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Item Extrusomas de células natural killer: Vesículas artificiales derivadas de células como una alternativa innovadora de encapsulamiento de fármacos para el cáncer de pulmón(Universidad del Desarrollo. Facultad de Medicina, 2024) Carrasco Rojas, Javiera Valentina; Schuh, ChristinaEl cáncer de pulmón (CP) tiene la tasa de mortalidad más alta a nivel mundial. Su patogénesis es multifactorial y, aunque se recomiendan terapias dirigidas, muchos pacientes deben depender de tratamientos clásicos (por ejemplo, quimioterapia), los cuales presentan limitaciones (efectos adversos, resistencias al tratamiento, entre otros). Los extrusomas (EXT), o vesículas derivadas artificialmente de células (ACDVs), han surgido como una herramienta prometedora para superar las barreras de la traslación hacia la clínica que presentan actualmente los tratamientos acelulares basados en vesículas. Este estudio propone la generación de EXTs encapsulados con docetaxel (DTX) a partir de células Natural Killer (NK) humanas (DTX-EXTs) y evaluar su internalización en células de CP y su efecto citotóxico. También se realizaron análisis preliminares utilizando macrófagos polarizados a fenotipo M1 (EXTs-M1) para validar este método como una estrategia prometedora en la producción de ACDVs para la liberación de fármacos en el tratamiento del CP. Los EXTs se generaron por extrusión celular y se evaluaron su morfología, estabilidad y composición. Se midió su efecto citotóxico en células de cáncer de pulmón, y se investigaron los mecanismos de captación celular mediante inhibidores de diferentes vías de endocitosis. Los EXTs-NK y EXTs-M1 mostraron una morfología en forma de copa (tamaño medio <200 nm), los EXTs-NK tuvieron composición estable y su análisis Proteómico reveló proteínas presentes de manera diferencial asociadas con las características de las vesículas extracelulares (EV) y los marcadores de células NK, además de un patrón de enriquecimiento proteico distintivo. Se observó un efecto citotóxico significativo con los tratamientos EXT-DTX en comparación con los EXTs y EVs en ambas líneas celulares, con mayor eficacia en las células A549; la inducción de apoptosis corroboró estos hallazgos. Los estudios de internalización identificaron una localización perinuclear y la participación de la endocitosis mediada por clatrina como mecanismo principal en todos los grupos de vesículas. Similar fueron los hallazgos obtenidos para los EXTs-M1, en donde los DTX EXTs-M1 fueron más eficaces en la reducción de la viabilidad celular en las líneas de CP H1975 y H1299. Asimismo, se identificó preliminarmente, la endocitosis mediada por clatrina como principal mecanismo de internalización en células H1975. En conjunto, los resultados de este estudio sugieren que el uso de EXTs derivados de células inmunes, ofrece un enfoque prometedor para mejorar la entrega de quimioterapéuticos como el DTX, permitiendo reducir las dosis necesarias y, en consecuencia, minimizar los efectos adversos, lo que podría traducirse en una mejora en la calidad de vida de los pacientes con CP.Item Saccharomyces cerevisiae as a model for the identification of modifier genes of paraquat toxicity(Universidad del Desarrollo. Facultad de Medicina, 2024) Rubilar Espinoza, Juan Carlos; Klein Posternack, Andrés David; Cubillos Riffo, Francisco AlbertoParaquat (PQ) is a potent herbicide that induces oxidative stress and mitochondrial dysfunction. In humans, it is highly toxic, and it can induce Parkinson's Disease (PD). PD is a chronic and progressive neurodegenerative disease, with a worldwide prevalence of 315 per 100,000 people of all ages. Recent studies have revealed that neurons from PD patients exhibit stress responses, mitochondrial dysfunction, and metabolic deficits involving ATP and nicotinamide adenine dinucleotide (NAD+). While studies have demonstrated the potential of nicotinamide riboside (NR) to mitigate age- and disease-related metabolic decline in PD, the specific effects of NR on PD induced by pesticide exposure, such as paraquat, have not been extensively explored. Saccharomyces cerevisiae is a model organism that has allowed the study of relevant biological processes over time. It exhibits remarkable genetic diversity, making it an ideal model for studying the genetic basis of phenotypic variation. In this study, we hypothesized that genetically diverse Saccharomyces cerevisiae strains exposed to paraquat exhibit cellular and molecular responses that reveal potential modifier genes associated with its toxicity. We treated 1,011 isolates and 96 segregants from the cross of SA x WE founder Saccharomyces cerevisiae strains with PQ (75 μg/mL). We measured their growth curves and calculated the specific growth rate (μMax), used as a phenotypic trait for the genome-wide association studies (GWAS) and quantitative trait loci (QTL) mapping. We performed mixed-model association analysis using FaST-LMM for GWAS and linkage analysis using R/qtlsoftware, calculating LOD scores with a nonparametric model. We used diploid strains (603 isolates) for GWAS and 96 segregants for QTL mapping, identifying variants and markers that exceeded the significance threshold. Using these variants and markers, we identified candidate genes for validation. We validated NRT1 in a yeast mutant and subsequently showed that NR treatment provided significant protection against PQ-induced damage in both S288C yeast and iPSC-derived dopaminergic neurons from GBA-PD patients. In conclusion, this study provides valuable insights into the genetic and metabolic factors underlying PQ resistance in S. cerevisiae. The findings highlight the importance of NAD+ metabolism and mitochondrial function in mitigating the toxic effects of PQ. The identification of Nrt1 as a key transporter of NR, suggests potential therapeutic targets for interventions aimed at preventing or treating PQ-induced toxicity in PD.Item Formulación tópica a base de compuestos bioactivos naturales y nanopartículas de cobre estabilizadas en miel, para el tratamiento de heridas crónicas.