Maternal immune activation (MIA) can affect fetal brain development and thus behavior of young and adult offspring. Reports have shown that increased Interleukin-6 (IL-6) in the maternal serum plays a key role in altering fetal brain development, and may impair social behaviors in the offspring. Interestingly, these effects could be attenuated by blocking IL-6. The current study investigated the effects of luteolin, a citrus bioflavonoid, and its structural analog, diosmin, on IL-6 induced JAK2/STAT3 (Janus tyrosine kinase-2/signal transducer and activator of transcription-3) phosphorylation and signaling as well as behavioral phenotypes of MIA offspring. Luteolin and diosmin inhibited neuronal JAK2/STAT3 phosphorylation both in vitro and in vivo following IL-6 challenge as well as significantly diminishing behavioral deficits in social interaction. Importantly, our results showed that diosmin (10 mg/kg day) was able to block the STAT3 signal pathway; significantly opposing MIA-induced abnormal behavior and neuropathological abnormalities in MIA/adult offspring. Diosmin's molecular inhibition of JAK2/STAT3 pathway may underlie the attenuation of abnormal social interaction in IL-6/MIA adult offspring.

Das Ziel der vorliegenden Arbeit war die Quantifizierung JAK2 p.V617F mutierter Allele zur Verlaufskontrolle von Patienten mit JAK2 p.V617F positiven MPN nach allogener Stammzelltransplantation (SCT). Dabei sollte insbesondere untersucht werden, ob sich frühzeitig nach SCT ein höheres Rezidivrisiko der MPN vorhersagen lässt und zu welchen Zeitpunkten molekulare Untersuchungen nach SCT sinnvoll sind. Wir analysierten retrospektiv den Krankheitsverlauf von 30 Patienten. Dafür verwendeten wir die ARMS-QPCR und WTB-AS QPCR als zwei allel-spezifische Amplifikationsmethoden und untersuchten 142 Proben der ersten Kohorte (n=14) und 32 Proben einer zweiten Kohorte (n=16) im direkten Vergleich. Aus unseren Ergebnissen konnten folgende Rückschlüsse gezogen werden: 1. Die beiden allel-spezifischen Amplifikationsmethoden ARMS-QPCR und WTB-AS QPCR zur Quantifizierung der JAK2 p.V617F Mutation sind vergleichbar. 2. Als Ausgangsmaterial sind antikoaguliertes Vollblut oder auch Beckenkammbiopsien gleichermaßen geeignet. 3. Der Nachweis von > 1% JAK2 p.V617F Allele 28 Tage nach allogener SCT ist assoziiert mit einem signifikant höheren Rezidivrisiko einer JAK2 positiven MPN und einem schlechteren Gesamtüberleben.

Caractérisée par la présence de la mutation JAK2 V617F, la polyglobulie primitive voit son développement contenu par des saignées mais est associée à une incidence plus élevée de cancers. Une exploration globale de l'immunité des patients a été réalisée par la quantification des sous populations lymphocytaires de l'immunité innée et adaptative. Ceci a permis la mise en évidence d'une diminution des lymphocytes B et d'une augmentation des cellules NK. Les cellules NK sont réputées pour leurs propriétés antitumorales mais elles ne sont pourtant pas capables d'éradiquer la PV, posant la question de leurs capacités fonctionnelles. Si les cellules NK des patients présentent une activité cytotoxique basale inférieure aux témoins, elles ne présentent pas d'anomalies de l'expression de leurs récepteurs, de la production de molécules cytolytiques ou de prolifération. Par contre, les cellules NK d'un patient ayant développé une érythroleucémie ou des cellules NK de sujets âgés par rapport à des témoins plus jeunes présentent des anomalies d'expression des récepteurs. L'augmentation des cellules NK pourrait être liée à la mutation JAK2 V617F. Si cette mutation est présente dans les lymphocytes de tous les patients, il existe des arguments pour sa présence dans les cellules NK de certains patients. Enfin, une analyse transcriptomique a permis de définir un profil d'expression propre aux cellules NK des patients. / Characterized by the presence of the JAK2 V617F mutation, polycythemia vera's development is content by phlebotomy but is associated with a higher incidence of cancer. A global exploration of the immunity of patients was performed by quantification of lymphocyte subpopulations of innate and adaptive immunity. This allowed the detection of a decrease in B cells and an increase in NK cells. NK cells are known for their antitumor properties but they are not yet able to eradicate PV, raising the question of their functional abilities. If NK cells of patients have a lower basal cytotoxic activity than healthy donors, they do not show abnormal expression of their receptors, the production of cytolytic molecules or proliferation. On the contrary, NK cells from a patient who developed erythroleukemia or NK cells from elderly healthy donors compared with younger healthy donors exhibit abnormalities of receptors expression. The increase in NK cells could be related to the JAK2 V617F mutation. If the mutation is present in cells of all patients, there are arguments for its presence in the NK cells of some patients. Finally, transcriptome analysis has identified an expression profile specific to NK cells of patients.

