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Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-05-21T13:12:10Z No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-05-28T17:29:36Z (GMT) No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) / Made available in DSpace on 2018-05-28T17:34:08Z (GMT). No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) Previous issue date: 2018-03-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is a major cause of diseases of the respiratory tract in humans being mainly associated with bronchiolitis, chronic obstructive pulmonary disease (COPD) and asthma exacerbation. RSV infection occurs primarily in pulmonary epithelial cells and, once infection is established, an innate immune response is triggered and mainly neutrophil recruitment is induced. Neutrophils can extrude neutrophil extracellular traps (NETs) capable of entrapping and inactivate a multitude of microorganisms because of its composition and due to the stringy nature of DNA fibers. Recently, was demonstrated that RSV particles and its fusion (F) protein were able to induce the release NETs coated with neutrophil elastase and myeloperoxidase, both antimicrobial peptides. Also, was observed that the excessive formation of NETs can have negative consequences to the host, such as airway obstruction during RSV infection. Therefore, the aim was to evaluate the mechanisms involved in NET formation induced by RSV infection of neutrophils, alveolar epithelial cells (A549) or lung fibroblasts (MRC5). Human neutrophils were infected with RSV and were able to induce NETs release only after 3 hours of stimulation indicating classical NETosis. Next was characterized NETs formation during infection associating DNA extrusion with MPO, NE and F protein of RSV. Was also observed NADPH oxidase and PAD4 dependence and PI3K/AKT, ERK and p38 MAPK pathways during infection. The inhibition of these signaling pathways, PAD4 and ROS production abolished NET formation. Considering a possible involvement of necroptosis during NETs production, were tested MLKL and RIPK inhibitors and evaluated LDH release in the supernatant of infected neutrophils. Neutrophils released LDH and depend on necroptosis induction to produce NETs. Likewise, neutrophils were co-cultured with A549 or MRC5 cells infected with RSV. Both A549 and MRC5 cells triggered NET release by human neutrophils in a virus concentration-dependent manner, the opposite occurs when used UV-inactivated virus. Briefly, RSV induces the classical/ROS-dependent NETosis by human neutrophils, and this effect relies on specific kinases activity. Furthermore, neutrophils are able to recognize pulmonary cells infected by RSV, releasing NETs. Thus, NETs release control could be crucial for minimizing tissue inflammation caused by RSV infection. / O v?rus sincicial respirat?rio (VSR) ? uma das principais causas de doen?as do trato respirat?rio em humanos sendo associado principalmente com bronquiolite, doen?a pulmonar obstrutiva cr?nica (DPOC) e exacerba??o de asma. O VSR infecta principalmente c?lulas epiteliais pulmonares e, uma vez que a infec??o ? estabelecida, uma resposta imune inata ? desencadeada e ocorre o recrutamento de c?lulas do sistema imune, principalmente neutr?filos. Os neutr?filos podem liberar redes extracelulares de neutr?filos (NETs) capazes de capturar e inativar uma grande quantidade de microrganismos devido ? sua composi??o e natureza fibrosa das fibras de DNA. Recentemente, foi demonstrado que part?culas do VSR al?m da prote?na de fus?o (F) do v?rus foram capazes de induzir a libera??o de NETs revestidas com elastase neutrof?lica e mieloperoxidase, ambos pept?deos com atividade antimicrobiana. Al?m disso, observou-se que a forma??o excessiva de NETs pode ter consequ?ncias negativas para o hospedeiro, como a obstru??o das vias a?reas durante a infec??o por VSR. Portanto, o objetivo foi avaliar os mecanismos envolvidos na forma??o de NET induzida pela infec??o por RSV em neutr?filos humanos, c?lulas epiteliais alveolares (A549) ou fibroblastos pulmonares (MRC5). Neutr?filos humanos foram infectados com VSR e foram capazes de induzir a libera??o de NETs somente ap?s 3 horas de infec??o, indicando uma NETose cl?ssica. Em seguida, foi caracterizada a forma??o de NETs durante a infec??o associando a extrus?o de DNA com as prote?nas MPO, NE e com a prote?na F do VSR. Tamb?m se observou a depend?ncia de NADPH oxidase e PAD4 e das vias de sinaliza??o PI3K / AKT, ERK e p38 MAPK durante a infec??o. A inibi??o dessas vias de sinaliza??o, da produ??o de PAD4 e de EROs aboliu a forma??o de NET. Considerando um poss?vel envolvimento da necroptose na produ??o de NETs, foram utilizados inibidores de MLKL e RIPK1 e foi avaliada a libera??o de LDH no sobrenadante de neutr?filos infectados. Os neutr?filos liberaram LDH e dependeram da ativa??o da necroptose para produzir NETs. Do mesmo modo, os neutr?filos foram co-cultivados com c?lulas A549 ou MRC5 infectadas com VSR. Ambas as c?lulas A549 e MRC5 desencadearam a libera??o de NET por neutr?filos humanos de uma maneira dependente da concentra??o de v?rus, o oposto ocorreu quando usado um v?rus UV-inativado. Resumidamente, o VSR induz a NETose cl?ssica / dependente de EROs em neutr?filos humanos, e este efeito depende de atividade espec?fica de quinases. Al?m disso, os neutr?filos s?o capazes de reconhecer c?lulas pulmonares infectadas pelo VSR, induzindo a libera??o NETs. Assim, o controle de libera??o de NETs pode ser crucial para minimizar a inflama??o do tecido causada pela infec??o por VSR.