(Universidad del Desarrollo. Facultad de Medicina, 2024) Jaldin Crespo, Limberg; Martínez Arenas, Jessica; Ezquer, MarceloLas complicaciones en el tratamiento de heridas crónicas representan un desafío significativo para la medicina moderna. En este sentido, la necesidad de enfoques terapéuticos integrales y potentes en el cuidado de heridas es cada vez más crítica, dado que una herida infectada, con una colonización bacteriana mayor a 105 UFC/ml, puede desencadenar inflamación crónica y estrés oxidativo, resultando en tiempos de curación prolongados o incluso en complicaciones severas como amputaciones y shock séptico. Al respecto, la presente tesis se enfocó en evaluar el potencial de combinar de manera sistemática y racional productos naturales, como el extracto de propóleo (EP) y el extracto de hoja de olivo (EHO), con nanopartículas de cobre estabilizadas en miel (H-CuNP) como una posible alternativa terapéutica. En primer lugar, partimos con la síntesis de dos lotes de H-CuNP mediante un método verde de reducción asistida por ultrasonido, estableciendo un protocolo reproducible y obteniendo partículas con características controladas. Las nanopartículas fueron caracterizadas utilizando técnicas como espectroscopia UV, TEM/SEM, NTA, DLS y DRX, confirmando la presencia de H-CuNP metálicas con propiedades fisicoquímicas similares a las descritas en la literatura. Simultáneamente, se estableció un proceso de estandarización para asegurar la calidad del EP y EHO, se evaluó el contenido fenólico y la capacidad antioxidante del EP y el EHO, demostrando que el EHO posee un mayor contenido de polifenoles totales y mayor capacidad antioxidante en comparación con el EP. En segundo lugar, se evaluó la actividad antimicrobiana (IC50) de los extractos naturales (EP y EHO) y las H-CuNP, frente a bacterias prevalentes en heridas crónicas, como S. aureus (Gram+) y P. aeruginosa (Gram-). Aquí se evidenció una mayor efectividad de las H-CuNP (IC50:67 μg/ml) y EP (IC50:33 μg/ml) frente a P. aeruginosa y S. aureus respectivamente. Los estudios de viabilidad celular in vitro revelaron que las H-CuNP exhiben un IC50 más bajo (60 μg/ml), indicando mayor toxicidad celular comparada con el EP (IC50: 125 μg/ml) y el EHO (IC50: 250 μg/ml). La evaluación del cierre de herida in vitro demostró que las H-CuNP (50 μg/ml), EP (125 μg/ml) y EHO (250 μg/ml) inhiben la migración de fibroblastos dérmicos humanos (HDF). Para la optimización de combinaciones, se utilizaron dos metodologías: el enfoque del Mayor Agente Único y el Diseño de Compuesto Central rotatorio (DCC). Lo cual permitió identificar combinaciones de H-CuNP, EP y EHO, con una capacidad superior para: inhibir radicales DPP, inhibir bacterias tanto ATCC como multi-drogo-resistentes y manteniendo una viabilidad celular similar a la de sus componentes individuales. Estos hallazgos sugieren que la combinación de H-CuNP con extractos de propóleo y hoja de olivo puede ser una opción efectiva y multifuncional para el tratamiento de heridas crónicas, donde es crucial reducir la colonización bacteriana y regular el estrés oxidativo. Esta estrategia innovadora presenta un potencial tratamiento para heridas infectadas, contribuyendo a la lucha contra la resistencia antimicrobiana y previniendo la cronicidad de las heridas.Publication A yeast cell-based precision medicine platform for the identification of modifier genes of Niemann-Pick Type C severity(Universidad del Desarrollo. Facultad de Medicina, 2024) Las Heras Parraguez, Macarena; Klein, AndrésNiemann-Pick Type C (NPC) disease is a devastating neurovisceral disorder caused by loss-of-function mutations in the NPC1 and NPC2 genes. It is characterized by the buildup of non-esterified cholesterol, sphingomyelin and other lipids in endosomes and lysosomes. The clinical presentations are highly heterogeneous and vary from a rapidly progressing neonatal form to an adult-onset chronic neurodegenerative condition. Currently, no effective treatments exist for NPC disease, prompting an urgent need for new therapeutic options. While some disease-causing variants have been associated to specific clinical outcomes, there exist numerous exceptions that highlight the complex nature of NPC, even among individuals of the same family. Differences in disease presentation and severity have also been observed in NPC mouse models of different genetic backgrounds, suggesting that other genetic factors besides the NPC1 mutation might act as disease modifiers. In this thesis, we aimed identify and validate putative modifier genes of NPC disease severity through studies in human and yeast. Our initial strategy involved using genomic and clinical data from a family affected by the disease, whose members exhibited a broad range of signs and symptoms with varying degrees of severity, to perform linkage analyses (cohort of identification). Typically, such findings are validated using a replication cohort; however, there is no publicly available genomic data of NPC patients that includes clinical information. To overcome this challenge and study the potential modifier role of the genes identified from human linkages, we developed a precision medicine platform using yeast cells. This platform included four already sequenced Saccharomyces cerevisiae strains. We treated these yeast of four different genetic backgrounds with U18, an NPC1 protein inhibitor, to mimic NPC disease severities. The strains exhibited varying responses to the U18 treatment. Then, to map additional genes potentially involved in modifying disease severity, we used segregants derived from parental yeast strains with divergent phenotypes to perform linkage analyses. Subsequently, we obtained lists of putative modifiers from both human and yeast linkages. Finally, we xiv selected several of these candidates for loss-of-function validation experiments in yeast. The results obtained revealed that yeast knockouts for two of the selected genes treated with U18 showed a partial rescue of the evaluated phenotypes (µmax and vacuolar phenotypes) compared to the wild-type strain treated with U18. These findings support the potential of these genes as modifiers of NPC disease severity and position them as potential therapeutic targets or biomarkers of severity. This study represents the first successful human linkage conducted to identify potential modifier genes of NPC severity, as well as the first linkages in a model organism with high resolution. This yeast cell-based platform could help to increase our understanding of some diseases, identify new therapeutic targets, and serve as an invaluable tool for