Les dernières avancées dans les traitements des hémopathies aboutissent à un meilleurs taux de rémission complète ainsi qu' à de meilleurs taux de survie après traitement. Cependant les risques de rechutes restent élevés. Notre projet s'inscrit dans la compréhension du rôle des cellules NK dans l'évolution de ce type de pathologies. Dans une première partie nous nous sommes intéressés à la polyglobulie de Vaquez. Cette pathologie présente une évolution lente et progressive, et elle est caractérisée par une mutation de JAK2 présente dans la lignée myéloïde chez plus de 95% des patients. Nous avons cherché à détecter la mutation dans les cellules NK de patients, puis, pour savoir si la mutation avait un effet sur les NK, nous avons exploré leurs fonctions in vitro. Nos résultats ont montré que, bien que la mutation soit présente dans les cellules NK, elle ne semble pas avoir d'impact sur les fonctions des cellules NK que nous avons pu tester. Nous en avons conclu que l'évolution de la polyglobulie de Vaquez en leucémie n'était peut-être pas due à une perte de fonction des NK mais plutôt à leur inhibition par l'environnement cellulaire.Dans une deuxième partie nous avons étudié la régulation des natural cytotoxicity receptors dans la leucémie aiguë myéloïde. D'apres des travaux antérieurs nous avons émis l'hypothèse que l'expression des trois NCR aurait une régulation commune s'effectuant au niveau de transcription de leurs gènes. Nos recherches bio-informatiques ainsi que notre expérimentation d'immunoprécipitation de la chromatine (Chip) montrent que le facteur de transcription ETS-1 semble être impliqué dans la régulation commune aux trois NCR. / The latest advances in blood disorders treatments lead to a better complete remission rate and a better survival rate after treatment. However, the risk of relapse remains high. Our project is included in the understanding of NK cells role in the development of these diseases.In a first part, we focused on polycythemia Vera for several reasons: the pathology has a slowly progressive disease, and it is characterized by the presence of JAK2 mutation for > 95% patients. We wanted to know if this mutation was found in NK cells from PV patients and what effects the mutation had on NK cells functions. Our results have shown that although the mutation was found in NK cells, it appears to have no impact on NK cells functions. We conclude that the evolution of PV to leukemia is not due to a loss of NK cell functions but to their inhibition by cellular environment.In a second part, we investigated the regulation of natural cytotoxicity receptors in acute myeloid leukemia because previous works have shown that NCR are weakly expressed in AML patients, that this down-regulation is acquired during evolution of AML and reversible after complete remission, ant that NCR weak expression is related to poor prognosis. We supposed that the expression of the three NCR has a common regulation at genes transcription level. Our bioinformatic researches and our experiment of chromatin immunoprecipitation show that ETS-1 transcription factor is a good candidate involved in the common regulation of the three NCR.