S. aureus sécrète de nombreux facteurs de virulence qui lui permettent de lutter efficacement contre le système immunitaire, afin de favoriser la dissémination de la bactérie dans l’organisme hôte. Parmi ces molécules, les leucotoxines ciblent principalement les cellules myéloïdes comme les neutrophiles, les macrophages ou encore les monocytes, et sont formées par deux sous-unités : une de classe S et une de classe F. La Leucodine de Panton et Valentine (LPV) et l’Hémolysine γ HlgC/HlgB sont deux leucotoxines dont le composant de classe S se fixe sur l’un des récepteurs du système du complément, le C5aR. Naturellement activé par l’anaphylatoxine C5a, le C5aR voit son activité modifiée lors d’une interaction avec la LPV ou HlgC/HlgB, tout du moins pour la libération du calcium intracellulaire. Ces deux leucotoxines, à l’instar du C5a, sont internalisées dans le neutrophile humain et utilisent le transport rétrograde pour atteindre l’appareil de Golgi. Elles peuvent rester dans la cellule jusqu’à 3h sans susciter la mort pour le neutrophile. Plus tard, à 6h, seule la LPV induit de l’apoptose et de la NETose. / S. aureus secretes many virulent factors which allow to efficiently fight the immune system, in a way to promote the bacterial spreading inside the host. Among these molecules, the leukotoxins target myeloid cells such as neutrophils, macrophages and monocytes, and are composed of two subunits: one of class S and one of class F. Panton and Valentine Leukocidin (PVL) and γ-Haemolysin HlgC/HlgB are two leukotoxins whose S-component binds to the C5aR, one of the complement system receptors. Naturally activated by the C5a anaphylatoxin, the activity of the C5aR is modified by the PVL and HlgC/HlgB interaction, for the intracellular calcium release. These two leukotoxins, as C5a, are internalised inside the human neutrophils and use the retrograde transport to reach the Golgi apparatus. These can rest inside the cells until 3h without neutrophil dead. Later, at 6h, only PVL induces apoptosis and NETosis.

