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[Resumo] Esta tese de doutoramento trata de identificar variantes xenéticas capaces de alterar o risco de desenvolver o fenotipo de artrose de xeonllo con progresión rápida. Para iso utilizáronse técnicas de secuenciación para atopar polimorfismos presentes no ADN nuclear e mitocondrial que poidan favorecer o desenvolvemento do fenotipo de interese. Detectouse unha variante mitocondrial, presente na posición m.16519C, que estivo significativamente asociada coa rápida progresión. Despois de realizar un pequeno estudo funcional, detectáronse niveis máis altos de interleucina 6 e cicloxixenasa 2 nos cíbridos portadores da variante, indicando que, o aumento da resposta inflamatoria podería ser un mecanismo desencadeante para unha progresión rápida. Por outra banda, detectouse por primeira vez unha interacción entre o ADN nuclear e o mitocondrial no campo da artrose. A presenza do haplogrupo mitocondrial UK e do polimorfismo nuclear rs12107036 tamén aumenta o risco de progresión rápida na artrose do xeonllo. Por último, xerouse un modelo preditivo de gran relevancia clínica e farmacolóxica, baseado en variables xenéticas e clínicas capaces de predecir o risco dun individuo de desenvolver o fenotipo de artrose de xeonllo con progresión rápida., [Resumen] Esta tesis doctoral trata de identificar variantes genéticas capaces de alterar el riesgo de desarrollar el fenotipo de artrosis de rodilla con progresión rápida. Para ello, se han empleado técnicas de secuenciación para buscar polimorfismos presentes en el ADN nuclear y en el mitocondrial que puedan favorecer el desarrollo del fenotipo de interés. Se detectó una variante mitocondrial, presente en la posición m.16519C, que se asoció significativamente con la progresión rápida. Tras realizar un pequeño estudio funcional, se detectaron mayores niveles de interleuquina 6 y ciclooxigenasa 2 en los cíbridos portadores de la variante, pudiendo ser el aumento de la respuesta inflamatoria un mecanismo desencadenante en la progresión rápida. Por otro lado, se ha detectado por primera vez en el campo de la artrosis una interacción entre el ADN nuclear y el mitocondrial. La presencia del haplogrupo mitocondrial UK y el polimorfismo nuclear rs12107036 también aumenta el riesgo de progresión rápida en la artrosis de rodilla. Finalmente, se generó un modelo predictivo de gran relevancia clínica y farmacológica, basado en variables genéticas y clínicas capaz de predecir el riesgo que tiene un individuo de desarrollar el fenotipo de artrosis de rodilla con progresión rápida., [Abstract] This thesis project tries to identify genetic variants capable of altering the risk of developing the rapidly progressive phenotype in knee osteoarthritis. To achieve this, sequencing techniques have been used to search for polymorphisms present in nuclear and mitochondrial DNA that may favor the development of the phenotype of interest. A mitochondrial variant, present at m.16519C, was detected and was significantly associated with rapid progression. After carrying out a small functional study, higher levels of interleukin 6 and cyclooxygenase 2 were detected in cybrids carrying this variant, and the increased inflammatory response could be a triggering mechanism for rapid progression. On the other hand, an interaction between nuclear and mitochondrial DNA has been detected for the first time in the field of osteoarthritis. The presence of the mitochondrial haplogroup UK and the rs12107036 nuclear polymorphism also increases the risk of rapid progression in knee osteoarthritis. Finally, a predictive model of great clinical and pharmacological relevance was generated, based on genetic and clinical variables, which is capable of predicting the risk of developing the rapidly progressive phenotype of knee osteoarthritis.

[Abstract] Objectives: To identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids. Methods: Three prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing. Results: mtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes. Conclusions: The presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended.

[Abstract] Osteoarthritis (OA) is a chronic musculoskeletal disease with a polygenic and heterogeneous nature. In addition, when clinical manifestations appear, the evolution of the disease is usually already irreversible. Therefore, the efforts on OA research are focused mainly on the discovery of therapeutic targets and reliable biomarkers that permit the early identification of different OA-related parameters such as diagnosis, prognosis, or phenotype identification. To date, potential candidate protein biomarkers have been associated with different aspects of the disease; however, there is currently no gold standard. In this sense, genomic data could act as complementary biomarkers of diagnosis and prognosis or even help to identify therapeutic targets of the disease. In this review, we will describe the most recent advances in genetic biomarkers in OA over the past three years.