La maladie de Huntington (MH) est une maladie neurodégénérative causée par une extension de répétitions du codon CAG dans le gène de la Huntingtine (Htt). Cette maladie est caractérisée par la mort des neurones striataux et la présence d’agrégats de Htt mutée (mHtt). De plus, au cours de la MH, les astrocytes, qui sont essentiels au bon fonctionnement neuronal, changent d’état et deviennent réactifs. La réactivité astrocytaire est caractérisée par des changements morphologiques et transcriptomiques mais l’impact fonctionnel de cette réactivité reste peu compris.Afin d’étudier le rôle des astrocytes réactifs dans la MH, nous avons utilisé des vecteurs viraux récemment développés par notre équipe, qui induisent ou bloquent la réactivité astrocytaire in vivo en ciblant la voie JAK2-STAT3. Nous avons montré que les astrocytes réactifs diminuent le nombre et la taille des agrégats de mHtt majoritairement présents dans les neurones. Ceci est associé à l’amélioration de plusieurs altérations neuronales observées dans ces modèles. Une analyse transcriptomique réalisée sur des astrocytes réactifs révèle des changements majeurs d’expression de gènes liés aux systèmes de protéostasie. De plus, l’activité du lysosome et du protéasome est augmentée dans les astrocytes réactifs de souris modèles de la MH. Nous montrons également que les astrocytes réactifs éliminent plus efficacement leurs propres agrégats de mHtt, suggérant qu’au cours de la MH, ces cellules pourraient dégrader plus efficacement la mHtt provenant des neurones. De plus, certaines protéines chaperonnes sont induites dans les astrocytes réactifs. En particulier, la co-chaperonne DNAJB1/Hsp40 est surexprimée dans les astrocytes réactifs et est retrouvée dans les exosomes isolés à partir de striata de souris MH. Des expériences de gain et perte de fonction suggèrent que cette chaperonne est impliquée dans les effets bénéfiques des astrocytes réactifs sur l’agrégation de la mHtt et l’état des neurones. Les astrocytes réactifs pourraient donc libérer des protéines anti-agrégantes qui favorise l’élimination de la mHtt dans les neurones.Notre étude montre que les astrocytes peuvent, en devenant réactifs au cours de la MH, acquérir des propriétés bénéfiques pour les neurones et favoriser, via un dialogue complexe avec les neurones, l’élimination des agrégats de mHtt. / Huntington’s disease (HD) is a hereditary neurodegenerative disease caused by an expansion of CAG codons in the Huntingtin gene. It is characterized by the death of striatal neurons and the presence of mutant Huntingtin (mHtt) aggregates. In pathological conditions, as in HD, astrocytes change and become reactive. Astrocyte reactivity is characterized by morphological and significant transcriptomic changes. Astrocytes are essential for the proper functioning of neurons but the functional changes associated with reactivity are still unclear.To better understand the roles played by reactive astrocytes in HD, we took advantage of our recently developed viral vectors that infect selectively astrocytes in vivo and either block or induce reactivity, through manipulation of the JAK2-STAT3 pathway. We used these vectors in two complementary mouse models of HD and found that reactive astrocytes decrease the number and the size of mHtt aggregates that mainly form in neurons. Reduced mHtt aggregation was associated with improvement of neuronal alterations observed in our mouse models of HD. A genome-wide transcriptomic analysis was performed on acutely sorted reactive astrocytes and revealed an enrichment in genes linked to proteolysis. Lysosomal and proteosomal activities were also increased in reactive astrocytes in HD mice. Moreover, we show that reactive astrocytes degrade more efficiently their own mHtt aggregates, suggesting that these cells could siphon mHtt away from neurons. Alternatively, several chaperones were induced in reactive astrocytes. In particular, the co-chaperone DNAJB1/Hsp40 was upregulated in reactive astrocytes and was present in exosomal fraction from HD mouse striatum. Loss and gain of function experiments suggest that this chaperone is involved in the beneficial effects of reactive astrocytes on mHtt aggregation and neuronal status. Therefore, reactive astrocytes could release anti-aggregation proteins that could promote mHtt clearance in neurons.Overall, our data show that astrocytes, by becoming reactive in HD, develop a protective response that involves complex bidirectional signaling with neurons to reduce mHtt aggregation.

As neoplasias mieloproliferativas (NMPs) são um grupo de doenças derivadas de uma transformação clonal de célula tronco hematopoiéticas no qual a linhagem celular mielóide é predominantemente expandida no sangue periférico. As NMPs Philadelphia-negativas incluem policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) que compartilham muitas características hematológicas, clínicas e evolutivas. A mutação da JAK2 (JAK2V617F) está presente em cerca de 95% dos pacientes com PV, entre 50 a 70% com TE e 40 a 50% com MFP. No entanto, os testes moleculares para diagnóstico são muitas vezes um desafio devido ao alto custo e a disponibilidade de equipamentos especializados. Objetivo: Verificar o impacto do teste molecular da mutação JAK2V617F para o diagnóstico de NMPs nos pacientes atendidos no Hospital de Clínicas de Porto Alegre. Métodos: Foram avaliados 87 pacientes com