assessing the efficacy of potential treatments. The findings generated from this study could have implications not only for NPC, but also for other LSDs and neurodegenerative diseases, including Alzheimer’s disease.Item Therapeutic effects of mesenchymal stem cell-derived secretome administration on morphine-induced consumption, relapse, and withdrawal in rats: Role of brain oxidative stress and neuroinflammation(Universidad del Desarrollo. Facultad de Medicina, 2023) Quezada Diez, Mauricio Alejandro; Ezquer, Fernando; Berríos-Cárcamo, PabloThis thesis presents the results obtained after the simultaneous intranasal and intravenous administration of the antioxidant and anti-inflammatory secretome generated from preconditioned hMSC on the precipitated (naloxone-induced) and spontaneous (oral model) withdrawal syndrome, the reduction in its chronic consumption, and the relapse to voluntary oral consumption (oral model) in rats exposed to continuous doses of opioids. Additionally, the association and role of oxidative stress and neuroinflammation in the different stages of morphine addiction were evaluated.Item Identification of modifier genes/networks of lysosomal biology(Universidad del Desarrollo. Facultad de Medicina, 2023) Durán Mojica, Anyelo Alberto; Klein Posternack, Andrés David; Calderón, Juan FranciscoLysosomal storage diseases (LSDs) are a heterogeneous group of ~70 rare inherited metabolic diseases caused by loss-of-function variants in genes encoding for lysosomal enzymes, their activators, or transport proteins. Clinical symptoms manifest during early childhood or adolescence, causing varying degrees of disability and short life expectancies. At a cellular level, LSD cells show a progressive accumulation of undegraded substrates. In a subset of LSDs, called sphingolipidosis, the primary buildup material corresponds to lipids. These diseases can affect several organs, including the liver, brain, heart, peripheral nervous system, haematologic, skeletal, gastrointestinal system, lung, muscle, and others. On the other hand, defects in processing sphingolipids have also been observed in patients with common diseases such as neurodegenerative disorders and cancer. To date, the phenotypic variability observed in monogenic conditions is thought to be influenced by genomic loci variation other than in the primary disease locus. These genes are called modifiers. We harnessed the natural genetic variation between different strains of healthy mice to identify modifier genes/networks of lysosomal biology. We used a systems genetics approach. We measured the hepatic activity of 12 lysosomal enzymes and several of their natural substrates in livers derived from a panel of inbred mouse strains, followed by genetic regulators mapping by genome-wide association studies (GWAS), transcriptome associations, Bayesian integration, and pathway enrichment analysis. The GWA study identified 137 non-redundant genes associated with changes in lysosomal enzyme activities and 1744 modifiers for GSLs. Among these genes, 30 are shared between the enzymes and lipid groups. They are clustered into three pathways and associated with other nonLSD diseases. Surprisingly, they are under the regulation of ten common transcription factors, suggesting a common regulation of sphingolipid metabolism. In summary, we have identified novel regulators of lysosomal enzymes and GSL levels that may serve as therapeutic targets for LSD and implicated GSL metabolism in other diseases.Item Healthy migrant effect on cardiovascular disease and risks factors: Analysis from the social determinants of health approach(Universidad del Desarrollo. Facultad de Medicina, 2023) Rada Ramírez, Isabel Cristina; Cabieses, BálticaThe international migration has steadily increased in Chile during the recent decades reaching an estimation of 1.452.104 international migrants. It has been described an intra-regional pattern called south-south migration. The health of this population is recognized as a global public health priority, mainly chronic diseases given the epidemiological transition that also has impacted migrant population. Currently, there is controversial evidence a possible healthy migrant effect on cardiovascular diseases and its risk factors, which suggest to lower prevalence of this conditions in comparison to local population. It has been postulated that the effect might be modulated by social determinants of health. The prevalence of cardiovascular conditions among migrant population residing in Chile is unknown, as well as the existence of the healthy migrant effect on cardiovascular health has not been investigated yet. In order to analyze the existence of this effect in migrant population compared to locals four sub-studies were conducted. The first substudy aimed to describe the scientific evidence of the last five years on healthy migrant effect on cardiovascular risk factors and cardiovascular diseases in the international migrant population vs. locals. A narrative review was conducted following a systematic search. The second substudy analyze the existence of EMS in self-reported chronic morbidity in the international migrant population vs. local, and its related social determinants (demographic, socioeconomic, health insurance, migratory process). From wich a secondary analysis of CASEN 2017 survey was performed with complex samples analyses. The third substudy aimed to analyze the existence of the healthy migrant effect on cardiovascular diseases and risk factors in the international migrant population vs. locations, and their related social determinants (demographic, socioeconomic, health system, psychosocial, and migration). The primary data collection included n=6.626 participants (n=3.324 Chileans and n=3.302 migrants) residing in La Pintana, La Granja and San Ramón who responded a survey of cardiovascular health and social determinants of heath. The second and third substudy analyzed the crude prevalence of health outcomes as proportions, the Pearson’s chi-square test was used to compared health outcomes between migrants and locals. Multivariate logistic regression was used to test the association of social determinants of health and health outcomes. The healthy migrant effect was tested by multivariate logistic regression sequentially adjusted by social determinants of health (reference=Chilean). The