1 has an important pro-life function linked to its disaggregase activity and its ability to form SGs. In Chapter 2, we explored the role of MC1 in heat stress responses. Heat shock proteins (HSPs) are crucial for protecting cells from heat-induced damage by maintaining correct protein folding and unfolding, and promoting aggregate degradation. In Arabidopsis, HSP101 is a key component in thermotolerance that participates in aggregate clearance and localizes to SGs under heat stress. Given the similarities between MC1 and HSP101 in terms of their localization and function, we examined their interplay. Our findings demonstrate that MC1 and HSP101 colocalize in SGs under heat stress, and that MC1 is necessary to stabilize and maintain the structural properties of HSP101 SGs. Furthermore, we elucidated that MC1 facilitates HSP101 degradation via autophagy. As HSP101 is essential for seedling thermotolerance, we assessed whether MC1 influences this process. Indeed, MC1 overexpression significantly enhances seedling survival under heat stress, confirming the pro-life function of this metacaspase. In Chapter 3, we expanded our investigation to encompass all type I metacaspases in Arabidopsis and examined their potential functional redundancy. We observed that all three type I metacaspases were recruited to SGs under heat stress. Additionally, MC2, but not MC3, was capable of clearing Serpin1, a known substrate of MC1. However, genetic analyses revealed that the autoimmune phenotype observed in mc1 mutant plants is not exacerbated by additional mutations in MC2 or MC3, nor does the deletion of all three genes affect seedling thermotolerance. These results suggest that Arabidopsis type I metacaspases have diverged functionally over the course of evolution, acquiring specialized roles rather than exhibiting redundancy. In conclusion, this thesis provides novel insights into how SGs contribute to plant stress tolerance. Our findings underscore the significance of SGs in plant proteostasis and elucidate new avenues for research on plant stress biology. Moreover, the discovery that MC1 can disassemble human protein aggregates opens the door for potential therapeutic uses in human diseases.
Biofilms microbianos: Virulencia e interacciones hongo-bacteria
(Universitat de Barcelona, 2024-11-13) Arévalo Jaimes, Betsy Verónica; Torrents Serra, Eduard; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[spa] En la naturaleza la mayoría de los microorganismos viven en comunidades protegidas por una matriz extracelular. Estas agrupaciones, denominadas biofilms, generalmente albergan múltiples especies de microorganismos que interactúan entre sí. En el contexto biomédico, los biofilms se asocian con infecciones comunes, infecciones causantes de morbilidad severa e incluso infecciones causantes de mortalidad. Una vez adquiridas, estas infecciones son difíciles de erradicar, generando típicamente enfermedades recurrentes y/o crónicas. La consecuente prolongación en las hospitalizaciones genera una carga económica considerable e impacta negativamente en la calidad de vida de los pacientes. A pesar de esto, actualmente existen varias limitaciones que dificultan el manejo adecuado de las infecciones causadas por biofilms. La presente tesis estudió interacciones ambientales e inter-especies que influencian las características de los biofilms de hongos del género Candida y bacterias patógenas, siendo de particular interés aquellas relacionadas con la susceptibilidad y la virulencia. En primer lugar, se evaluó el impacto de cuatro medios de cultivo comúnmente usados en las principales características patogénicas de Candida parapsilosis. Las variaciones obtenidas en la tasa de crecimiento, morfología, susceptibilidad y virulencia asociadas al medio de cultivo resaltan el papel de las adaptaciones metabólicas en la patogenicidad de esta levadura y señalan la importancia de elegir adecuadamente las condiciones de los diseños experimentales. Luego, se estudiaron biofilms polimicrobianos de Candida albicans y Pseudomonas aeruginosa. Los resultados demuestran que el orden de colonización de las especies es un factor importante en el establecimiento de las interacciones entre estos microorganismos. En este escenario, los efectos de prioridad no sólo influencian la biomasa de las especies, sino también la estructura, susceptibilidad y virulencia del biofilm resultante. Por otra parte, el estudio de las interacciones no físicas entre C. albicans y Staphylococcus aureus permitió identificar que cambios ambientales relacionados con el pH y la disponibilidad de recursos resultan en nuevos fenotipos de S. aureus. Estas variantes presentan cambios en la expresión de factores de virulencia pertenecientes al sistema agr, que impactan negativamente la supervivencia de larvas de Galleria mellonella. I Finalmente, conscientes de la necesidad de contar con métodos y plataformas confiables para la realización de pruebas de susceptibilidad de biofilms, se evaluó el uso de diferentes tinciones en el estudio de los biofilms de C. parapsilosis. De esta manera, se encontró que las tinciones Syto 9, Naranja de tiazol y Diacetato de fluoresceína tenían un bajo rendimiento en el marcaje de C. parapsilopsis, a diferencia de la tinción FUN-1. Adicionalmente, se diseñó un dispositivo de microfluídica para el estudio de biofilms bacterianos en condiciones dinámicas. El BiofilmChip permite la formación de biofilms partir de cepas de laboratorio, aislamientos clínicos y muestras de pacientes, proporcionando condiciones que se asemejan a las encontradas en infecciones naturales. Además, posee un método de lectura rápido y sencillo basado en la impedancia, que permite monitorear el crecimiento y los cambios de biomasa asociados a la efectividad del tratamiento. En conclusión, el presente trabajo demuestra la importancia de caracterizar las respuestas de los microorganismos que crecen en biofilms frente a variaciones ambientales, para así definir mejores protocolos para su estudio. Además, resalta la necesidad de estudiar las coinfecciones hongo-bacteria como una comunidad, donde las interacciones físicas y químicas y los efectos de prioridad pueden aumentar el potencial patogénico de los microorganismos. Esto permitirá la formación de biofilms en condiciones más realistas, lo que se traducirá en avances de conocimiento en el campo. Por último, el desarrollo conjunto de métodos para la evaluación de susceptibilidad en biofilms, brindará las herramientas necesarias para ofrecer un tratamiento con mayor probabilidad de éxito en el manejo de las infecciones.