suspeita de NMPs. As amostras de sangue periférico foram analisadas para a mutação JAK2V617F pelo método genético molecular de PCR alelo-específico e os resultados correlacionados com os dados clínico-laboratoriais. Para estabelecimento do diagnóstico, foram utilizados os critérios da Organização Mundial da Saúde (OMS) de 2016. Resultados: Dos 87 pacientes avaliados, 27,6% foram diagnosticados como PV, 39,1% como TE, 4,6% como MFP e 28,7% não contemplavam os critérios para o diagnóstico NMPs. A comparação da utilização do teste da mutação JAK2V617F mostrou que, apenas 41,7% dos pacientes com PV sem utilizar o teste, teriam sido diagnosticados comparados a 91,7% utilizando este teste como um dos critérios no diagnóstico final (p = 0,004). Na TE e na MFP, este critério não foi estatisticamente significativo. Conclusão: O teste molecular para a mutação de JAK2V617F no nosso hospital teve um impacto significativo no diagnóstico dos pacientes com PV, mostrando ser uma ferramenta importante para o diagnóstico final desta NMP. / Myeloproliferative neoplasms (MPNs) are a group of disorders derived from a clonal transformation of stem cell on which myeloid cell lineage is predominantly expanded in the peripheral blood. Philadelphia-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) which share many hematological, clinical, and evolutionary characteristics. The JAK2 mutation (JAK2V617F) is present in about 95% of patients with PV, between 50 to 70% with ET and 40 to 50% PMF. However, the molecular diagnostic tests are often a challenge due to the high cost and the availability of specialized equipment. Objective: To verify the impact of molecular testing of the JAK2V617F mutation for the diagnosis of MPNs in patients attended at Hospital de Clinics, Porto Alegre. Methods: A total of 97 patients were evaluated with suspected of MPNs. The peripheral blood samples were analyzed for the JAK2V617F mutation by the molecular genetic allelespecific PCR method and the results correlated with the clinical-laboratory data. To establish the diagnosis, the 2016 World Health Organization (WHO) criteria were used. Results: Of the 87 patients evaluated, 27.6% were diagnosed as PV, 39.1% as ET, 4.6% as PMF and 28.7% did not meet criteria for MPNs diagnosis. Comparison of the use of the JAK2V617F test showed that only 41.7% of patients with PV without the mutation test were diagnosed compared to 91.7% using this test as one of the criteria for the final diagnosis (p = 0.004). In the ET and the PMF, this criterion was not statistically significant. Conclusion: The molecular test for the JAK2V617F mutation in our hospital had a significant impact in the diagnosis of patients with PV, showing to be an important tool for the final diagnosis of this MPN.

The Mevalonate Pathway: A Potential Therapeutic Target for JAK2-driven Myeloproliferative Neoplasms Lori Nicole Griner Abstract Myeloproliferative neoplasms (MPNs) are diseases of hematopoietic stem cell origin and are characterized by uncontrolled growth of cells of the myeloid compartment. The Philadelphia chromosome negative classical MPNs, including polycythemia vera, essential thrombocythemia, and myelofibrosis, are diseases of dysregulated JAK2 signaling. In fact, the majority of MPN patients have activating mutations in JAK2 (e.g JAK2-V617F), a tyrosine kinase that contributes to the growth and survival of myeloid cells. While MPNs were first described over sixty years ago, a significant need remains to develop therapeutic strategies for them. Inhibitors of JAK2 are currently being developed, and one inhibitor, ruxolitinib, was recently approved for certain MPN patients. Ruxolitinib has made profound impacts on improving splenomegaly and constitutional symptoms in MPN patients, but it and other JAK2 inhibitors have not significantly reduced the JAK2 mutant allele burden, and thus such inhibitors have not induced remission in these patients. The current consensus in the MPN field supports JAK inhibition for the treatment of patients, but a further understanding of MPNs and JAK2 signaling, as well as improved JAK2 inhibitors, may be necessary for treating MPN patients. The work described in this dissertation has uncovered novel requirements for JAK2-V617F-driven signaling and transformation. We demonstrate that JAK2-V617F co-localizes with lipid rafts, cholesterol-rich microdomains within the plasma membrane that function to serve as platforms for signaling complex formation. Signaling complex formation is a necessary component for dysregulated signaling induced by JAK2-V617F. We provide evidence that cholesterol altering-lipid raft disrupting agents attenuate JAK2-V617F-driven signaling. We also show that cholesterol-lowering statins are effective at downregulating JAK2 signaling and inducing apoptosis in JAK2-V617F-driven cell lines. Importantly, we show that statins, inhibitors of the mevalonate pathway, inhibit the growth of primary MPN cells, while the same statin doses have no effect on healthy controls. Impressively, we demonstrate that statins cooperate with multiple JAK inhibitors, including ruxolitinib, to inhibit cell growth and induce apoptosis of JAK2-V617F-driven cells. This report establishes statin-mediated inhibition of the mevalonate pathway as a potential approach to improve MPN therapeutics. We propose future studies with statins and JAK2 inhibitors in the treatment of MPNs.

