Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-08-30T19:28:55Z No. of bitstreams: 1 Disserta??o Final Gabriela Fabiano de Souza.pdf: 1295606 bytes, checksum: 81a673082dab9c871a133af1f8517135 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-09-03T12:11:04Z (GMT) No. of bitstreams: 1 Disserta??o Final Gabriela Fabiano de Souza.pdf: 1295606 bytes, checksum: 81a673082dab9c871a133af1f8517135 (MD5) / Made available in DSpace on 2018-09-03T12:19:46Z (GMT). No. of bitstreams: 1 Disserta??o Final Gabriela Fabiano de Souza.pdf: 1295606 bytes, checksum: 81a673082dab9c871a133af1f8517135 (MD5) Previous issue date: 2018-03-19 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Respiratory syncytial virus (RSV) is the major cause of infection in children up to five years of age. Reinfection is very common among patients, causing symptoms such as cold or allergy. However, in children, immunosuppressed patients and elderly infection is exacerbated leading to hospitalization and in some case, even death. The number of hospitalizations each year is alarming, even more so because up to now there is still no vaccine for RSV. Tissue damage in the lung caused by RSV leads to an immune response, where infected cells signal activation of signaling pathways, production of reactive oxygen species (ROS), and massive production of inflammatory mediators. Among this production is the macrophage migration inhibitory factor (MIF), which is a pro-inflammatory cytokine, which has been shown to play an important role in the immune response. Knowing this importance, we evaluated MIF expression macrophages from BALB/c mice. The cells were infected with different concentrations of RSV and analyzed by western blot, real-time PCR and Cytometric Bead Array (CBA). After confirmation of MIF expression by the infection, different inhibitors of signaling pathways and ROS were used to evaluate its importance for the expression of MIF. From the results obtained, we showed the dependence of ROS, 5-lipoxygenase (5-LOX), COX, PI3K and partially of P38 MAPK, for MIF expression, besides the need for viral activity. MIF was shown to be important for the release of cytokines such as TNF?, MCP-1 and IL-10. Based on this information MIF may play an important role in the exacerbation of infection, so it was extremely important to explore mechanisms involved in the expression of MIF in relation to RSV. / O V?rus sincicial respirat?rio (VSR) ? a maior causa de infec??o em crian?as at? os cinco anos de idade. A reinfec??o ? muito comum entre os pacientes, causando sintomas como de uma gripe ou alergia, no entanto, em crian?as, pacientes imunossuprimidos e idosos a infec??o ? muito mais exacerbada, o que acaba levando a necessidade de interna??o, podendo levar o paciente a ?bito. O n?mero de interna??es a cada ano ? alarmante, ainda mais que at? os dias atuais ainda n?o se tem uma vacina para o VSR. O dano tecidual no pulm?o, causado por VSR leva a uma resposta imune, onde c?lulas infectadas sinalizam para que ocorra a ativa??o de vias de sinaliza??o, produ??o de esp?cies reativas de oxig?nio (EROs) e tamb?m uma produ??o massiva de mediadores inflamat?rios. Dentre essa produ??o, est? o fator inibidor de migra??o de macr?fagos (MIF), que ? uma citocina pr?-inflamat?ria, que tem demonstrado um importante papel na resposta imune. Sabendo dessa import?ncia, avaliamos a express?o de MIF em macr?fagos de camundongos BALB/c. As c?lulas foram infectadas com diferentes concentra??es de VSR e analisadas por western blot, PCR em tempo real e Cytometric Bead Array (CBA). Ap?s a confirma??o da express?o de MIF pela infec??o, foram utilizados diferentes inibidores de vias de sinaliza??o e de EROs, para que fosse poss?vel avaliar sua import?ncia para a express?o de MIF. A partir dos resultados obtidos mostramos a depend?ncia de EROS, 5-lipoxigenase (5-LOX), COX, PI3K e parcialmente de P38 MAPK, para a express?o de MIF, al?m da necessidade de atividade viral. MIF se mostrou importante para a libera??o de citocinas como TNF ?, MCP-1 e IL-10. Baseado nessas informa??es MIF pode desempenhar um papel importante na exacerba??o da infec??o, sendo assim, foi de extrema import?ncia explorar mecanismos envolvidos na express?o de MIF em rela??o ao VSR.

Abstract available through The FASEB Journal.

Abstract available through The FASEB Journal.