Population genomics of the nesting and foraging areas of the loggerhead turtle (Caretta caretta) facing climate change
(Universitat de Barcelona, 2024-11-18) Luna Ortiz, Patricia Astrid; Pascual Berniola, Marta; Carreras Huergo, Carlos; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Genomic techniques are increasingly being utilized in conservation biology, providing critical insights into the behavior, reproductive success, and various threats faced by vulnerable species. The loggerhead sea turtle (Caretta caretta) is particularly sensitive to climate change and presents a complex life cycle characterized by long migrations from nesting populations to foraging grounds. The threats and characteristics of the species highlight the importance of understanding its population dynamics for informed management decisions and effective conservation strategies. In this study, we focused on loggerhead turtles at different life stages. We first established a robust genomic baseline using the 2bRAD technique, analyzing 278 individuals from three regional management units (RMUs): the Mediterranean RMU (MED), represented by individuals from 11 nesting sites; the North West Atlantic RMU (NWA), represented by individuals from two nesting sites; and the North East Atlantic RMU (NEA), represented by individuals from one nesting site. We revealed significant genetic differentiation among the three RMUs and subsequently within the Mediterranean RMU identified three genetically differentiated SubRMUs: Greece (GRE), Levantine (LEV), and Sirte (SIR). Additionally, we tested our baseline assessing the natal origin of juvenile loggerhead turtles in foraging areas employing a hierarchical approach, moving from a global perspective that included all three RMUs to a regional level that considered the three SubRMUs within the Mediterranean. We genotyped 103 individuals from four Mediterranean foraging areas: the Catalan region (CAT), Lampedusa (LAM), Eastern Aegean Sea (EAS), and Western Aegean Sea (WAS). Only 3 individuals were assigned to NWA and 1 to NEA, thus indicating that these foraging areas are primarly used by Mediterranean individuals. Among the 99 individuals assigned to the Mediterranean RMU, the individuals assigned to the Lebantine SubRMU were predominant, with some exhibiting mixed assignments, indicating some admixture among subregions. Finally, to assess the rapid increase in the number of nesting events observed in Spain, we sampled 45 hatchlings from eight nests laid between 2016 and 2019. We found that nests were laid by different females, except for two nests attributed to the same female but within the same season. This suggests that the nesting activity is due to a surge of colonizing individuals rather than a return of females born in the region. We hypothesize that the rising number of colonizers results from successful conserva- tion efforts, a feminization of the originating populations, and earlier sexual maturation among individuals. Using our baseline, we identified in the emerging nesting area, one hybrid nest between an Atlantic female and a Mediterranean male assigned to the Sirte SubRMU, as well as several nests resulting from the genetic admixture among different Mediterranean SubRMUs. Thus, in response to climate change, the expansion into new nesting sites may be promoting genetic mixing between previously isolated populations, with potential implications for the species’ conservation. Our results not only clarify the current status of this colonization, but also highlight the need for ongoing efforts to monitor returning individuals to confirm the establishment of a stable resident population. In conclusion, the results obtained in this study provide critical information for understanding the ecological and evolutionary processes affecting loggerhead sea turtles. Our findings will contribute to enhancing conservation strategies in long lived species with high and complex dispersal behavior by identifying populations impacted by threats beyond nesting areas and facilitating the study of specific origins in mixed foraging habitats.
Less, but more: Massive gene losses and expansions in appendicularians stretch the evolutionary limits of the FGF signalling pathway within chordates
(Universitat de Barcelona, 2024-10-14) Sánchez Serna, Gaspar; Cañestro García, Cristian; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] The extensive diversity of forms in the animal kingdom arises from a limited set of evolutionarily conserved genes that coordinate during embryonic development to build complex bodies. Consequently, minor variations in this conserved set can lead to morphological innovations. The impact of gene loss on the evolution of developmental mechanisms has become an important topic in EvoDevo, especially since genomics revealed a high prevalence of gene loss across the tree of life. Over the past decade, the appendicularian Oikopleura dioica has emerged as a key model for studying this phenomenon in chordates. Despite a drastic reduction in its genome size and the loss of many developmental genes, O. dioica retains a typical chordate body plan, oﬀering a unique opportunity to investigate the organization of gene regulatory networks (GRNs) in a biological system heavily aﬀected by gene loss. Previous research has shown that O. dioica has experienced signiﬁcant losses in developmental signalling pathways, including the complete dismantling of the retinoic acid (RA) signalling machinery and a major reduction in the number of Wnt families. The extensive interactions between these pathways and the Fibroblast Growth Factor (FGF) signalling pathway in other chordates raise an interest in investigating the evolution of FGF signalling in this species. This study characterizes the FGF signalling pathway in O. dioica, revealing an unprecedented remodelling compared to other chordates. The species has lost six of the eight Fgf subfamilies present in the last common chordate ancestor, but the remaining two subfamilies have expanded along with the FgfR gene. Phylogenetic analyses, combined with comparative studies on gene structure and conserved protein motifs, provide robust evidence that the 10 Fgf genes identiﬁed in O. dioica belong exclusively to two subfamilies: Fgf9/16/20 and Fgf11/12/13/14. Analyses of gene structure, putative functional motifs, and developmental expression paherns indicate functional diversiﬁcation of these paralogs, as well as of the FgfR gene, following gene expansion. Additionally, examination of the three main intracellular transduction pathways associated with FgfR activation—MAPK, PLCγ/PKC, and PI3K/AKT—reveals that their main components are preserved and expressed throughout O. dioica development. However, structural rearrangements in FGF signal transduction have occurred, including the loss of classical Ras and Spred genes. Functional studies using the pharmacological inhibitor SU5402 demonstrate that the FGF signalling is crucial for embryonic development in O. dioica. Phenotypic analyses via whole-mount in situ hybridization of tissue-speciﬁc genes and omic approaches reveal that FGF signalling is involved in gastrulation and cellular lineage diﬀerentiation. Finally, by comparing the developmental expression domains of O. dioica Fgf genes with those in the ascidian Ciona robusta, an evolutionary scenario is proposed where the evolution of FGF signalling in O. dioica is related to the transition from an ascidian-like biphasic lifestyle to a fully free-living one. This scenario categorizes expression domain changes into three types: extinction of ancestral domains due to gene loss, functional shuﬄing among surviving paralogs, and innovation of novel expression domains in new paralogs. Overall, this doctoral thesis presents a comprehensive study of the FGF signalling pathway in a chordate with unprecedented levels of developmental gene loss. Our ﬁndings unveil the evolution of the Fgf family in appendicularians as a paradigmatic example of what we call “less, but more”, where massive gene losses, but also extensive duplications, result in the loss, conservation, and innovation of Fgf expression domains. This research provides new insights into the ﬂexibility and resilience of the FGF signalling pathway in chordates and raises questions about the evolutionary signiﬁcance of the Fgf9/16/20 and Fgf11/12/13/14 subfamilies. These ﬁndings contribute to a broader understanding of gene loss in EvoDevo, highlighting the complex interplay between genetic conservation and innovation.
Characterization of Hippo downstream effectors in the maintenance of planarian cellular identity
(Universitat de Barcelona, 2024-10-30) Font Martín, Daniel; Adell i Creixell, Teresa; González Estévez, Cristina; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] The study of tissue renewal in adult organisms has widely been viewed as a source to uncover the triggers for the disruption of cell lineages in pathologies such as cancer. One of pathways involved in this process is the Hippo pathway. The Hippo pathway acts as a mechanosensor of extracellular physical forces. These inputs lead to the control of organ growth through the regulation of cellular fate. However, the exact mechanisms and biological processes that underly this maintenance of cellular fate remain elusive and require further characterization. One of this biological processes is cellular senescence. Cellular senescence has been linked with the onset of tumoral transformation and other age-associated disorders similar to dysregulations of the Hippo pathway. Other processes that share similarities with Hippo in regards to tumorigenesis include the regulation of cell cycle progression, cytoskeleton maintenance and transcriptional regulation. Nonetheless, the connection between the Hippo pathway and these biological processes have been poorly characterized so far. To better understand these mechanisms we use planarians (Schmidtea mediterranea). These are ageless flatworms able to regenerate any body part and constantly resize according to food availability. The aim of this thesis revolves around highlighting the importance of cellular senescence, transcriptomic and cytoskeletal regulation and cell cycle progression downstream of the Hippo pathway to properly maintain cellular differentiation in vivo. In order to assess the role of these biological processes we use a RNAi screening approach by inhibiting putative effectors of the pathway representative of these biological functions. All in all, we found that cellular senescence predates and contributes to the loss of cellular differentiation. Moreover senescence rescues are able to prevent such phenotype. Furthermore we show that defects in maintenance of the cytoskeletal architecture impair proper metaphase progression of the cell cycle and induce dedifferentiation. We also detect that deregulation in transcription also impairs the progression of the cell cycle and is associated with loss of cellular identity. Finally we also correlate defects in the progression of the cell cycle with proliferation and alterations in growth and degrowth dynamics according to the nutrient status of the planarian. In conclusion we affirm that the biological processes of cellular senescence, cytoskeletal maintenance, transcriptional regulation and cell cycle progression are linked with the Hippo molecular pathway and their physiological regulation are key for maintaining the cellular fate.