As neoplasias mieloproliferativas (NMPs) são um grupo de doenças derivadas de uma transformação clonal de célula tronco hematopoiéticas no qual a linhagem celular mielóide é predominantemente expandida no sangue periférico. As NMPs Philadelphia-negativas incluem policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) que compartilham muitas características hematológicas, clínicas e evolutivas. A mutação da JAK2 (JAK2V617F) está presente em cerca de 95% dos pacientes com PV, entre 50 a 70% com TE e 40 a 50% com MFP. No entanto, os testes moleculares para diagnóstico são muitas vezes um desafio devido ao alto custo e a disponibilidade de equipamentos especializados. Objetivo: Verificar o impacto do teste molecular da mutação JAK2V617F para o diagnóstico de NMPs nos pacientes atendidos no Hospital de Clínicas de Porto Alegre. Métodos: Foram avaliados 87 pacientes com suspeita de NMPs. As amostras de sangue periférico foram analisadas para a mutação JAK2V617F pelo método genético molecular de PCR alelo-específico e os resultados correlacionados com os dados clínico-laboratoriais. Para estabelecimento do diagnóstico, foram utilizados os critérios da Organização Mundial da Saúde (OMS) de 2016. Resultados: Dos 87 pacientes avaliados, 27,6% foram diagnosticados como PV, 39,1% como TE, 4,6% como MFP e 28,7% não contemplavam os critérios para o diagnóstico NMPs. A comparação da utilização do teste da mutação JAK2V617F mostrou que, apenas 41,7% dos pacientes com PV sem utilizar o teste, teriam sido diagnosticados comparados a 91,7% utilizando este teste como um dos critérios no diagnóstico final (p = 0,004). Na TE e na MFP, este critério não foi estatisticamente significativo. Conclusão: O teste molecular para a mutação de JAK2V617F no nosso hospital teve um impacto significativo no diagnóstico dos pacientes com PV, mostrando ser uma ferramenta importante para o diagnóstico final desta NMP. / Myeloproliferative neoplasms (MPNs) are a group of disorders derived from a clonal transformation of stem cell on which myeloid cell lineage is predominantly expanded in the peripheral blood. Philadelphia-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) which share many hematological, clinical, and evolutionary characteristics. The JAK2 mutation (JAK2V617F) is present in about 95% of patients with PV, between 50 to 70% with ET and 40 to 50% PMF. However, the molecular diagnostic tests are often a challenge due to the high cost and the availability of specialized equipment. Objective: To verify the impact of molecular testing of the JAK2V617F mutation for the diagnosis of MPNs in patients attended at Hospital de Clinics, Porto Alegre. Methods: A total of 97 patients were evaluated with suspected of MPNs. The peripheral blood samples were analyzed for the JAK2V617F mutation by the molecular genetic allelespecific PCR method and the results correlated with the clinical-laboratory data. To establish the diagnosis, the 2016 World Health Organization (WHO) criteria were used. Results: Of the 87 patients evaluated, 27.6% were diagnosed as PV, 39.1% as ET, 4.6% as PMF and 28.7% did not meet criteria for MPNs diagnosis. Comparison of the use of the JAK2V617F test showed that only 41.7% of patients with PV without the mutation test were diagnosed compared to 91.7% using this test as one of the criteria for the final diagnosis (p = 0.004). In the ET and the PMF, this criterion was not statistically significant. Conclusion: The molecular test for the JAK2V617F mutation in our hospital had a significant impact in the diagnosis of patients with PV, showing to be an important tool for the final diagnosis of this MPN.