fourth substudy aimed to offer a regional perspective of the growing challenges faced by international migrants in Latin America in accessing hypertension preventive care from human rights, equity and universal primary health care approaches. The results of the first substudy revealed scarce reports of intraregional in Latin America, heterogeneous evidence which difficults consensus for healthy migrant effect on cardiovascular health. The second substudy showed a lower crude prevalence of chronic diseases that remained significant after adjusting by social determinants of health. Meanwhile the third substudy revealed an unadjusted lower prevalence of cardiovascular diseases, metabolic risk factors, tobacco and alcohol consumption. The advantage on acute myocardial infarcation disappeared after adjusting by demographic determinants of health, while the effect on cerebrovascular accident disappeared after adjusting by access to health care determinants of health. The healthy migrant effect on metabolic risk factors, alcohol and tobacco consumption remained significant after adjusting by all social determinants of health. The present study contributes to the knowledge of health and migration, make visible challengues on cardiovascular health from social determinants of health perspective and provide evidence for local and regional public health.Item Evaluation of the promotion of pro-metastatic capacities mediated by sEVs secreted by MDA-MB-231 metastatic breast cancer cells and identification of “EMT-promoter” sEV-miRs present in their cargo(Universidad del Desarrollo. Facultad de Medicina, 2023) Durán Jara, Eduardo Felipe; Lobos-González, Lorena; Ezquer, MarceloBreast cancer (BC) is one of the most common and deathly cancers worldwide. However, despite the improvements in screening and treatment, there is a high probability of local recurrence and distant metastasis to occur; the latter being the main cause of the patient’s death. Communication between heterogeneous tumor cells mediated by small extracellular vesicles (sEVs) is essential to promote tumorigenesis and metastasis. sEVs are nanosized vesicles secreted by all cell types that mediate intercellular communication through their cargo, which include nucleic acids, proteins and other biomolecules. However, the mechanisms and specific molecules involved in these phenomena are still not completely defined and vary between different cancer types. Among the molecules described in the cargo of sEVs are microRNAs (sEV-miRs); small non-coding, single-stranded RNA molecules of approximately 20 nucleotides, which are master regulators of gene expression. It is widely demonstrated that cellular miRNA dysregulation can promote tumor growth, progression and metastasis. These findings have positioned miRNAs, and particularly sEV-miRs as a new research focus worldwide. Epithelial-mesenchymal transition (EMT) is a complex and dynamic process that involves many cellular and molecular changes. Cells undergoing EMT can increase their tumorigenic and pro-metastatic capacities, such as cell migration and invasion, cytoskeleton remodeling, increased anchorageindependent growth, among others. Some miRNAs have been implicated in the regulation of EMT in BC, such as members of the let-7 and miR-200 family, as well as miR-105, miR-21 and miR-10b. However, to date there are very few studies that consider BC tumor cells-secreted sEVs as vehicles for “EMTpromoter” sEV-miRs, favoring the EMT and EMT-related phenotypic and functional changes (such as increased migration), promoting the tumorigenic and/or metastatic potential of recipient cells. Therefore, we hypothesize that the EMT and the migration of cells with no metastatic potential is favored by specific sEV-miRs in the cargo of metastatic BC cell-secreted sEVs. The aim of this project is to characterize the sEV-miRs profile of metastatic BC cells and identify specific sEV-miRs that could induce EMT and/or migration in cells with no metastatic potential. The findings of this thesis could be relevant in order to identify new possible BC biomarkers in sEVs, as well as the possible use of specific sEV-miRs as therapeutic options to treat this disease.Publication Reconstruction of the evolution of the Chilean-Cordobes clone of methicilin resistant: Staphylococcus aureus in Latin America(Universidad del Desarrollo. Facultad de Medicina, 2023) Martínez Solís, José Rodrigo Waldemar; Munita, José; Calderón Giadrosic, Juan FranciscoThe global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The MRSA Chilean-Cordobes clone (ChC) predominated widely throughout several countries of Latin America, but during the mid-2000s the USA300-SAE clone quickly replaced the ChC in Colombia and Ecuador. Most notably, this replacement was not observed in Peru or Chile, where the ChC remains as the most frequent clone. Here, we aimed to perform a phylogenomic reconstruction of the evolution of the ChC in Latin America and to estimate the effect on the fitness of genetic features that are particular to the ChC clone from Chile. To achieve this aim, we sequenced 925 genomes of MRSA strains from Latin America. First, we performed whole genome sequencing and confirmed that the ChC clone remains the most frequent clone in our country. However, this lineage has been gradually replaced, in a nontypical MRSA clonal replacement event, by new emerging clones (ST105-SCCmecII and ST72-SCCmecVI). Then, we explore the impact of heavy metal resistance genes in the evolution of the ChC clone and provide evidence to suggest a possible link between the release of high quantities of heavy metals in the aftermath of an environmental disaster and the divergent evolution of the ChC in Latin America. Finally, we found evidence that the Sau1 restriction modification system plays a key role in the genomic evolution of the Chilean-Cordobes clone in Latin America by increasing the acquisition of foreign DNA. Overall, these results have the potential to greatly enhance our understanding of MRSA dissemination and contribute to improved approaches for combating this pathogen in Chile and Latin America.Item Conexina 46 es un regulador transcripcional en células HeLa(Universidad del Desarrollo. Facultad de Medicina, 2022) Fernández-Olivares, Ainoa; Retamal, Mauricio A.; Acuña, RodrigoCancer is the second leading cause of