Study of early signaling events governing epithelial regeneration
(Universitat de Barcelona, 2024-09-13) Esteban Collado, José; Serras Rigalt, Florenci; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] The mechanisms through which cells communicate to initiate a regenerative response remains unsolved. It is established that Reactive Oxygen Species (ROS) are early signals released from injured cells that propagates to stimulate the replacement of lost tissue. ROS triggers the activation of the stress activated protein kinases (SAPKs) p38 and JNK via phosphorylation. Prolonged or elevated activation of these kinases may induce apoptosis, while brief or minimal activation can foster regeneration. Nonetheless, the precise connection between ROS generation and the initiation of regenerative signaling pathways remains incompletely elucidated. To this aim, we used Drosophila wing imaginal discs as a model system due to its well-characterized regenerative ability aCer injury or genetic ablation. We found that The Apoptosis signal-regulating kinase 1 (Ask1) emerges as a pivotal factor for driving regenerative growth. It exhibits the remarkable ability to sense ROS both in dying and living cells. However, in living cells, its activation is intricately regulated by nutrient sensitivity through Akt, a core kinase of the insulin (PI3K/Akt) pathway. Akt phosphorylates Ask1 at Ser83, an indispensable event for Ask1 to catalyze the activation of p38, albeit not JNK. Moreover, nutrient restriction or mutations targeting Ser83 of the Drosophila Ask1 block regeneration. Impediments that can be rescued by the ectopic activation of p38, but not JNK. Besides, ROS plays a critical role in the ligand-independent TNF receptor (TNFR) Wengen (Wgn) activation in response to apoptosis. Wgn, but not Grindelwald (Grnd), exhibits a protective mechanism mediated by the signaling molecule TRAF1, ultimately leading to the activation of the Ask1-p38 axis. This stress-response mechanism could be evolutionary conserved as Wgn cysteine rich domain (CRD) shows greater similarity with TNFR families found in other deuterostomes. Our ﬁndings underscore a non-autonomous activation of a ROS sensing mechanism orchestrated by Ask1, Akt1 and Wgn that results in the activation of a p38-dependent regenerative response.
L’exposició a virus presents en l’ambient: descripció del viroma i avaluació del risc
(Universitat de Barcelona, 2024-07-19) Itarte Sorolla, Marta; Bofill Mas, Silvia; Rusiñol Arantegui, Marta; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[cat] Existeix una gran diversitat de virus presents en l’ambient als quals els humans estem exposats, constituint l’exposoma víric. Les eines de vigilància ambiental, com ara l’epidemiologia basada en aigües residuals, han posat de manifest el seu potencial per monitoritzar la circulació vírica en una comunitat, sobretot amb la recent pandèmia de la COVID-19. Per aquest motiu, aquesta tesi s’ha centrat, primerament, en l’estudi de l’aigua residual com a font de contaminació i eina per a estudis epidemiològics. Concretament, s’han avaluat diferents mètodes de detecció i caracterització dels coronavirus presents en les aigües residuals en un context de pandèmia, principalment mitjançant tècniques de seqüenciació de nova generació (NGS). Els virus excretats a les aigües residuals poden ser resistents als tractaments de les estacions depuradores d’aigües residuals (EDARs) i acabar contaminant les fonts d’aigua que s’utilitzen per beure, per regar o per a ús recreatiu, suposant així potencials vies de transmissió d’aquests virus. En aquesta tesi, s’ha estudiat l’exposició a virus patògens presents en aigües i aliments mitjançant la detecció i caracterització vírica, especialment aplicant tècniques de NGS en aliments de producció orgànica i les seves fonts d’aigua de reg. A més, també s’ha avaluat la contaminació vírica i s’ha explorat la diversitat de virus presents en escorrenties i aigües subterrànies d’un entorn urbà, les quals, en l’actual context de sequera, són de gran interès per a diferents usos. Finalment, considerant que l’exposició ocupacional a patògens pot comportar riscos per a la salut dels treballadors, s’ha avaluat l’exposició vírica en entorns laborals. S’ha quantificat la contaminació vírica i s’han identificat virus presents en aerosols i superfícies d’una EDAR i una granja de porcs mitjançant tècniques de NGS. A més, s’han realitzat estimacions del risc ocupacional associat a l’exposició a virus amb l’avaluació quantitativa del risc microbiològic (QMRA), seleccionant l’adenovirus humà (HAdV) per a l’EDAR i un virus hipotèticament zoonòtic amb característiques similars a l’adenovirus porcí (PAdV) per a la granja de porcs com a patògens de referència. Les vies d’exposició considerades en els models són la inhalació d’aerosols i la ingestió oral a través de superfícies contaminades i el contacte mà-boca. Aquesta tesi ha permès la identificació de virus presents en diverses matrius ambientals, incloent-hi aigües residuals, aigües d’escorrentia, aigües subterrànies, mostres d’origen animal, aliments de producció orgànica i aigües de reg, així com superfícies i aerosols d’entorns de treball. S’ha avaluat el nivell de contaminació vírica mitjançant la quantificació de virus indicadors de contaminació fecal i altres virus patògens. S’han identificat virus humans i animals d’interès aplicant tècniques de NGS, com la seqüenciació massiva d’amplicons i la seqüenciació amb enriquiment de dianes, utilitzant panells específics dirigits a la captura de seqüències de virus de vertebrats o també de coronavirus. Finalment, els resultats de QMRA han revelat un potencial risc ocupacional associat a l’exposició a HAdV per als treballadors d’EDARs, si no es prenen mesures. La simulació de QMRA a la granja de porcs també suggereix un potencial risc en cas d’exposició a un virus potencialment zoonòtic. Els resultats d’aquesta tesi ofereixen una visió rellevant sobre la presència de virus en diverses matrius ambientals i els potencials riscos associats amb l’exposició a aquests, contribuint al desenvolupament de mesures que ajudin a reduir aquests riscos i destacant la importància del monitoratge i la vigilància ambiental per a la detecció de virus emergents i zoonòtics, així com per a entendre les seves rutes de transmissió i identificar possibles fonts de contaminació.