As neoplasias mieloproliferativas (NMPs) são um grupo de doenças derivadas de uma transformação clonal de célula tronco hematopoiéticas no qual a linhagem celular mielóide é predominantemente expandida no sangue periférico. As NMPs Philadelphia-negativas incluem policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) que compartilham muitas características hematológicas, clínicas e evolutivas. A mutação da JAK2 (JAK2V617F) está presente em cerca de 95% dos pacientes com PV, entre 50 a 70% com TE e 40 a 50% com MFP. No entanto, os testes moleculares para diagnóstico são muitas vezes um desafio devido ao alto custo e a disponibilidade de equipamentos especializados. Objetivo: Verificar o impacto do teste molecular da mutação JAK2V617F para o diagnóstico de NMPs nos pacientes atendidos no Hospital de Clínicas de Porto Alegre. Métodos: Foram avaliados 87 pacientes com suspeita de NMPs. As amostras de sangue periférico foram analisadas para a mutação JAK2V617F pelo método genético molecular de PCR alelo-específico e os resultados correlacionados com os dados clínico-laboratoriais. Para estabelecimento do diagnóstico, foram utilizados os critérios da Organização Mundial da Saúde (OMS) de 2016. Resultados: Dos 87 pacientes avaliados, 27,6% foram diagnosticados como PV, 39,1% como TE, 4,6% como MFP e 28,7% não contemplavam os critérios para o diagnóstico NMPs. A comparação da utilização do teste da mutação JAK2V617F mostrou que, apenas 41,7% dos pacientes com PV sem utilizar o teste, teriam sido diagnosticados comparados a 91,7% utilizando este teste como um dos critérios no diagnóstico final (p = 0,004). Na TE e na MFP, este critério não foi estatisticamente significativo. Conclusão: O teste molecular para a mutação de JAK2V617F no nosso hospital teve um impacto significativo no diagnóstico dos pacientes com PV, mostrando ser uma ferramenta importante para o diagnóstico final desta NMP. / Myeloproliferative neoplasms (MPNs) are a group of disorders derived from a clonal transformation of stem cell on which myeloid cell lineage is predominantly expanded in the peripheral blood. Philadelphia-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) which share many hematological, clinical, and evolutionary characteristics. The JAK2 mutation (JAK2V617F) is present in about 95% of patients with PV, between 50 to 70% with ET and 40 to 50% PMF. However, the molecular diagnostic tests are often a challenge due to the high cost and the availability of specialized equipment. Objective: To verify the impact of molecular testing of the JAK2V617F mutation for the diagnosis of MPNs in patients attended at Hospital de Clinics, Porto Alegre. Methods: A total of 97 patients were evaluated with suspected of MPNs. The peripheral blood samples were analyzed for the JAK2V617F mutation by the molecular genetic allelespecific PCR method and the results correlated with the clinical-laboratory data. To establish the diagnosis, the 2016 World Health Organization (WHO) criteria were used. Results: Of the 87 patients evaluated, 27.6% were diagnosed as PV, 39.1% as ET, 4.6% as PMF and 28.7% did not meet criteria for MPNs diagnosis. Comparison of the use of the JAK2V617F test showed that only 41.7% of patients with PV without the mutation test were diagnosed compared to 91.7% using this test as one of the criteria for the final diagnosis (p = 0.004). In the ET and the PMF, this criterion was not statistically significant. Conclusion: The molecular test for the JAK2V617F mutation in our hospital had a significant impact in the diagnosis of patients with PV, showing to be an important tool for the final diagnosis of this MPN.

[EN] Idiopathic pulmonary fibrosis (IPF) is the pulmonary disease with higher incidence and worse prognosis. Recent evidence suggests that cucurbitaceae, selective inhibitors of the JAK2/STAT3 pathway, may improve the pathogenesis of IPF, as anti-inflammatory and antioxidative properties have been confirmed in other diseases. However the role in IPF is unknown. Two pharmacological models were investigated in the present study. In the preventive model, Wistar rats were instilled intratracheally with a single dose of bleomycin (BLM)(3.75 U/kg; n=12) to induce lung injury. CuI (20mg/kg/day; n=6) or CuI vehicle (control and IPF group) was administered intraperitoneally daily for 21 days. The therapeutic model was exactly the same starting CuI administration on day 7 until day 21. Animal evolution together with the ventilation-perfusion ratio was controlled through CT/SPECT imaging. Cellular count and characterization was performed in broncoalveolar lavage (BAL) together with the total protein content and the IL-6 and IL-13 concentration in BAL and lung tissue. Hematoxilin-eosin