Dans le présent travail qui s'inscrit dans le cadre général de la protection du tissu cardiaque, nous avons mis en évidence les effets protecteurs d'un oligo-élément antioxydant, le sélénium, au cours du post-infarctus et tenté d'analyser les mécanismes cellulaires mis en jeu dans cet effet. <br />Dans une première partie, sur un modèle d'ischémie/reperfusion ex-vivo, nous démontrons qu'une alimentation enrichie en sélénium (1,5 mg/kg, po) limite significativement les arythmies ventriculaires malignes (AVM) dues à l'ischémie/reperfusion en améliorant le statut rédox cellulaire et en limitant la déphosphorylation de la connexine 43 (Cx43). Cette déphosphorylation, proportionnelle à l'intensité et à la sévérité du stress ischémique, entraîne des anomalies de l'excitabilité myocardique, responsables de l'apparition des AVM. Dans une deuxième partie, nous confirmons cet effet cardioprotecteur in-vivo. Dans ces conditions, le sélénium réduit significativement la mortalité due à l'ischémie myocardique et la taille de l'infarctus, et améliore le remodelage cardiaque précoce post-infarctus. Dans la troisième partie, nous montrons que ces effets protecteurs sont associés à une diminution significative du niveau cardiaque de TNF- et à une amélioration de la capacité antioxydante tissulaire à 8 jours post-infarctus. Enfin, nos résultats montrent que le statut en sélénium est inversement corrélé à la taille de l'infarctus et peut être modulé par l'ischémie/reperfusion. <br />Le statut préischémique en sélénium semble donc conditionner la sensibilité du myocarde à l'ischémie/reperfusion et déterminer le pronostic post-infarctus.

The aims were to investigate oxidative stress (I), angiogenesis (II) and inflammation (III-IV) in healthy women during pregnancy and postpartum. Oxidative stress was estimated by measurement of 8-iso-PGF2α and the antioxidants α- and γ-tocopherol. The angiogenic factors PlGF, VEGF-A and the antiangiogenic factor sFlt1 were measured to estimate angiogenesis. PTX3, IL-6, TNF-α and a PGF2α metabolite were measured to estimate inflammation. Out of 52 included women, 15 had minor pregnancy complications and 37 were classified as normal. In study III data from all 52 women were used. For the other studies (I, II and IV) only data from the 37 women with normal pregnancy were used. Pregnancy was associated with increased levels of 8-iso-PGF2α with advancing gestational age. The median postpartum value corresponded to values observed in early gestation and a significant decrease was observed from late pregnancy to postpartum. Lipid-adjusted α- and γ-tocopherol levels decreased with advancing gestational age (I). PlGF increased from early pregnancy until weeks 29–30 and thereafter decreased until week 40. sFlt1 levels were relatively constant until weeks 29–30, when they increased, reaching a peak at weeks 39–40. Postpartum levels were low. The sFlt1:PlGF ratio decreased from weeks 9–12, was constantly low from weeks 19–20 to 37–38 and then increased to weeks 39–40. VEGF-A was detectable in only 8 % of the samples during pregnancy and in 64 % postpartum (II). There was a continuous increase of PTX3 as pregnancy progressed. The increase was most evident after week 31 with the highest levels just before delivery (III). IL-6 increased throughout pregnancy and remained high postpartum. No change in TNF-α could be seen with advancing gestational age or postpartum. The PGF2α metabolite levels increased throughout pregnancy and decreased postpartum (IV). In conclusion, normal pregnancy is associated with mild oxidative stress and inflammation. This might have physiological effects for normal pregnancy development. By delineating how these mediators of oxidative stress, angiogenesis and inflammation fluctuate throughout normal pregnancy and postpartum, we have established a reference for studies of these factors in pregnancy complications.

Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. / <p>QC 20121122</p>