Of flatworms and genes: Systematics, hybridization and evolutionary history of the Corso-Sardinian Dugesia (Platyhelminthes, Tricladida)
(Universitat de Barcelona, 2024-07-19) Dols Serrate, Daniel; Riutort León, Marta; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Planarian flatworms (Platyhelminthes, Tricladida), commonly referred to as triclads, constitute a diverse taxonomic group encompassing a wealth of species exhibiting a broad spectrum of phenotypic diversity. Triclads are present in all biogeographical realms and have colonized terrestrial, marine, and freshwater environments distributed all across the globe, bearing testament to their evolutionary success. Free-living freshwater planarians of the family Dugesiidae encompass some of the most well studied taxa in the group and stand out due to their outstanding regenerative capabilities and diversity of reproductive strategies. Among the 12 genera presently recognized in the family, Dugesia is possibly one of the most emblematic. This taxon constitutes the most speciose genus of Dugesiidae, probably even of Tricladida, and is a well-defined taxonomic group that has silently colonized freshwater environments spread across Africa, Europe, the Middle East and Australasia, in spite of the low vagility of its species. In recent years, numerous studies have placed European Dugesia in the limelight as rising model systems for phylogeography and, ultimately, evolutionary biology. Understanding the processes by which populations differentiate and evolve into new species is fundamental to the field of evolutionary biology and holds significant implications for the underpinning of novel conceptual frameworks and the generation of both local and global patterns of species-level biodiversity. This thesis aims to contribute to the pursual of this knowledge by gaining insights into the processes that have shaped the genetic diversification, speciation and evolutionary history of a seemingly small taxonomic group of the longstanding genus Dugesia, namely: the Corso-Sardinian clade. This particular group, which only comprises endemics from Corsica and Sardinia, caught my interest for several reasons: (i) no phylogenetic or phylogeographic studies dedicated to its taxa were ever carried out; (ii) its diversity had not been evaluated under the light of integrative taxonomy approaches; (iii) this group harbors the only species with a haploid chromosome complement of 7 chromosomes in the entire Western Palearctic region, posing a possible case of speciation by chromosomal re- arrangement; (iv) the only putative case of interspecific hybridization in Dugesiidae (and Tricladida) was reported in this group. To unravel the evolutionary history and processes that have shaped the diversification of this group, I employed an integrative approach, incorporating morphological, karyological and molecular data. This approach has enabled the reevaluation of the taxonomic status of several Dugesia populations, resulting in the description of 3 new species for the group and the uncovering of multiple species candidates whose status and pertinent taxonomic decisions will be addressed in a forthcoming paper. Furthermore, divergence time estimation analyses indicated that most species within this group diverged during the Pleistocene, probably due to population fragmentation, contraction, and extinction events caused by the glacial phases. While it cannot be claimed that the phylogenetic relationships within the Corso-Sardinian clade have been fully resolved, this thesis has significantly contributed to elucidating its hidden diversity and phylogeography, thus laying the foundations for future research on this group. Most notably, by using phylogenomic markers and combining phylogenetic, genetic clustering and introgression analyses, this thesis reports and characterizes the first case of interspecific hybridization in Dugesiidae, and likely Tricladida. Furthermore, I examine the potential origin of the hybrids, their status as a potential hybrid species, and their seemingly mixoploid chromosome set. In addition, I also debate on the established concepts of hybrid speciation and assess their suitability for the current case presented within this thesis. Finally, this thesis has contributed to the planarian research community by presenting the first chromosome-scale genome assemblies of Dugesia. The results obtained from the analysis of these genomes have elucidated the origin of Dugesia hepta’s unique karyotype, while shedding light on the evolution of genome size in the genus. These results highlighted a differential expansion of transposable elements (TEs) in Dugesia involving large DNA TEs as the likely cause for the increment of genome size in this genus with respect to Schmidtea, while discarding a whole-genome duplication event as the underlying cause. Most importantly, these new genome assemblies will benefit the planarian research community and contribute to improving the understanding of the structure, organization, and genome evolution in planarians.
CERKL as a modulator gene of mitochondrial dynamics and stress response in the retina
(Universitat de Barcelona, 2024-04-19) García Arroyo, Rocío; Marfany i Nadal, Gemma; Mirra, Serena; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Inherited Retinal Dystrophies (IRDs) are a clinically and genetically highly heterogeneous group of genetic pathologies characterized by progressive attrition of photoreceptor cells and other retinal neurons, which eventually leads to vision loss. The retina, the most affected tissue in IRDs, is the specialized region of the central nervous system capable of transducing light into neural signals. This neurosensory tissue is particularly susceptible to genetic and environmental alterations due to its highly active metabolism, external location, and daily light irradiation. Therefore, the disturbance of the balance between retinal resilience systems and endogenous and exogenous stress factors eventually leads to several alterations that underlie the pathogenesis of many IRDs, such as mitochondrial dysfunction, misregulated autophagy, and activation of cell death pathways. CERKL (CERamide Kinase-Like) mutations cause two different IRDs in humans: Retinitis Pigmentosa and Cone-Rod Dystrophy. While the precise role of CERKL remains unclear, numerous studies have proposed CERKL as a resilience gene against oxidative stress, by participating in the formation of stress granules, regulation of the antioxidant protein TRX2 and inhibition of oxidative stress-induced apoptosis, among other functions. Previous work from our research group led to the generation of the CerklKD/KO mouse model, characterised by a strong deficiency in the expression of Cerkl. This model mimics the disease progression of CERKL-associated Retinitis Pigmentosa-affected patients, showing a slow and progressive loss of photoreceptors and, ultimately, vision impairment. Taking advantage of the CerklKD/KO mouse model, we aimed to dissect CERKL function in mitochondrial metabolism and dynamics. Our findings describe a pool of CERKL isoforms colocalizing with mitochondria. In addition, we observed accumulation of fragmented mitochondria and mitochondrial bioenergetics dysfunction in CerklKD/KO retinas. Moreover, mitochondrial distribution and trafficking were reduced in retinal and hippocampal neurons upon Cerkl depletion, reflecting the important role of CERKL in the regulation of mitochondrial network morphology and energy production. Furthermore, we sought to analyse the impact of CERKL downregulation on stress response and activation of photoreceptor death mechanisms upon light/oxidative stress. Data collected from CERKL silencing and overexpression experiments in ARPE-19 cells (derived from retinal pigment epithelium) revealed that CERKL exerts a protective role maintaining the mitochondrial network morphology against oxidative damage. Additionally, using CerklKD/KO albino mouse models, we assessed immediate (early) or after two weeks (late) retinal stress response to light injury, using data from transcriptomics, metabolomics, and immunohistochemistry images. Our results showed that Cerkl depletion causes an exacerbated response to stress in basal conditions, through alterations in glutathione metabolism and stress granule production. Consequently, upon light-stress exposure, CerklKD/KO retinas cannot cope with additional stress factors, resulting in increased ROS levels and the subsequent activation of several cell death mechanisms. To sum up, our studies indicate that Cerkl gene is a novel player in regulating mitochondrial organization and metabolism, together with light-challenged retinal homeostasis, thus suggesting that CERKL mutations cause blindness by impairing the mitochondrial homeostasis and oxidative stress response in the retina. These findings contributed to determine early phenotypic biomarkers of the Cerkl-depleted mouse retina, which will be compared and confirmed with those observed in retinal organoids derived from CERKLR257X patient-derived hiPSCs (currently under differentiation). Altogether, these studies will allow us to test the feasibility of genetic rescue using a proof-of-principle AAV-based gene augmentation therapy for CERKL-associated IRDs, as well as novel gene delivery systems using gold nanoparticles as vectors. The principal area of cooperation of this Thesis in the Sustainable Development Goals (SDGs) is related to the SDG 3: “Good health and well-being” according to the point 3.8: “Achieve universal health coverage, including financial risk protection, access to quality essential health-care services and access to safe, effective, quality and affordable essential medicines and vaccines for all”. Moreover, an important part of our findings was result of fruitful collaborations with groups with high expertise in other areas. Therefore, the objectives of this work are also related to SDG 17: “Partnerships for the goals” according to the point 17.6: “Knowledge sharing and cooperation for access to science, technology and innovation”. Finally, this PhD Thesis has been directed, supervised and tutorized by women, Dr Gemma Marfany and Dr Serena Mirra, and our research group has also been mostly composed of female scientists during many years, contributing to SDG 5: “Gender equity and empowering of girls and women” according to the point 5.5: “Assure effective and full participation of women and equity in leadership opportunities at all deciding levels in politic, economic and public life”.