[Abstract] The association between obesity and osteoarthritis (OA) in joints not subjected to mechanical overload, together with the relationship between OA and metabolic syndrome, suggests that there are systemic factors related to metabolic disorders that are involved in the metabolic phenotype of OA. The aim of this work is study the effects of palmitate and oleate on cellular metabolism in an “in vitro” model of human chondrocytes. The TC28a2 chondrocyte cell line was used to analyze the effect of palmitate and oleate on mitochondrial and glycolytic function, Adenosine triphosphate (ATP) production and lipid droplets accumulation. Palmitate, but not oleate, produces mitochondrial dysfunction observed with a lower coupling efficiency, maximal respiration and spare respiratory capacity. Glycolytic function showed lower rates both glycolytic capacity and glycolytic reserve when cells were incubated with fatty acids (FAs). The production rate of total and mitochondrial ATP showed lower values in chondrocytes incubated with palmitic acid (PA). The formation of lipid droplets increased in FA conditions, being significantly higher when the cells were incubated with oleic acid (OL). These results may help explain, at least in part, the close relationship of metabolic pathologies with OA, as well as help to elucidate some of the factors that can define a metabolic phenotype in OA.

[Abstract] During recent years, the significant influence of mitochondria on osteoarthritis (OA), the most common joint disease, has been consistently demonstrated. Not only mitochondrial dysfunction but also mitochondrial genetic polymorphisms, specifically the mitochondrial DNA haplogroups, have been shown to have an important influence on different OA-related features, including the prevalence, severity, incidence, and progression of the disease. This influence could probably be mediated by the role of mitochondria in the regulation of different processes involved in the pathogenesis of OA, such as energy production, the generation of reactive oxygen and nitrogen species, apoptosis, and inflammation. The regulation of these processes is at least partially controlled by the bi-directional communication between the nucleus and mitochondria, which permits the regulation of adaptation to a wide range of stressors and the maintenance of cellular homeostasis. This bi-directional communication consists of an “anterograde regulation” by which the nucleus regulates mitochondrial biogenesis and activity and a “retrograde regulation” by which both mitochondria and mitochondrial genetic variation exert a regulatory signaling control over the nuclear epigenome, which leads to the modulation of nuclear genes. Throughout this mini review, we will describe the evidence that demonstrates the profound influence of the mitochondrial genetic background in the pathogenesis of OA, as well as its influence on the nuclear DNA methylome of the only cell type present in the articular cartilage, the chondrocyte. This evidence leads to serious consideration of the mitochondrion as an important therapeutic target in OA.

[Resumen] El objetivo de este proyecto será dar los primeros pasos que permitan el diseño y la realización de un estudio citogenético para las especies Drosera intermedia Hayne y Drosera rotundifolia L. Los principales problemas que pretendemos resolver serían: (i) asegurar el mantenimiento de las plantas en el laboratorio de forma que se tenga una fuente constante de ápices radiculares y (ii) asegurar mediante un estudio filogenético que las especies de estudio son un buen modelo para el estudio citogenético. Asimismo se realizaron las primeras pruebas para la observación de los cromosomas. El cultivo in vivo en vermiculita con agua destilada ofreció los mejores resultados, mientras que los in vitro resultaron complejos y no ofrecieron resultados satisfactorios. El análisis filogenético mostró que las especies a estudio se encuentran en la misma sección del género Drosera, siendo taxa próximos pero diferenciados. Se trata de especies adecuadas para realizar estudios citogenéticos que permitirán clarificar la taxonomía y evolución del grupo. Las primeras pruebas citogenéticas realizadas han permitido la observación de cromosomas pero con un elevado nivel de artefactación. Será necesario optimizar el protocolo utilizado para continuar con el estudio., [Abstract] The aim of this proyect is to take the inital steps in order be able to conduct a cytogenetic study focused on Drosera intermedia Hayne and Drosera rotundifolia L. The main problems that we try to solve are: (i) to ensure the maintenance of these plants in the laboratory, obtaining a constant source of root tips and (ii) to ensure, using a phylogenetic study, that these species are a suitable model for a cytogenetic study. In addition to this, we have also performed the first attempts at observing chromosomes in Drosera. The in vivo culture with distilled water yielded the best results, in stark contrast with the in vitro cultivation, which were very complex and didn’t produce any successful results. The phylogenetic analyses showed that these species belong to the Drosera section, being closely related but different taxa. These species are suitable to develop cytogenetic studies that can help us to unravel the taxonomy and evolution of the group. The first cytogenetic tests allowed us to observe highly altered chromosomes. It would be necessary to improve the used protocols in order to continue with this study.