Στην επίκτητη απλαστική αναιμία (ΑΑ) ο υποκυτταρικός μυελός και η πανκυτταροπενία στο περιφερικό αίμα είναι αποτέλεσμα βλάβης των αρχέγονων αιμοποιητικών κυττάρων. Προηγούμενες μελέτες έχουν δείξει ότι κύριο χαρακτηριστικό γνώρισμα της νόσου είναι η ποσοτική αλλά και ποιοτική διαταραχή της stem cell δεξαμενής. Κλινικά και εργαστηριακά ευρήματα προτείνουν το σημαντικό ρόλο του ανοσοποιητικού συστήματος και ειδικά των Τ λεμφοκυττάρων στην ανάπτυξη της απλαστικής αναιμίας. Σήμερα, πλέον, είναι ευρύτερα αποδεκτό ότι η καταστολή του μυελού, που παρατηρείται στην ιδιοπαθή απλαστική αναιμία, είναι αποτέλεσμα της υπερπαραγωγής των μυελοκατασταλτικών κυτταροκινών IFN-γ και TNF-α από διεγερμένα CD8+ κυτταροτοξικά λεμφοκύτταρα, τα οποία συναντούμε τόσο στο περιφερικό αίμα όσο και στο μυελό ασθενών με απλαστική αναιμία. Οι κυτοκίνες αυτές παρουσιάζουν μάλλον προσθετική αντί συνεργική δράση, η οποία σχετίζεται με την IFN-γ-εξαρτώμενη αύξηση της έκφρασης του Fas στα CD34+ κύτταρα και από την IFN-γ-επαγώμενη έκκριση του TNF-α από τα μακροφάγα. Μελέτες έχουν δείξει ότι τόσο τα CD34+ όσο και τα BMMNC κύτταρα του μυελού των ασθενών με ΑΑ είναι περισσότερα αποπτωτικά σε σχέση με φυσιολογικούς μυελούς. Στόχος της διατριβής ήταν η περαιτέρω μελέτη των μοριακών μηχανισμών που εμπλέκονται στην ανοσολογικής προέλευσης απλαστική αναιμία. Λόγω, όμως, του ότι η απλαστική αναιμία είναι μια σπάνια νόσος, η παρουσία ενός εύκολα αναπαραγώγιμου in vitro μοντέλου μυελικής απλασίας θα βοηθούσε περισσότερο στη μοριακή μελέτη αυτής. Στη μελέτη μας, η αναπαραγωγή του καταλληλότερου μοντέλου μυελικής απλασίας επιτεύχθηκε με την προσθήκη των μυελοκατασταλτικών κυτταροκινών IFN-γ και TNF-α σε φυσιολογικό σύστημα μακράς διάρκειας καλλιέργειας μυελού των οστών. Στο μοντέλο αυτό έγινε διερεύνηση των μοριακών μονοπατιών των σχετιζομένων με την Fas και TRAIL επαγόμενη απόπτωση. Παράλληλα, έγινε σύγκριση των δεδομένων από το in vitro μοντέλο με τα αποτελέσματα της παράλληλης μελέτης των αντίστοιχων μοριακών παραμέτρων σε κύτταρα μυελού ασθενών με απλαστική αναιμία. Στο IFN-γ/ΤNF-α μοντέλο παρατηρήθηκε σημαντική μείωση τόσο των πιο άωρων LTC-IC όσο και των πιο δεσμευμένων προγονικών κυττάρων, σε σχέση με τις καλλιέργειες-μάρτυρες. Επιπλέον, τα αποτελέσματα των πρωτογενών και δευτερογενών καλλιεργειών βραχείας διάρκειας σε μυελικά κύτταρα ασθενών με ενεργό νόσο, επιβεβαίωσαν το χαρακτηριστικό γνώρισμα της απλαστικής αναιμίας, δηλαδή, το μειωμένο αριθμό προγονικών αιμοποιητικών κυττάρων. Η επώαση φρέσκων φυσιολογικών μυελικών κυττάρων, σε 5-6 εβδομάδων LTBMC σύστημα, με συνδυασμό των TNF-α/IFN-γ παραγόντων οδηγεί σε αύξηση της Fas mRNA έκφρασης στα CD34+ κύτταρα, κάτι που δεν παρατηρείται στα, αντίστοιχα, φρέσκα και στα 5-6 εβδομάδων κύτταρα-μάρτυρες. Το εύρημα αυτό σε συνδυασμό με τη χαμηλή mRNA έκφραση της caspase 3 καθώς και την απουσία έκφρασης των Bcl-2, Bax και της caspase 8 στον ίδιο πληθυσμό, προτείνουν το σημαντικό ρόλο του external μονοπατιού επαγωγής της απόπτωσης, όπως αυτό ρυθμίζεται από την δράση των μυελοκατασταλτικών κυτοκινών TNF-α/IFN-γ. Παράλληλα, η παρουσία χαμηλής Bcl-2 mRNA έκφρασης, στα CD34+ κύτταρα, τονίζει τη σημασία της αναλογίας προ-αποπτωτικών/αντι-αποπτωτικών σημάτων στη κυτταρική έκβαση. To σημαντικότερο, πάντως, εύρημα του TNF-α/IFN-γ μοντέλου είναι η συνεχής TRAIL mRNA έκφραση, στα CD34+ κύτταρα αυτού, κάτι το οποίο δεν έχει αναφερθεί, ως τώρα, στη βιβλιογραφία. Η μοριακή ανάλυση των μυελικών κυττάρων των ΑΑ ασθενών απεκάλυψε, εκτός της Fas mRNA έκφρασης στα BMMNC και/ή στα CD34+ κύτταρα, την αυξημένη TRAIL mRNA έκφραση στο CD34+ κυτταρικό πληθυσμό των ασθενών με ενεργό νόσο. Αντίθετα, στους ασθενείς σε ύφεση, απουσιάζει η έκφραση και των δύο γονιδίων στον ίδιο πληθυσμό. Ενώ, στα BMMNC η έκφραση του TRAIL mRNA παραμένει, ένα συνεχές εύρημα, ακόμη και στους ασθενείς σε ύφεση. Επιπρόσθετα, η μειωμένη έκφραση των αντι-αποπτωτικών γονιδίων Bcl-xl και/ή Bcl-2 στα BMMNC όλων των ασθενών και του Bcl-xl στα CD34+ κύτταρα των ασθενών με ενεργό νόσο, δείχνει «ανίκανη» να αναστείλλει το μηχανισμό της απόπτωσης στους AA ασθενείς. Το γεγονός ότι η έκφραση του TRAIL mRNA είναι συνεχής στο CD34+ κυτταρικό πληθυσμό του TNF-α/IFN-γ μοντέλου και μόνο στα CD34+κύτταρα ΑΑ ασθενών με ενεργό νόσο, δείχνει τη σημαντικότητα του συγκεκριμένου μορίου στην απόπτωση των προγονικών κυττάρων στη