Tesis por compendio / [ES] El cáncer constituye la segunda causa de muerte en España. El tromboembolismo venoso (TEV), una complicación del cáncer, conlleva gran gasto del presupuesto sanitario y representa la segunda causa de muerte en estos pacientes. Sin embargo, las herramientas actuales disponibles para la identificación de pacientes oncológicos con elevado riesgo trombótico son limitadas. Adicionalmente, no existen métodos simples, mínimamente invasivos y económicos de diagnóstico de cáncer vesical. Por este motivo, se utilizan técnicas dañinas como la tomografía computarizada la cual implica una elevada dosis de exposición a radiación y procedimientos invasivos como la cistoscopia. Además, un estado hipercoagulable parece tener una relación directa con una mayor carga tumoral y un peor pronóstico. El objetivo principal de la presente Tesis Doctoral es explorar la utilidad clínica de nuevos métodos diagnósticos y pronósticos para el cáncer y sus complicaciones trombóticas. En la primera parte de la Tesis, nos hemos centrado en el papel de miRNAs en orina como biomarcadores de cáncer vesical. Hemos identificado al miR-29c-3p como el miRNA más estable por lo que fue utilizado como normalizador. Hemos ajustado un modelo de regresión logística ordinal para el diagnóstico y estratificación de cáncer vesical utilizando la expresión de miRNAs en orina de pacientes y controles. Este modelo incluyó la expresión de 7 miRNAs: miR-221-3p, miR-93-5p, miR-362-3p, miR-191-5p, miR-200c-3p, miR-192-5p y miR-21-5p. En la segunda parte de la Tesis, nos centramos en el estudio de nuevos biomarcadores para la trombosis asociada a cáncer. Analizamos el potencial predictivo de los miRNAs y de marcadores de activación de neutrófilos en pacientes con cáncer pancreático y pacientes con glioma y meningioma. En cáncer pancreático, obtuvimos un perfil de 7 miRNAs (miR-486-5p, miR-106b-5p, let-7i-5p, let-7g-5p, miR-144-3p, miR-19a-3p y miR-103a-3p) capaz de estimar el riesgo de TEV al diagnóstico con dianas incluidas en las rutas pancreatic cancer y complement and coagulation cascades. En el estudio de los marcadores de activación de neutrófilos, obtuvimos un nuevo modelo predictivo de TEV con la calprotectina como variable predictora. Respecto al estudio de trombosis asociada a cáncer en tumores intracraneales, en pacientes con glioma, ajustamos y validamos un modelo predictivo de embolismo pulmonar (EP) postquirúrgico con 6 miRNAs: miR-363-3p, miR-93-3p, miR-22-5p, miR-451a, miR-222-3p y miR-140-3p y otro con cfDNA y mieloperoxidasa como predictores. Además, hemos combinado los dos tipos de marcadores y hemos obtenido un modelo con mayor capacidad predictiva que incluye a miR-140-3p y a la mieloperoxidasa como predictores. En pacientes con meningioma, ajustamos y validamos un modelo predictivo de EP postquirúrgico con 6 miRNAs: miR-29a-3p, miR-660-5p, miR-331-3p, miR-126-5p, miR-23a-3p y miR-23b-3p. En conclusión, proponemos diferentes perfiles de biomarcadores para el diagnóstico de cáncer de vejiga y para la identificación de pacientes oncológicos con elevado riesgo de trombosis. / [CA] El càncer constitueix la segona causa de mort a Espanya. El tromboembolisme venós (TEV), una complicació del càncer, representa la segona causa de mort en aquests pacients i comporta una gran despesa sanitària. No obstant això, les eines disponibles actualment per a la identificació de pacients oncològics amb elevat risc trombòtic són limitades. Actualment, no existeixen mètodes diagnòstics per al càncer de bufeta senzills, mínimament invasius i econòmics. Per aquest motiu, s'utilitzen tècniques nocives com la tomografia computada la qual implica una elevada dosi d'exposició a radiació i procediments invasius com la cistoscòpia. A més, un estat hipercoagulable sembla tindre una relació directa amb una major càrrega tumoral i un pitjor pronòstic. L'objectiu principal de la present Tesi Doctoral fou explorar la utilitat clínica de nous mètodes diagnòstics i pronòstics per al càncer i les seues complicacions trombòtiques. En la primera part de la Tesi, ens hem centrat en el paper dels microRNAs (miRNAs) en orina com biomarcadors de càncer de bufeta. Hem identificat al miR-29c-3p com el miRNA més estable per la qual cosa va ser utilitzat com a normalitzador. Hem ajustat un model de regressió logística ordinal per al diagnòstic i estratificació de càncer de bufeta utilitzant l'expressió de miRNAs en orina de pacients i controls. Aquest model va incloure l'expressió de 7 miRNAs: miR-221-3p, miR-93-5p, miR-362-3p, miR-191-5p, miR-200c-3p, miR-192-5p i miR-21-5p. En