Modificación enzimática de la celulosa para la producción de biomateriales
(Universitat de Barcelona, 2023-10-19) Cabañas Romero, Lourdes Verónica; Valenzuela Mayorga, Susana Valeria; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[spa] La creciente preocupación por la sostenibilidad y el impacto ambiental de los derivados del petróleo ha impulsado el desarrollo de nuevos materiales basados en fuentes renovables y amigables con el medio ambiente. Entre estos materiales, los polímeros naturales, como la celulosa, han surgido como una de las alternativas más prometedoras para reemplazar los a los plásticos y otros polímeros contaminantes. La celulosa, que es el polímero más abundante en la tierra, ha demostrado ser un recurso valioso debido a su carácter biodegradable, renovable e insoluble en muchos solventes, atribuido a sus enlaces de hidrógeno y su estructura cristalina. Esta tesis se enfoca en la modificación de la celulosa con dos propósitos diferentes. Primeramente, se exploró la funcionalización de la celulosa, es decir dotar este material de nuevas propiedades. Luego, se estudió el uso de celulasas como agentes biorefinadores para la producción de papel. La primera funcionalización consistió en añadir a la celulosa bacteriana un biopolímero, el quitosano. Se produjeron nanocomposites de celulosa bacteriana y quitosano mediante dos métodos diferentes: sumergiendo hojas de celulosa bacteriana en una solución acuosa de quitosano (BC−ChI) y empapando la pulpa de celulosa bacteriana con quitosano antes de producir hojas de papel (BC−ChM). Estos nanocomposites se investigaron para evaluar sus características físicas, sus propiedades antimicrobianas y antioxidantes, así como su capacidad para inhibir la formación de biofilms en su superficie. Ambos nanocomposites conservaron el carácter hidrofóbico y las propiedades de barrera de la celulosa bacteriana. Además, los nanocomposites mostraron actividad antimicrobiana Staphylococcus aureus y Pseudomonas aeruginosa, así como contra la levadura Candida albicans. También se encontró que la incorporación de quitosano aumentó la actividad antioxidante de la celulosa bacteriana. La segunda funcionalización se centró en la modificación enzimática de la celulosa bacteriana y vegetal mediante el uso de la enzima oxidativa SamLPMO10C, una monooxigenasa lítica de polisacáridos (LPMO). La celulosa bacteriana y vegetal fueron funcionalizadas por oxidación enzimática, lo que aumentó la cantidad de grupos carboxilo en ambas. A continuación, se añadió una solución de nitrato de plata y se produjeron soportes de papel que contenían nanopartículas de este metal, lo que permitió la interacción entre los iones de plata y los grupos hidroxilo o carboxilo de las celulosas. La formación de nanopartículas de plata se dio mediante reducción térmica. Estos soportes de papel funcionalizados con plata exhibieron propiedades antibacterianas contra Staphylococcus aureus. SamLPMO10C demostró su potencial como enzima modificadora de celulosa y, por tanto, impulsó la investigación de estrategias de expresión que permitieran producir este tipo de enzimas de manera más eficiente, rápida y económica. Se ha producido exitosamente dos LPMOs activas, SamLPMO10C y ShaLPMO10A, en Escherichia coli y Streptomyces lividans. Seguidamente, se observó que ambas mostraron actividad oxidativa en un amplio rango de temperaturas, lo que las convierte en candidatas prometedoras para ser utilizadas a nivel industrial. La segunda modificación de la celulosa consistió en la evaluación del potencial de la enzima Cel6D, una exocelulasa recientemente descubierta, como agente biorefinador para la pasta de lino. Se compararon los efectos de Cel6D con otra enzima, Cel9B, y una combinación de ambas. Los resultados mostraron que los tratamientos con las enzimas Cel6D y Cel9B, tanto por separado como combinadas, mejoraron la permeabilidad al aire de las hojas de pasta de lino. Además, las enzimas tuvieron efectos variados en las propiedades mecánicas de las hojas de papel, como el índice de resistencia a la tensión, el índice de resistencia a la tracción y el índice de resistencia al rasgado, lo que sugiere que las exocelulasas y endocelulasas pueden tener aplicaciones diferenciadas en procesos de biorefinado y en otras aplicaciones biotecnológicas
Estudi de l’efecte dels canvis socials sobre la conducta de tres grups familiars de goril·les de plana occidental (Gorilla gorilla gorilla) captius. A la recerca d’indicadors conductuals de benestar
(Universitat de Barcelona, 2024-01-24) Martínez Gutiérrez, Raquel; Cubedo Culleré, Marta; Riba, Carles, 1949-; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[cat] Un dels pilars bàsics que componen la raó de ser dels parcs zoològics actuals és la conservació ex situ d’espècies amenaçades. Aquesta comesa, comporta el compliment d’una sèrie de requisits, entre ells, el de mantenir un bon estat de salut psicològic dels animals que acullen, especialment dels grans simis, donada la seva complexitat cognitiva i sensibilitat emocional. La present tesi doctoral amplia el coneixement sobre l’avaluació del benestar en grans simis captius, i més concretament, en l’espècie Gorilla gorilla gorilla (Goril·la de plana occidental) la qual presenta una important població captiva arreu del món, proposant tres indicadors conductuals de benestar, descrits a partir de l’estudi de l’efecte de diferents tipus de canvis socials sobre la conducta de tres famílies de goril·les que habiten en dues institucions zoològiques europees, Zoo de Barcelona i Zoo de Leipzig. L’anàlisi de l’afectació del canvi social sobre la conducta, ha revelat que les nostres famílies d’estudi han dut a terme una estratègia basada en la flexibilitat conductual a l’hora de fer front a la situació d’estrès viscuda. Aquesta flexibilitat s’ha estudiat en tres conductes: Manteniment bàsic (Autoesplugar), Inactivitat i Joc individual, les quals es troben presents tant en la situació prèvia al canvi social com en la situació posterior al canvi social, sent doncs, les més importants per a les nostres famílies. S’ha comprovat, a més, que la funció de cadascuna d’aquestes conductes com a instruments de gestió de l’estrès depèn de diversos factors, sent els més importants: l’individu, el grau de parentiu entre l’individu i el subjecte participant del canvi social, el tipus de canvi social, la laxitud de la jerarquia de les femelles i la dinàmica del mascle dominant dins el grup. Aquests factors causen variacions tant a nivell individual com a nivell grupal a l’hora d’afrontar el canvi social. Donat que les conductes que ens ocupen han format part d’una resposta a l’estrès, aquestes són susceptibles a esdevenir indicadors conductuals de benestar empobrit, ampliant així els tipus ja existents, el quals es basen principalment en conductes anòmales, com per exemple la Regurgitació/Reingesta (R/R) o bé el hair plucking, en el cas dels goril·les.