and masson trichrome stains allowed the study of the lung's tissue histology. TGF-ß1, CTGF, COL1A and ET-1 gene and protein expression were both measured by real time PCR and WB as pulmonary vascular remodeling markers. P-STAT3, P-SMAD3 and P-JAK2 proteins were determined by protein and immunohistochemistry quantification. Finally, JAK2 and STAT3 expression and distribution was studied in lung tissue of fibrotic patients with pulmonary hypertension. CT/SPECT quantification showed reduction of the fibrotic areas in the CuI treated group by reestablishing the air space in the lungs back to day 0 levels for the preventive and the therapeutic model. Furthermore, ventilation/perfusion correlation was restored in the therapeutic model after administrating CuI during 14 days. Hematoxilin-eosine stains demonstrated how the group treated pharmacologically presented an improvement in the lung tissue architecture, reversing vascular remodeling and right heart hypertrophy. Masson trichrome stain revealed a reduction of the collagen deposits. Ashcroft score, used to determine the severity of pulmonary fibrosis, was measured and diminished significantly in the CuI treated group. The results showed a gene and protein overexpression of TGF-ß1, CTGF, COL1A and ET-1 in BLM relative to Control rats. This was counteracted with CuI treatment which reduced the expression back to control. In terms of immunohistochemistry, the results demonstrated a decrease in the COL1A deposits in the treated group versus the absence of treatment group. Protein and immunohistochemistry analysis of JAK2, STAT3 and SMAD3 demonstrated an overexpression in bleomycin rats while the protein expression was inhibited in the CuI treated group. In accordance to the results obtained, the immunohistochemistry analysis of the lung parenchyma in patients with pulmonary hypertension related to pulmonary fibrosis showed and overexpression of the phosphorylated forms of JAK2 and STAT3, lacking its expression in healthy lung tissue. The preventive and therapeutic administration of JAK2/STAT3 inhibitor can be a potential treatment for pulmonary fibrosis, as it improves the parameters related to the disease. / [ES] La fibrosis pulmonar idiopática (FPI) es la enfermedad pulmonar con mayor incidencia y peor pronóstico. Estudios recientes sugieren que la familia de las cucurbitaceae, inhibidores selectivos de la ruta JAK2/STAT3, pueden mejorar la patogénesis de la enfermedad, al haberse confirmado sus propiedades antinflamatorias y antioxidantes en otras enfermedades. Sin embargo se desconoce su papel en FPI. En el presente trabajo se estudiaron dos modelos farmacológicos. En el modelo preventivo las ratas Wistar fueron instiladas con una dosis única de bleomicina intratraqueal (BLM)(3.75 U/kg; n=12) para inducir las lesión pulmonar. Durante 21 días se administró CuI (20mg/kg/día; n=6) o vehículo de CuI (control y grupo FPI) por vía intraperitoneal. El modelo curativo se diferencia del preventivo en el comienzo de la administración farmacológica a los 7 días de inducir la enfermedad. El seguimiento de la evolución animal y el ratio de ventilación-perfusión se realizó mediante las técnicas de imagen TC/SPECT. Se realizó el recuento y caracterización de las células totales extravasadas en lavado broncoalveolar (LBA) así como el contenido de proteína total y la concentración de IL- 6 e IL-13 en LBA y tejido pulmonar. Las tinciones de hematoxilina-eosina y masson tricrómico permitieron el estudio de la histología del tejido pulmonar. Se determinó la expresión génica y proteica de TGF-ß1, CTGF, COL1A y ET-1 mediante las técnicas de real time PCR y WB como marcadores de remodelado vascular. Las proteínas P-STAT3, P-SMAD3 y P-JAK2, fueron determinadas mediante cuantificación proteica e inmunohistoquimia. Por último se estudió la expresión y distribución de JAK2 y STAT3 en tejido de pacientes con fibrosis pulmonar e hipertensión pulmonar. La cuantificación de las imágenes TC/SPECT mostraron una reducción de las áreas fibróticas en el grupo tratado con CuI. El tratamiento farmacológico permitió el restablecimiento del espacio aéreo pulmonar hasta valores control en ambos modelos estudiados. El grupo con tratamiento farmacológico restauró el ratio de ventilación/perfusión tras administrar CuI durante 14 días. Las tinciones de hematoxilina eosina revelaron como el grupo animal tratado farmacológicamente presenta una mejora de la histología pulmonar, revirtiendo el remodelado vascular y la hipertrofia del ventrículo derecho. La tinción de masson tricrómico mostró una disminución de los depósitos de colágeno. Se determinó el valor de Ashcroft, evaluador del grado de fibrosis pulmonar, que descendió significativamente en el grupo