death in the world and was responsible for around 10 million deaths in 2020. Metastatic colonies are of greatest interest to clinical oncologists as they are responsible for approximately 90% of cancer-associated deaths. Therefore, understanding the molecular mechanisms underlying tumor progression, local invasion, and metastasis formation represents one of the great challenges in cancer research. Connexins are membrane proteins that form ion channels that participate in cell communication, which is essential for tissues to maintain the ability to grow in a controlled manner and respond to their environment. However, this process is frequently impaired in cancer cells. This is why during the last 50 years, the role of connexins and cell communication has been highlighted in the area of cancer and much of the research effort has been directed to understanding their dysfunction in malignant neoplasms in humans. Within the Connexin family, Cx46 has been proposed as a biomarker of malignancy in breast cancer, it has also been directly correlated with the presence of cancer stem cells in glioblastoma. In addition, it has been positively correlated with invasion, self-renewal capacity and metastasis independently of GJC formation. Each of these processes of transformation towards a malignant phenotype requires the reprogramming of gene transcription. Based on the data collected in the literature, the following hypothesis is proposed, cx46 produces phenotypic changes in HeLa cells through gene regulation networks. For this, a stable cell line of HeLa cells over-expressing human Cx46 was generated. The following objectives were studied in it, 1: To evaluate phenotypic characteristics associated with the expression of Cx46 in HeLa cells by means of proliferation, self-renewal and invasion assays. 2: Determination of the subcellular localization of Cx46 by purification of nuclear proteins and confocal microscopy. 3: Determine the nuclear role of the Cx46 protein through ChIP-seq and RNA-seq studies. The results obtained show that HeLa cells transfected with human Cx46 undergo phenotypic changes such as an increase in their capacity for self-renewal proliferation, migration and invasion. In addition the nuclear presence of the protein, which is found interacting with DNA, could be evidenced. In this context, 77 differentially expressed genes could be found, of which 26 have a Cx46 peak in their promoter region. The expression network analysis determined that those genes that did not have Cx46 in their promoter region were being activated by transcription factors with which Cx46 was interacting. The data obtained in this thesis allow us to propose a new role for Cx46 as a regulator of gene transcription in the HeLa cell line. Based on the number of tumors that overexpress Cx46 with respect to their surrounding healthy tissue it is plausible to propose Cx46 as a tumor biomarker and therefore as a possible therapeutic target, in accordance with the literature.Item Evaluación del efecto antidepresivo del secretoma derivado de células madre mesenquimáticas precondicionadas en un modelo animal de trastorno depresivo mayor(Universidad del Desarrollo. Facultad de Medicina, 2022) Ávila Suárez, Alba Liliana; Ezquer, Fernando; Riveros, María EugeniaEl trastorno depresivo mayor es un desorden heterogéneo y multifactorial caracterizado por episodios de al menos dos semanas de duración, en los cuales se presente al menos uno de los dos síntomas centrales. Estos son: 1.- Estado de ánimo depresivo con pérdida de interés por las actividades cotidianas y 2.- Ausencia de la capacidad de sentir placer (anhedonia). Estos síntomas traen consigo trastornos anímicos, cognitivos y fisiológicos y en los casos más severos, pueden asociarse a conductas suicidas. Si bien en la actualidad la etiología del trastorno depresivo mayor es poco conocida, se ha reportado que tanto la presencia de neuroinflamación como la reducción de la neurogénesis hipocampal adulta son eventos claves en el desarrollo de esta patología. Las terapias actuales para el trastorno depresivo mayor no están enfocadas en estas alteraciones, lo que podría explicar el alto porcentaje de refractariedad al tratamiento de pacientes con trastorno depresivo mayor frente a la terapia convencional. Por este motivo, se ha propuesto como una opción de tratamiento el uso de células madre mesenquimáticas (MSC), las cuales son capaces de producir de manera paracrina, una gran variedad de moléculas terapéuticas entre las que se encuentran moléculas anti-inflamatorias y factores neuroprotectores inductores de la neurogénesis. Estas moléculas secretadas por las MSC se pueden recuperar desde el medio de cultivo celular y en conjunto reciben el nombre de secretoma. Este trabajo se basó en la evaluación del potencial antidepresivo de la administración intranasal repetida de secretoma derivado de MSC en un modelo animal de trastorno depresivo mayor instaurado en el bioterio de la Universidad del Desarrollo. En conjunto, los datos obtenidos muestran que el tratamiento intranasal con secretoma derivado de MSC fue capaz de revertir algunas de las conductas asociadas a estados depresivos en animales, disminuyendo significativamente el puntaje global de depresión en comparación a los animales tratados con vehículo. A nivel estructural el modelo animal de depresión utilizado no mostró presencia de alteraciones del proceso de neurogenesis hipocampal adulta pero sí mostró neuroinflamación, evidenciada en un aumento de la densidad de astrocitos a nivel del hipocampo en comparación a los animales control. Este aumento no se observó en los animales tratados con secretoma intranasal, sugiriendo un efecto anti-inflamatorio del biofármaco. Adicionalmente, se observó que el secretoma derivado de MSC ejerció un efecto protector sobre los astrocitos inhibiendo el proceso de atrofia de sus prolongaciones. Los datos pre-clínicos obtenidos sugieren que la administración intranasal de secretoma derivado de células madre mesenquimáticas representa un tratamiento potencialmente eficaz para reducir los síntomas de depresión en humanos y una posible alternativa terapéutica a los tratamientos actuales para esta devastadora patologíaItem Genetic modifiers associated with the variability of the aortic phenotype in patients with inheritable connective tissue disorders(ICTDs)(Universidad