An Omics World: Colonization and Adaptive Processes of Styela plicata
(Universitat de Barcelona, 2024-01-22) Galià Camps, Carles; Pascual Berniola, Marta; Carreras Huergo, Carlos; Turon Barrera, Xavier; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] In the last decades biological invasions have emerged as a global concern, as they impact local biodiversity by disrupting ecosystem functioning and ultimately affect human health, economy and wellness. In a climate change scenario, catalyzed by globalization, the issue of invasive species is expected to become magnified in the near future. However, little is known about the intrinsic mechanisms allowing invasive species to overcome new conditions and become a major threat worldwide. This PhD thesis aims to study the colonization and adaptive processes of the invasive tunicate S. plicata worldwide using ‘-omic’ approaches, including population genomics and microbiome. By combining multiple sequencing techniques such as PacBio Long Reads, Illumina Whole Genome Sequencing (WGS), Illumina RNAseq and Illumina amplicon sequencing, we have adopted multiple approaches to the study of this species. First, by using 80 publicly available genomes, we evaluated general output trends when using the 2b-RADseq technique depending on the genomic architecture of target species, demonstrating that overall the technique does not generate biases although it slightly enriches exonic regions. Furthermore, by empirically testing the effect of 2b-RADseq base-selective adaptors on genotyping using four S. plicata individuals, we paved the way for cost-effective population studies on this species. Next, we built and annotated a “de-novo” reference genome for S. plicata, and combined it with Illumina WGS of 24 individuals worldwide. The resulting pangenome revealed the presence of 4 potentially adaptive inversions, the first evidence of population structure, SNPs related to local adaptation, and mito-nuclear interactions. These methodological improvements and the generation of a reference genome assembly allowed us to analyze 87 individuals from 18 localities worldwide using 2b-RADseq to further reveal its population genomic structure, finding clear signals of local adaptation and recent demographic bottlenecks related to invasive events. Finally, using Illumina sequencing of the V4 region of the 16S gene of several tissues of juvenile and adult individuals and water samples from three harbors, in addition to adult samples on two of these harbors over two years, we shed light on the dynamics of the microbiome hosted by S. plicata. We revealed that Methylocaeanibacter sp. ASV0 is the most important bacteria in the tunic, whereas Gammaproteobacteria ASV1 and the unidentified bacteria ASV2 are so in the gill for holobiont survival. Moreover, we found evidence of abundance shifts through adulthood for these bacteria, suggesting active enrichment by S. plicata towards its symbiotic bacterial community. Finally, we revealed potential adaptive bacteria responding to seasonality and trace elements such as Candidatus Hepatoplasma sp. ASV11 and Endozoicomonas sp. ASV50, among others. This multilevel approach provides a solid basis to properly understand the mechanisms of Styela plicata and other invasive marine species to colonize and quickly adapt to new habitats.
Role Of The Innate Immunity In The Etiopathogenesis of Primary Sjögren Syndrome: Influence Of Viral And Immunogenetic Factors Related To Cell Adhesion Molecules In Systemic Disease And Autoantibody Profile
(Universitat de Barcelona, 2024-01-26) Gheitasi, Hoda; Brito Zerón, María del Pilar; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Sjögren Syndrome is a systemic autoimmune disease that affects the exocrine glands, most notably the salivary and lacrimal glands, although it can affect other mucosal surfaces. Inflammation at these sites leads to a spectrum of sicca symptoms, thereby the predominantly symptoms are dryness of the mouth and the eyes. It is not limited to exocrine gland dysfunction; it may involve any other extraglandular systems as well (kidneys, lungs, central and peripheral nervous systems, among others). It is called primary SjS when it exists as an isolated diagnosis, and associated SjS when it co-occurs with another systemic autoimmune disorder. It is characterized by the presence of anti-Ro/La antibodies and/or the presence of focal lymphocytic sialadenitis. SjS is a chronic, systemic autoimmune disease for which no cure currently exists and the management should be organ-specific. The etiopathogenetic factors contributing to SjS remain enigmatic. The development of SjS can be considered as occurring in three stages: 1) an environmental trigger incites autoimmunity against a specific genetic background, b) the autoimmune response becomes chronic due to aberrant regulatory mechanisms within the immune system, and c) lymphoepithelial lesions and subsequent tissue damage arise from persistent inflammation. The prevailing theory, termed 'autoimmune epithelitis,' posits that the epithelial cells of the exocrine glands are the primary sites of inflammation in SjS. According to this model, lymphocytic infiltrates develop in epithelial cells surrounding or invading organs. These epithelial cells act as central players in the autoimmune response by functioning as atypical antigen-presenting cells. Extensive research has been devoted to investigating the role of salivary gland epithelial cells and epithelial cells of the lacrimal glands in SjS. These studies have demonstrated that epithelial cells are capable of orchestrating both innate and acquired immune responses, thereby affirming their crucial role in the disease process. The study of innate immunity in the context of autoimmune diseases like SjS is still in its infancy. Emerging evidence suggests that innate immune components may play a critical role in SjS etiopathogenesis. In this thesis, we focus on the role of HCV and innate immunity components (pattern recognition receptor SP-D, scavenger receptors CD5 and CD6 and adhesion molecule ALCAM) in SjS etiopathogenesis. Individuals infected with HCV often present sicca symptoms that mimic those of SjS. The virus is known to be associated with autoimmune phenomena and histopathological evidence shows that HCV can be isolated in the salivary glands of infected individuals. SP-D is found in epithelial cells and luminar material in glandular tissues, potentially implicating it in the pathogenesis of primary SjS. CD5, CD6, and ALCAM/CD166 are expressed in inflamed tissues of SjS, and their SNPs have been linked to other immune-mediated inflammatory diseases. Based on the evidence, we believe that individuals with HCV infection and those with specific SNPs in innate immunity components (SP-D, CD5/CD6, and ALCAM/CD166) may exhibit altered disease expression and immunological profiles in SjS. Understanding the role of these factors could provide critical insights into the onset and perpetuation of autoimmune responses in SjS. The overarching goal is to advance our understanding of how innate immunity machinery and specific etiopathogenic factors contribute to the complex landscape of SjS, thereby providing new avenues for diagnosis, prognosis, and therapeutic intervention.