tratado con CuI. Los resultados presentan una sobrexpresión génica y proteica de TGF-ß1, CTGF, COL1A y ET-1 en los grupos de bleomicina frente a las ratas control. Dicha condición fue revertida mediante el tratamiento con CuI que restableció los valores a niveles control. Los análisis proteicos e inmunohistoquíimicos de JAK2, STAT3 y SMAD3 revelaron una sobreexpresión en las ratas con bleomicina mientras que la expresión proteica fue inhibida en el grupo tratado con CuI. En consonancia con los resultados obtenidos, el análisis inmunohistoquímico del parénquima pulmonar de pacientes con FPI e HP asociada muestran una sobreeexpresión de las formas fosforiladas de JAK2 y STAT3 frente a la ausencia de expresión en tejido pulmonar sano. La administración curativa y preventiva de un inhibidor de la ruta JAK2/STAT3 puede ser un potencial tratamiento para la fibrosis pulmonar, ya que mejora parámetros indicativos de la patología. / [CAT] La fibrosi pulmonar idiopàtica (FPI) és la enfermetat pulmonar amb major incidència i pitjor pronostic. Estudis recents suggereixen que la família de les cucurbitàcies, Inhibidors selectius de la ruta JAK2 / STAT3, poden millorar la patogènesi de la malaltia, en haver-se confirmat les seues propietats antiinflamatòries i antioxidants En altres patologies. No obstant això es desconeix el seu paper en FPI. En el present treball es van estudiar 2 models farmacològics. En el model preventiu les rates Wistar foren instilades amb una dosi única de bleomicina intratraqueal (BLM) (3,75 U / kg; n = 12) per a induir les lesions pulmonars. Durant 21 dies es va administrar CuI (20 mg / kg / dia, n = 6) o Vehicle de CuI (control i grup FPI) per vía intraperitoneal. El model curatiu es diferència del preventiu en el començament de l'administració farmacológica als 7 dies d'induir l'enfermetat. El seguiment de l'evoluciò dels animals i el ratio de Ventilació-perfusió es va realitzar mitjançant tècniques d'imatge TC/SPECT. Es realitzà el recompte i caracterització de les cèl·lules totals extravasades en el llavat broncoalveolar (LBA). Així com el contingut de proteina total i la concentració d'IL-6 i IL-13 en LBA i teixit pulmonar. Les tincions d'hematoxilina-eosina i tricròmic de Masson van permetre l'estudi de la histologia del teixit pulmonar. Es va determinar l'expressió gènica i proteica de TGF-ß1, CTGF, COL1A i Et-1 mitjançant tècniques de PCR en temps real i WB com marcadors de remodelat vascular. Les proteïnes P-STAT3, P-SMAD3 i P-JAK2, varen ser determinades mitjançant quantificació proteica i inmunohistoquimia. Per últim se estudià l'expressió i distribució de JAK2 i STAT3 en teixit de pacients amb fibrosi pulmonar e hipertensió pulmonar. La quantificació de les imatges TC/SPECT mostraren una reducció de les àrees fibrótiques en el grup tractat amb CuI. El tractament farmacològic permet el restabliment de l'espai aeri pulmonar fins valors de control en els dos models estudiats. El grup amb tractament farmacològic va restaurar el ratio de ventilació/perfusió tras administrar Cul durant 14 dies. Les tincions d'hematoxilina eosina van revelar com el grup animal tractat farmacològicament presenta una Millora de la histologia pulmonar, revertint el remodelat vascular i la hipertròfia del ventricle dret. La tinció de tricròmic de Masson va mostrar una disminució dels dipòsits de col·lagen. Es va determinar el valor d'Ashcroft, avaluador del grau de fibrosi pulmonar, que va baixar significativament a el grup tractat amb CuI. Els resultats presenten una sobreexpressió gènica i proteica de TGF-ß1, CTGF, COL1A i Et-1 en els grups de bleomicina en comparacióa les rates control. Aquesta condició va ser revertida mitjançant el tractament amb CuI que va restablir els valors fins a nivel dels control. A nivell immunohistoquímic els resultats mostren una disminució dels dipòsits de COL1A en el grup tractat comparativament al grup sense tractament. Els anàlisi proteics i inmunohistoquíimics de JAK2, STAT3 i SMAD3 van revelar una sobreexpressió en les rates amb bleomicina mentre que l'expressió proteica va ser inhibida en el grup tractat amb CuI. D'acord amb els resultats obtinguts, l'anàlisi immunohistoquímic del parènquima pulmonar de pacients amb FPI i HP associada mostren sobreeexpresió de les formes fosforilades de JAK2 i STAT3 davant l'absència d'expressió en teixit pulmonar sa. La administració curativa i preventiva d'un inhibidor de la ruta JAK2 / STAT3 pot ser un potencial tractament per la fibrosi pulmonar, ja que millora paràmetres indicatius de la patologia. / Hernández Ribes, G. (2016). ESTUDIO DE LA RUTA CELULAR JAK2/STAT3 COMO POTENCIAL INHIBIDOR EN EL MODELO DE FIBROSIS PULMONAR [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/64087 / TESIS