del Desarrollo. Facultad de Medicina, 2022-05-27) Jiménez Bejarano, Yanireth José; Calderón Giadrosic, Juan FranciscoInheritable Connective Tissue Disorders (ICTDs) are rare genetic diseases that involve variants in genes that encode for proteins of the extracellular matrix (ECM). ICTDs affect patients from birth and their symptoms are present in the cardiovascular system, musculoskeletal system, the skin, the eye and the respiratory system. This family of diseases includes Marfan Syndrome (MFS), Loeys-Dietz Syndrome types I, II, III and IV (LDS), Ehlers- Danlos Syndrome type IV (vEDS), among others, and are caused by variants in genes that code for different effectors or regulators of the Transforming Growth Factor beta (TGFβ) signaling pathway, including genes that encode for ECM components that play active roles in the signaling activity of this pathway. The cardinal feature of these diseases is the enlargement and rupture of big vessels and, most prominently, the aorta. Aneurysm and dissection of the aorta is the main cause of mortality in these patients but the clinical course of MFS and other ICTDs differs considerably in terms of age of onset and severity, even among individuals who share the same causative variant. This led us to hypothesize the existence of genetic variants elsewhere in the genome that influence the severity of the cardiovascular phenotype in MFS. We recruited familial and sporadic MFS patients that were subsequently classified as having severe (n=8) or mild aortic phenotype (n=14) according to the age of presentation of the first cardiovascular manifestation and/or catastrophe related to the aorta. We used Exome Sequencing (ES) to identify genetic variants that may be associated with the severity of this clinical manifestation, and we performed linkage analysis using the software VAAST. We identified 5 genes associated with severe aortic phenotype and 3 genes that could be protective of this phenotype in MFS. These genes regulate components of the ECM, TGFβ pathway and other signaling pathways that are involved in the maintenance of the ECM and/or angiogenesis. Further studies will be required to understand the functional effect of these variants and explore novel, personalized risk management strategies and, potentially, new therapies for these patients.Item Characterizing the molecular mechanisms of immune dysregulation in proteasome-related interferonopathies and other autoinflammatory diseases(Universidad del Desarrollo. Facultad de Medicina, 2022-01) Jarur Chamy, Valentina Lucía; Poli, Cecilia; Carrión Arriagada, FlavioMonogenic Immune Dysregulatory Syndromes are rare life-threatening diseases in which a single genetic defect causes global immune dysregulation manifested by either immunodeficiency, autoinflammation or autoimmunity in a nonexclusive way. Particularly, Proteasome-associated autoinflammatory Syndromes (PRAAS) are a group of Immune Dysregulatory Syndromes in which loss of function variants in proteasome subunits cause Type I IFN-mediated autoinflammation, ulcerative skin lesions and lipodystrophy through still unknown mechanisms. Proteasome-related Autoinflammation and Immunodeficiency Disease (PRAID) is a novel PRAAS caused by pathogenic variants in POMP, a proteasome assembly chaperone. PRAID patients present PRAAS-like clinical manifestations but also, they have an increased susceptibility to pneumocystis and mycobacterial infections, delineating a combined immunodeficiency that paradoxically co-exist with the autoinflammatory features in this Immune Dysregulatory Syndrome. PRAID patients present low expression of proinflammatory cytokines in T cells in vitro and NFkB deficiency is a known cause of immunodeficiency. In this regard, the proteasome plays a critical role in the activation of NFkB pathway that could be underlying the immunodeficiency in PRAID patients. Conversely, here we identified a sustained up-regulation of NFkB signaling in PRAID patients, suggesting the contribution of NFkB to the Type I IFN-mediated autoinflammatory phenotype in PRAID patients. Additionally, we identified that Type I IFN and NFkB dysregulation in PRAID patients are mediated by the Protein Kinase R (PKR), a cytosolic RNA sensor that triggers Type I IFN and NF-kB signaling upon activation. The mechanisms by which PKR is up-regulated in PRAID still remains elusive and a constitutive activation of PKR does not explain the immunodeficiency in these patients. In this regard, we demonstrated that pathogenic variants in POMP causing PRAID impair the interaction of the chaperone POMP with the immune-proteasome catalytic subunits suggesting a potential role of impaired immune proteasome assembly in the combined immunodeficiency of PRAID patients. PRAID supports the notion of Immune Dysregulatory Syndromes as complex immune-related phenotypes with more than one compromised pathway, reinforcing the relevance of a complete characterization of the pro-inflammatory environment in each genetically-determined disease. In this regard, we used the Type I IFN Signature previously standardized in PRAID patient-derived cells as a translational application for the diagnosis of Immune Dysregulatory patients with clinical features compatible with Type I IFN dysregulation. Here, we empirically demonstrated the relevance of Type I IFN evaluation through the Type I IFN signature for the genetic interpretation and functional validation of novel genetic candidates in patients with type I IFN dysregulation with and without proteasome dysfunction.Item Rol de la conexina 46 en la estabilización de HIF-1α y la agresividad tumoral de la línea de melanoma SK-MEL-2(Universidad del Desarrollo. Facultad de Medicina, 2021) Orellana Villena, Viviana Paulina; Retamal, Mauricio A.; Godoy Sánchez, AlejandroEl melanoma es el cáncer de piel más agresivo, cuya tasa de incidencia se encuentra en constante aumento por sobre otros tipos de tumores sólidos. Recientemente la conexina 46 (Cx46) fue descrita en este tipo de cáncer, pero se desconoce su rol en la biología de este. En otros tipos de cáncer como son el cáncer de mama y glioblastoma la Cx46 está asociada con la metástasis y el fenotipo cancer stem cells. Por otro lado, se ha sugerido que la Cx46 actúa como un factor protector frente a la muerte celular inducida por hipoxia. Interesantemente, la hipoxia activa