Relevant deubiquitinating enzymes in cilium formation and function in the retina: USP48
(Universitat de Barcelona, 2023-12-15) Sánchez Bellver, Laura; Marfany i Nadal, Gemma; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Ciliopathies encompass a broad group of heterogeneous inherited disorders associated with dysfunction of the cilium, a ubiquitous microtubule-based organelle that translates extracellular stimuli into cellular responses. The retina is one of the most affected tissues by mutations in ciliary genes due to the highly specialised neurosensory cilium that photoreceptors possess, known as outer segment, where photoreception and phototransduction occur. To date, mutations in more than 100 ciliary genes have been associated with retinal degeneration, accounting for almost 25 % of inherited retinal dystrophy (IRD) cases. Proteins related to the ubiquitin-proteasome system play a pivotal role during retinal differentiation and ciliogenesis of photoreceptor cells. In fact, mutations in several genes involved in ubiquitination and proteostasis have been identified as underlying causes of IRDs and/or ciliopathies. USP48 is a deubiquitinating enzyme whose role in the retina is still unexplored. However, previous reports reveal its relevance for neurosensory organs since dominant mutations in this gene are causative of hearing loss. Building upon prior findings regarding USP48 expression in the retina, we sought to uncover USP48 involvement in retinal development and cilium function with the ultimate aim to gather evidences to nominate it as a new candidate gene for unsolved cases of retinal ciliopathies. We demonstrated that USP48 is highly expressed in cones in mouse retinas, in agreement with previous RNA-seq data. Furthermore, we described for the first time that a pool of endogenous USP48 localises to the basal body in retinal cell lines, independently of the cell cycle phase. Intriguingly, the overexpressed full-length and Cterminal isoforms fail to localise to the basal body in serum-starved hTERT-RPE1 cells, raising questions about which USP48 protein isoform precisely localises to the basal body and centrosome. Moreover, neither USP48 overexpression nor siRNA-based silencing affects ciliogenesis or cilium length. However, proteomic analysis of mouse retinas revealed that several USP48 interactors had cilium-related functions. Among USP48 partners, ARL3 and UNC119a drew our attention due to their association with retinal ciliopathies and involvement in protein transport in photoreceptor cilia. Subsequent in vitro analysis indicated that USP48 increases ARL3 and UNC119a protein levels through different mechanisms and interacts with them via distinct protein domains. Furthermore, USP48 is also able to increase the protein levels of different ARL3 mutants. These findings collectively indicate that USP48 could play a role in regulating key ciliary proteins, thereby modulating intracellular protein transport and ciliary trafficking to the photoreceptor outer segment, crucial to maintain photoreceptor homeostasis and function. Due to limitations of cell-based studies – which involve transient transgenesis with a short time frame for system manipulation – and the necessity for a retinal tissue context, we further aimed to generate a USP48-/- human induced pluripotent stem cell (iPSC) line by CRISPR/Cas9. The subsequent differentiation into retinal organoids would allow the study of the retinal and cilium alterations caused by the absence of USP48 during retinal development and differentiation. Unfortunately, we could not identify a homozygous iPSC colony for USP48 deletion, suggesting the deletion of both USP48 loci might lethal in iPSCs. Notably, USP48+/- iPSCs displayed a severe ciliary phenotype, indicating that USP48 haploinsufficiency is disrupting key cellular pathways for cilium formation and further hinting at the potential lethality due to the USP48 knockout. Finally, the differentiation for the USP48+/- iPSC line alongside the BJ iPSC control line was initiated and the phenotypical characterisation of the retinal organoids is still ongoing. In conclusion, our investigation demonstrates that USP48 is a new ciliary regulator that potentially modulates intracellular and ciliary protein transport in the retina. Consequently, we posit USP48 as a new candidate gene for retinal ciliopathies. Finally, the studies in retinal organoids promise to shed light on USP48 role in the retina and photoreceptor cilia.
Dimensionality reduction in multigroup data: applications in integrative omics
(Universitat de Barcelona, 2023-12-18) Millapán Toledo, Carolina Andrea; Reverter Comes, Ferran; Vegas Lozano, Esteban; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] The idea of this research is to propose dimensionality reduction methods considering multi- groups in a dataset. In multivariate analysis, there are many multigroup methods, but they have different objectives than the goals presented in this thesis. Classical principal component analysis (PCA) is considered in this research as it has some- thing similar to our objective which is the exploration and visualization of the dataset. However, this unsupervised method lacks in considering the multigroup configuration. The thesis presents two multivariate dimension reduction approaches under a multigroup configuration. Statistical simulation helps us better observe and control the parameters of interest with these new methods proposed in this research. Thus, in this way, it helps us to conclude how they contribute to the literature of multivariate techniques in the visualization and exploration of high-dimensional data analysis. The method, multigroup principal component analysis (mgPCA), is based on maximizing the interdistances between pairs of observations when the observations belong to different groups. The second method, multigroup dimension reduction (MDR), determines linear varieties that minimize overlap by comparing observations in one group with the rest of the observations in the other groups. It is worth mentioning that a package was created in the R statistical programming lan- guage called MultiGroupO containing our two dimensionality reduction methods, and with vignettes, for better explanation and visualization of our multivariate multigroup approaches on omics datasets or any data analysis.
Del cicle biològic a noves estratègies vacunals pel virus de l’hepatitis A
(Universitat de Barcelona, 2023-10-30) Chavarria Miró, Gemma; Pintó Solé, Rosa María; Costafreda Salvany, M. Isabel (Maria Isabel); Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[cat][cat] El virus de l'hepatitis A (HAV) s'allibera de cèl·lules infectades principalment embolcallat en membranes de la pròpia cèl·lula, formant partícules quasi embolcallades (eHAV). Es creu que la biogènesis d’eHAV podria utilitzar les vies pròpies que la cèl·lula utilitza per a la biogènesis dels exosomes cel·lulars. Les proteïnes RAB GTPases tenen un paper clau en l'alliberament d'exosomes mediat per syndecan-ALIX. En aquest treball, hem estudiat el patró d'expressió de diversos gens RAB que codifiquen proteïnes que poden estar implicades en l'alliberament d'exosomes a la línia cel·lular Huh7-AI derivada d'hepatòcits. El patró d'expressió gènic no ha diferit significativament entre els hepatòcits no polaritzats i els polaritzats, mostrant una expressió més alta de RAB11A, seguit de RAB35 i RAB7A. Per avaluar com la replicació viral afecta l'expressió gènica, s’han utilitzat dues soques d’HAV diferents en la seva capacitat de replicació: la soca HM175 (L0) i la de replicació ràpida HM175-HP (HP). Independentment de l'estat de polarització, l'expressió de RAB7A i RAB35 ha augmentat després de la infecció, sobretot amb HP. La microscòpia confocal ens ha permès identificar una co-localització clara entre les càpsides d'HP i RAB35. Al contrari, les càpsides L0 co-localitzen preferentment amb RAB7A. Aquests resultats suggereixen que s’utilitzen preferentment RAB35 i RAB7A per a la sortida d'eHAV a les cèl·lules infectades amb HP i L0, respectivament. En l’estudi amb cèl·lules polaritzades, RAB7A, RAB11A i RAB35 han co-localitzat amb marcadors tant de la membrana basolateral com apical i curiosament, RAB35 preferentment a la membrana basolateral. Amb els resultats obtinguts, la nostra hipòtesi planteja que RAB35 estaria implicat en la via de trànsit que es produeix a la membrana basolateral, la qual cosa ajudar a explicar l'alliberament més eficient de la població HP respecte la població L0 a través d'aquesta membrana. Per altra banda, tenint en compte la lenta replicació que presenten la majoria de soques d’HAV en diferents cultius cel·lulars i per tant, la baixa producció d’antigen necessari per una vacuna, el nostre objectiu és avaluar el potencial de la població HP com a candidata vacunal. El nostre estudi ha permès demostrar que la població HP mostra uns nivells de replicació més elevats i és més ràpida que la parental L0 en cèl·lules MRC-5, Vero i FRhK-4. Per tant, la població HP seria una bona candidata per produir una vacuna inactivada més barata i assequible. No obstant, un altre inconvenient present en la producció de vacunes és la falta d’adjuvants segurs i eficaços per immunopotenciar la resposta. Per aquest motiu, s’ha estudiat la immunogenicitat de les partícules del virus nues (HAV) i les partícules del virus quasi-embolcallades (eHAV) en presència o absència d’adjuvant en dos models animals diferents, ratolins i porcs. Els resultats demostren que els quasi-embolcalls serien una bona estratègia per potenciar la resposta immune de ratolins i porcs. Concretament, la nostra proposta vacunal utilitzant la soca de replicació ràpida HP i les partícules quasi- embolcallades seria bona per implementar noves estratègies vacunals per l’HAV que abaratirien els costos de producció. A més, l’ús de l’adjuvant CAF01, juntament amb les partícules quasi-embolcallades, aporta resultats molt prometedors, on s’ha observat una resposta accentuada de Th1.