la segona part de la Tesi, ens centràrem en l'estudi de nous biomarcadors per a la trombosi associada a càncer. Analitzàrem el potencial predictiu dels miRNAs i de marcadors d'activació de neutròfils en pacients amb càncer pancreàtic i pacients amb glioma i meningioma. En càncer pancreàtic, vàrem obtindre un perfil de 7 miRNAs (miR-486-5p, miR-106b-5p, let-7i-5p, let-7g-5p, miR-144-3p, miR-19a-3p i miR-103a-3p) capaç d'estimar el risc de TEV al diagnostic dels pacients els quals tenen dianes incloses en les rutes biològiques pancreatic cancer y complement and coagulation cascades. En el estudi dels marcadors d¿activació de neutròfils, vàrem obtenir un altre model predictiu de TEV amb la calprotectina com a variable predictora. Respecte a l'estudi de trombosi associada a càncer en tumors intracranials, en pacients amb glioma, ajustàrem i validàrem un model predictiu d'embolisme pulmonar (EP) incidental postquirúrgic amb 6 miRNAs (miR-363-3p, miR-93-3p, miR-22-5p, miR-451a, miR-222-3p i miR-140-3p) i un altre amb cfDNA i mieloperoxidasa com a predictors. A més, vàrem combinar els dos tipus de marcadors i vàrem obtenir un model amb major capacitat predictiva que inclou al miR-140-3p i la mieloperoxidasa com a predictors. En pacients amb meningioma, ajustàrem i validàrem un model predictiu d¿EP incidental postquirúrgic amb 6 miRNAs: miR-29a-3p, miR-660-5p, miR-331-3p, miR-126-5p, miR-23a-3p i miR-23b-3p. En conclusió, proposem diferents perfils de biomarcadors per al diagnòstic de càncer de bufeta i per a la identificació de pacients oncològics amb elevat risc de trombosi. / [EN] Cancer is the second leading cause of death in Spain. Collaterally, venous thromboembolism (VTE), as a complication of cancer, consumes a great part of its healthcare budget and, more importantly, it is the second cause of death in these patients. However, limited tools are available to identify high risk patients. Additionally, a simple, minimally invasive and economical diagnostic methods for bladder cancer are also lacking. For that aim, harmful techniques are used like CT scan with high radiation exposure and invasive procedures like cystoscopy. Moreover, a hypercoagulable state seems directly related to a large tumor burden and poor prognosis. The overall aim of this Doctoral Thesis is to explore the clinical utility of novel diagnostic and prognostic methods for cancer and its thrombotic complications. In the first part of this Doctoral Thesis, we focused on the role of urine miRNAs as bladder cancer biomarkers. We identified miR-29c-3p as the most stable miRNA and was therefore used as normalizer. We adjusted an ordinal logistic regression model for the diagnosis and stratification of BC using the urine miRNA expression levels of patients and controls. This model included 7 miRNAs: miR-221-3p, miR-93-5p, miR-362-3p, miR-191-5p, miR-200c-3p, miR-192-5p and miR-21-5p. In the second part of this Doctoral Thesis, we focused on the study of novel biomarkers for cancer-associated thrombosis. We analyzed the predictive potential of miRNAs and neutrophil activation markers of thrombotic events in patients with pancreatic cancer and patients with glioma and meningioma. In pancreatic cancer, we obtained a profile of 7 miRNAs (miR-486-5p, miR-106b-5p, let-7i-5p, let-7g-5p, miR-144-3p, miR-19a-3p and miR-103a-3p) able to estimate the risk of potential VTE at diagnosis with targets involved in the pancreatic cancer and complement and coagulation cascades pathways. In the study of the neutrophil activation makers, we obtained a new predictive model of VTE with calprotectin as predictor. Regarding the study of cancer-associated thrombosis in intracranial tumors, in glioma patients, we adjusted and validated a predictive model for post-surgical pulmonary embolism (PE) with 6 miRNAs: miR-363-3p, miR-93-3p, miR-22-5p, miR-451a, miR-222-3p and miR-140-3p, and another with cfDNA and myeloperoxidase as predictors. Furthermore, we combined both types of biomarkers and obtained an improved model using myeloperoxidase and miR-140-3p as predictors. In meningioma patients we fitted and validated a predictive model with 6 miRNAs: miR-29a-3p, miR-660-5p, miR-331-3p, miR-126-5p, miR-23a-3p and miR-23b-3p. In conclusion, we propose several profiles of biomarkers for the diagnosis of bladder cancer and for the identification of oncologic patients at high risk of suffering a thrombotic event. / Oto Martínez, AJ. (2022). Novel Diagnostic and Prognostic Methods for Cancer and Cancer Associated Thrombosis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/181510 / Compendio