al factor de transcripción HIF-1α el cual está relacionado con el desarrollo de tumores de mal pronóstico. La expresión de Cx46 y HIF-1α ha sido descrita de forma independiente en melanoma, y en este sentido, nuestra investigación tiene como foco evaluar la posible interrelación entre estas dos proteínas, y su posible efecto sobre la progresión tumoral en la línea celular de melanoma SK-MEL-2. Para este estudio, se estableció un modelo celular con reducción de la expresión de Cx46, en el cual se observó un aumento en la estabilización de HIF-1α mediado por la reducción de los niveles de las proteínas PHD2 y pVHL cuyo rol es degradar a HIF-1α en normoxia. Además, se observó que la reducción de Cx46 impacta negativamente sobre las características que favorecen la progresión tumoral, tales como la proliferación, migración y crecimiento de esferas tumorales. Este comportamiento menos agresivo, se explica por el estudio de proteómica el cual demuestra que la vía de señalización RAS se encuentra disminuida, específicamente por una reducción de las proteínas RAS y NFκβ y otras vías representadas por STAT3, CSPG4 e ICAM1. Todas estas relacionadas con favorecer la sobrevida celular mediante la evasión de la apoptosis, y el aumento de la proliferación y la metástasis. Finalmente, se realizó un estudio preliminar en modelo in vivo en ratones NSG, en donde se observó que la reducción de Cx46 inhibe el crecimiento tumoral al igual que en el estudio in vitro. Y junto con esto, pudimos observar a nivel de proteína, que los tumores reducidos en Cx46 presentan la diminución de los niveles de AKT, RAS y NFκβ, confirmando los datos de proteómica. Los datos obtenidos en esta tesis nos permiten proponer un mecanismo en el cual la Cx46 actúa aumentando los niveles de RAS y proteínas rio abajo (AKT y NFκβ). Lo que aumenta las características de proliferación, migración y crecimiento de esferas tumorales, asociadas al fenotipo CSCs. Lo que orientado a la aplicación clínica o traslacional nos permite pensar que la Cx46 es un target interesante de desarrollar con el fin de disminuir la agresividad tumoral del melanoma.Item Rol de conexina 40 como intermediario en la migración del trofoblasto extra velloso inducida por hipoxia(Universidad del Desarrollo. Facultad de Medicina, 2021-08) Rozas Villanueva, María Fernanda; Retamal, MauricioLa conexina 40 (Cx40) ha sido involucrada en la proliferación del trofoblasto extra velloso durante el proceso de placentación, el que ocurre bajo concentraciones de oxígeno de 1-2%. Sin embargo, se desconoce hasta la fecha cuál es el efecto directo de la hipoxia sobre la Cx40. Mediante el uso de la línea celular Jeg-3 derivada de coriocarcinoma, demostramos que la hipoxia promueve la traslocación de parte de la proteína a la membrana celular, aumentando la probabilidad de encontrar estructuras que asemejan canales de unión en hendidura en las membranas de aposición entre células vecinas. La presencia de estos canales se asoció a un aumento en el acoplamiento de las células demostrado por el traspaso de colorante entre ellas. Utilizando la línea Jeg-3 transfectada con Cx40 pudimos comprobar que la sobreexpresión de Cx40 es capaz de inducir una mayor proliferación en estas células, pero lo más interesante es que un aumento en la expresión de Cx40 es capaz de frenar el estímulo migratorio inducido por la hipoxia. A su vez, utilizando análogos de GMPc fuimos capaces de reproducir los hallazgos observados en hipoxia, y de revertirlos parcialmente utilizando un inhibidor especifico de PKG, sugiriendo que la vía NO-GMPc-PKG pudiese ser un mecanismo importante a través del cual el estímulo hipóxico comanda el tráfico de Cx40 a la membrana celular.Item Transcriptomic characterization provides insights into Hantavirus Cardiopulmonary Syndrome (HCPS) severity(Universidad del Desarrollo. Facultad de Medicina, 2020) Esteves Ribeiro, Grazielle; Vial Cox, CeciliaHantaviruses are important human pathogens that cause a severe zoonotic disease called hantavirus cardiopulmonary syndrome (HCPS), presenting a case fatality rate up to 40%. Clinical course of HCPS may present as a mild condition with moderate respiratory failure or progress rapidly to a severe condition with cardiopulmonary shock that can be fatal. However, the role of the host's responses in this progression towards HCPS and their association with severity remains elusive. In this study, a transcriptome approach combined with clinical laboratory data was applied to gain a better insight into factors associated with HCPS severity. For this, total RNA was extracted from peripheral blood mononuclear (PBMCs) isolated from hantavirus infected patients in acute and convalescent phases. Healthy subjects were used as control. Severe patients are defined as those who develop cardiopulmonary shock and need to receive vasoactive drugs and mechanical ventilation as a treatment and in the most severe cases, they also receive extracorporeal membrane oxygenation (ECMO). Mild clinical course is defined as a disease characterized only by prodromal symptoms or who progress to a minor cardiorespiratory failure that does not require mechanical ventilation and remains hemodynamically stable. Samples from 18 patients (12 severe and 6 mild) and 9 healthy controls were sequenced and analyzed. Differential expression was evaluated by comparing the patient samples grouped by severity in acute and convalescent phase versus the healthy controls. Our results showed that proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and serum levels are increased and could contribute to proinflammatory response in severe HCPS patients. PCSK9 has two FDA approved inhibitors and could be a potential therapeutic target for HCPS. Also showed that hantaviruses can become insensitive to type I IFN response, which contributes to a more severe HCPS outcomeItem Identification of germline genetic variants in miRNA genes targeting BRCA 1/2 genes and their role in chilean familial breast cancer risk : searching for novel findings(Universidad del Desarrollo. Facultad de Medicina, 2018) Mayo Glaser, Tomas Gabriel de; Ziegler Bonicalzi, Annemarie; Jara Sosa, LilianBreast cancer (BC) is the most common cancer in women worldwide and the second most common malignancy as a whole, therefore constituting an important cause of death and a severe burden for public