Study of expression levels in MECP2 related disorders using transcriptomics and proteomics: characterizing Rett syndrome and MECP2 duplication syndrome
(Universitat de Barcelona, 2023-07-14) Pascual Alonso, Ainhoa; Armstrong i Morón, Judith; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] MECP2 is a multifunctional gene involved in multiple processes such as transcription regulation, chromatin remodelling, splicing and miRNA regulation. Malfunction of MECP2 due to loss of function mutations leads to Rett syndrome (RTT) whereas its overexpression triggers MECP2 duplication syndrome (MDS). Besides, variants in MECP2 can cause a wide spectrum of phenotypes, from severe congenital encephalopathy with early death to mild intellectual disability (ID). RTT and MDS are two well characterized rare diseases with a partly overlapping phenotype consisting of neurodevelopmental delay, ID, impaired muscle tone, lack or unstable ambulation, little or absent speech, gastrointestinal problems, autism like behaviour and hand stereotypies. With next generation sequencing derived methodologies, gigantic breakthroughs have been done in diagnostics and research. These new omic strategies, such as transcriptomic or proteomic, can be applied to patient-derived samples to obtain answers to some of the still unknown aspects of the molecular effect of MECP2 in RTT and MDS. For the present thesis project, patients with alterations in MECP2 were gathered and three cohorts were created and thoroughly studied and characterized: a classic RTT girls group with large deletions within MECP2, a group of patients with MDS together with their duplication carrier mothers, and a group of boys with ID and neurodevelopmental delay with variants in MECP2. Genotype-phenotype correlations were also attempted for these cohorts. In order to further study patients with classic RTT and MDS we decided to use a multi-omic (transcriptomic and proteomic) approach. For that, 22 classic RTT, 17 MDS, 10 MECP2 duplication carriers and 13 healthy controls were gathered and primary cultured cell lines were established from their skin biopsies. DNA, RNA and proteins were extracted from them all and RNA sequencing and tandem mass tag-mass spectrometry (TMT-MS) experiments were performed. The obtained data was analysed in a case-control approach. The multi-omic analysis revealed shared and distinct altered biological processes for each cohort studied. The gene causing RTT and MDS is the same, but its downstream molecular effects might be opposite. Being able to obtain RNA and protein profiles from these patient cohorts seems to be a promising way to better understand MECP2’s role in the underlying pathomechanism triggering RTT and MDS. Differentially expressed genes and proteins involved in cytoskeleton, vesicular activity or immune system were found, and some of them are highlighted as potential biomarker and therapeutic target candidates. Altogether, we aimed to fill the gap by exploring the patients’ genetics, transcriptomics and proteomics in order to get closer to identifying therapeutic targets and biomarkers that could be used in future clinical trials.
Global evaluation of the fitness and virulence determinants in the phytopathogen Ralstonia solanacearum
(Universitat de Barcelona, 2023-05-19) Pedro Jové, Roger de; Valls i Matheu, Marc; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Losses to plant pathogens pose a major threat to global agriculture and food security worldwide. In the context of globalisation and climate change, the emergence and dispersion of pathogens resistant to conventional management strategies causes destructive outbreaks. One of the most important bacterial phytopathogen is R. solanacearum, the causal agent of the bacterial wilt disease, infecting over 200 plant species. R. solanacearum colonises the vascular system of the plants and blocks the water flow by secreting exopolysaccharides, which causes the wilting symptoms. Moreover, it can persist and easily disperse through contaminated soil and waterways. Many different virulence factors have been studied to date but a comprehensive understanding of the transcriptional regulation during the life cycle of this pathogen is lacking. The huge genetic and phenotypic variability of this traditionally tropical pathogen has led to its spread and establishment in temperate regions. To prevent its dispersal and design efficient management strategies, inexistent to date, a thorough understanding of the pathogen infection and dispersion process is of paramount importance. In this thesis we set to characterise the transcriptomic landscape of R. solanacearum to unravel novel virulence and fitness determinants deployed by the pathogen throughout its life cycle. In the first two chapters, we studied the gene expression profile of the bacterium during in different stages of plant infection (Chapter 1 or C1) and the environmental soil and water stages (Chapter 2 or C2). Overall, we have identified a dynamic expression profile of different metabolism and virulence genes along the life cycle of the pathogen. Consistent with previous analysis, we identified that the Type III secretion system (T3SS) is also transcriptionally active at late stages of infection but also in water. Interestingly, we identified the alkali pH as a cue triggering T3SS expression in water, which links to the pH alkalinisation along infection inside the plant. Moreover, we validated the expression of different virulence factors in planta such as the flagellar or T4P motility along infection. In soil, we identified the expression of multiple metabolic pathways and stress-related genes that are required for the life of the bacterium in the soil. Among them, we described the induction of genes related to lignin degradation, and alternative metabolic pathways to synthetise carbon molecules related to stress tolerance. The last two chapters have the objective to characterise and describe specific genes potentially involved in virulence and/or fitness of R. solanacearum. In Chapter 3 (C3), we studied the role of the catalase KatE in detail. We proved its importance for the detoxification of the hydrogen peroxide but discovered that, possibly to redundancy, its mutation has no biological effect on the virulence or the life of the bacterium inside the plant. Finally, in Chapter 4 (C4), we took a different approach studying the secretome of R. solanacearum inside the apoplast and xylem sap of the plant. Many potential proteins related to virulence were discovered but we focused on the description of the S8 serine protease protein family. Preliminary results suggest that highly accumulated S8 proteases might be involved in the life of the bacterium inside the plant. To sum up, this thesis provides with a solid background to further study and characterise virulence and fitness factors important for the life cycle of the bacterium. Additionally, we started the description and characterisation of different potential virulence factors important for the bacterium. All this information might be of use in the future to have a comprehensive knowledge of the pathogen and to design novel and efficient management and control strategies.
Determining the Three-dimensional Structure of Genomes and Genomic Domains Integrating Chromosome Conformation Capture Data and Microscopy Images
(Universitat de Barcelona, 2023-03-01) Castillo Andreo, David; Martí-Renom, Marc A.; Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística
[eng] Microscopy and Chromosome Conformation Capture (3C) are the two main techniques for studying the three-dimensional (3D) organization of the genome. Microscopy, allowing the visualization of genomic loci in individual nuclei, pioneered the field of structural genomics and became the gold-standard for the validation of new discoveries. 3C and 3C-based techniques, identifying the number of contacts between pairs of genomic loci, have already been key to unveil the importance of the 3D genome organization in many cellular processes. Both techniques are continuously evolving pushing forward the technologies and giving rise to innovative assays that require the support of new computational methods for data collection, analysis and modeling. In this thesis, I have contributed to provide these essential computational methods to the Structural Genomics community. In Microscopy, I participated in the design and implementation of OligoFISSEQ, a novel multiplexing imaging technology to visualize multiple genomic regions in hundreds and thousands of individual cells. In 3C-based techniques, I contributed to the development of a tool for the reconstruction of the 3D organization of chromatin from highly-sparse 3C-based datasets (e.g. Promoter Capture Hi-C). Finally, I have introduced pTADbit, a novel approach for the reconstruction of the 3D Genome organization integrating both Microscopy and 3C data via the application of Machine Learning methods.

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