health systems. During the year 2012, it was estimated that 1.67 million new BC cases were diagnosed, which accounts for 25% of all new cancers and 15% of all cancer deaths among females. In Chile, BC is the first cause of death from cancer in women, followed by gallbladder and bile duct adenocarcinoma. Family history of BC (genetic predisposition) is one of the most important non-modifiable risk factors, being responsible for 5-15% of all cases and up to 25% of cases in women fewer than 30 years. Some familial BC cases present a dominant autosomal inheritance due to germline mutations in BRCA1/BRCA2 genes, which are responsible for up to 25% of hereditary BC cases. For the remaining 75%, other susceptibility alleles (moderate or low penetrance), could be responsible for a significant percentage of hereditary BC risk in BRCA1/BRCA2-negative families, as proposed by the current polygenic inheritance model of breast tumors. Examples of these genes are, PALB2, BARD1, ATM, CHECK2, RAD51 and XRCC3, among others. Nevertheless, 50 to 60 percent of familial BC cases carry an unknown genetic etiology. Thus, it is of vital importance to search for other novel gene candidates in order to better elucidate its genetic structure. As low penetrance susceptibility genes may account for the excess of this risk, recent research evidence strongly suggests that single nucleotide variants (SNVs) in pre-microRNA (pre-miRNAs and its boundaries) or mature microRNAs (miRNAs) genes are new possible candidates, as it is well established that they can function as oncogenes or tumor suppressor genes. In this milieu, several genetic epidemiological studies show associations between SNVs in miRNAs and BC risk, mainly through down regulation of DNA repair genes such as BRCA1/BRCA2 genes, which can be regulated by miRNAs targeting their mRNA sequences mostly at their three prime untranslated regions (3´UTRs). Considering that genetic variation is ethnic-specific, some of these studies have shown conflicting results when comparing the effect on BC risk for same variant within different populations. Currently, there are very few studies concerning variants in miRNA genes and BC risk in South American population, making the available information in this arena very scarce. Thus, the purpose of this doctoral thesis project is to identify novel genetic variants (such as SNVs, deletions or insertions) in 10 selected and experimentally validated pre-miRNA genes targeting BRCA1/BRCA2 genes. This will be carried out studying BRCA1/BRCA2-negative (also known as BRCAX) Chilean families with a strong family history for hereditary breast cancer disease. New identified variants will be searched in genetic databases and in the available literature in order to identify whether they correspond to previously described or non-previously described variants and if there are previous studies regarding these variants and familial BC disease. Also,bioinformatics analyses will be performed in order to evaluate the impact of the variant on the miRNA secondary structure and its physicochemical parameters, and comparing these results with the wild type (WT) of the same miRNA gene. Finally, using a TaqMan genotyping assay, selected variants according to specific criteria will be subjected to a case control study in order to assess if there is any association with an increased BC risk in the studied population.Item Regulación de la enzima 11 beta hidroxiesteroide deshidrogenasa tipo 2 [11ß-HSD2] por miRNA y su asociación con hipertensión arterial(Universidad del Desarrollo. Facultad de Medicina, 2018) Tapia Castillo, Alejandra; Carvajal Maldonado, Cristian Andrés; Repetto, GabrielaClassical apparent mineralocorticoid excess (AME) is a rare recessive disorder, caused by 11β-HSD2 deficiency. AME manifests as low-renin pediatric hypertension, hypokalemia and high cortisol/cortisone (F/E) ratio. The study of mild AME cases (NC-AME) requires more clinical suspicion. It has been suggested that the partial deficit of 11βHSD2 can be negatively regulated by microRNAs. miRNA expression can be evaluated in exosomes found in biofluids, which are nanovesicles released from different cell types and though as potential reporters of local metabolic conditions. Aim: To evaluate the activity of 11βHSD2 and its epigenetic regulation by miRNA, in urinary exosomes, through a clinical, biochemical and molecular study model. Subjects and Methods: We recruited 127 adolescents and adults. Subjects with secondary hypertension were excluded. Serum F/E was measured by HPLC/MS-MS. Renal, vascular and inflammatory damage were evaluated with validated biomarkers. Sequencing of HSD11B2 gene was performed in all subjects. RNA was isolated from spot urinary exosomes by ultracentrifugation and analyzed by Nanosight NS300. The miRNAs expression were assayed by Taqman-qPCR. The associations are analyzed by Spearman and comparisons were performed Mann-Whitney test (p<0,05). Results: F/E ratio was positively associated with systolic Blood Pressure (SBP), microalbuminuria and high sensitive c reactive protein (hsCRP). Serum cortisone correlated with MR activation parameters even when adjusted for age, BMI and sex: lower cortisone with higher potassium excretion (partial r= -0.29, p=0.002) and with lower plasma renin activity (PRA, partial r= 0.29, p=0.001). Consistently, we identified, 9/127 subjects (7.1%) with high F/E ratio (First quartile) and low cortisone (Last quartile), suggestive of NC-AME.These subjects had higher SBP (mmHg): 141.4 ± 25.7 vs 127.3 ± 18.1 p=0.03; lower PRA (ng/L*s): 0.36 ± 0.19 vs 0.64 ± 0.47, p<0.0001) and greater potassium excretion, microalbuminuria, hs-CRP and PAI-1. We only found in 2/9 NC-AME subjects heterozygous mutations in HSD11B2 gene. The urinary exosomes concentration was lower in subjects with 11βHSD2-PD vs controls (6.8x1011 [3.4x1011 - 7.8x1011] vs 7.5 x1011 [5.3x1011 - 9.8x1011] particles/ml; p<0.05) and the expression of miR-488 in urinary exosomes is higher in subjects with NCAME vs controls (p = 0.01). Conclusion: We describe a new spectrum of partial deficiency of 11β-HSD2 (NC-AME), which represent 7.1% of the population. The miRNAs could have a role in the negative regulation of 11βHSD2 expression and could be evaluated in urinary exosomes, which would be potential biomarkers of local metabolism at the renal level.