μυελική απλασία. Συμπερασματικά, το μοντέλο μυελικής απλασίας που τελικά επιλέξαμε επιβεβαιώνει προηγούμενη γνώση της συμμετοχής των TNF-α και IFN-γ στη παθοφυσιολογία της απλαστικής αναιμίας. Παράλληλα, τα μοριακά δεδομένα, τόσο του μοντέλου όσο και των ασθενών με απλαστική αναιμία, ενισχύουν την εμπλοκή του Fas/FasL στη παθογένεση της νόσου και προτείνουν την πιθανή συμμετοχή του TRAIL/Apo2L στην όλη διαδικασία. / In aplastic anemia (AA) the hypocellular bone marrow and blood pancytopenia occur as a result of damage to hematopoietic stem cells . Previous studies have shown that a profound quantitative and qualitative defect in the stem cell compartment is a common feature in most patients with AA. Clinical and laboratory data suggest that the immune system, especially T lymphocytes, have an important role in the development of AA. It is well established that IFN-γ and TNF-α mediate hematopoietic stem cell suppression in aplastic anemia. These proinflammatory cytokines exhibit additive rather than synergistic effect, which may be mediated by the IFN-γ-dependent increase in Fas expression on CD34+ progenitor cells and by the IFN-γ-inducible secretion of TNF-α by macrophages. Bone marrow total mononuclear and progenitor cells from aplastic anemia patients are more apoptotic than cells from normal donors, indicating that apoptosis may be a major mechanism of stem cell loss in aplastic anemia. The aim of our study was to investigate the molecular mechanisms involved in the immune-mediated pathology of aplastic anemia. Since aplastic anemia is a rare disease the existence of an easily reproducible model of in vitro hematopoietic cell suppression can facilitate studies concerning the molecular pathways of this disease. In our study, we reproduced such a model with the addition of the myelosuppressive cytokines IFN-γ and TNF-α in a normal long term bone marrow culture system. In this model, we examined the Fas mediated pathway of apoptosis and especially the correlation between TRAIL expression and myelosuppression. We, also, studied these parameters in marrow cells from aplastic anemia patients. The IFN-γ and TNF-α inhibitory cytokines appeared to affect both immature LTC-ICs and more commited progenitor cells capable of lineage-specific colony formation (CFCs). In addition our progenitor cell assays results in patients, supported this unifying feature of reduced haematopoietic progenitor cells in aplastic anemia. TNF-α and IFN-γ treatment up-modulated Fas expression and induced apoptosis of 5-6 weeks cultured normal CD34+ cells, while normal freshly isolated and 5-6 weeks untreated cultured CD34+ cells showed no Fas mRNA expression. This finding, along with the low mRNA expression of caspase 3 and the absence of Bcl-2, Bax and caspase 8 expression, proposes the major role for activation of the extrinsic apoptosis pathway due to treatment of BMMNC with TNF-α and IFN-γ. In parallel, the existence of the low Bcl-xl mRNA expression in the same cell compartment points to the importance of the ratio of pro-apoptotic to anti-apoptotic signals, in cell fate. The most interesting finding is the constant TRAIL mRNA expression on CD34+ cells in TNF-α/IFN-γ treated LTBMC, something not mentioned before. Molecular analysis of patients’ marrow cells revealed, apart from Fas mRNA expression in BMMNC and/or CD34+ cells, TRAIL mRNA expression in CD34+ cell population in active disease. Ιn contrast, in patients in remission, both Fas and TRAIL mRNA expression does not exist. Instead, in BMMNC’s cell compartment TRAIL mRNA expression remains a constant finding even in patients in remission. Additionally, the decreased expression of anti-apoptotic bcl-xl and/or bcl-2 in all patients’ BMMNCs and bcl-xl expression in CD34+ cells from patients with active disease, seems unable to inhibit the mechanism of apoptosis in aplastic anemia patients. In our study, the fact that the expression of TRAIL was constant on CD34+ cells in TNF-α/IFN-γ treated LTBMC and only in CD34+ cells of patients with active disease, points out its significance in apoptosis of progenitor cells. In conclusion, our in vitro model of hematopoietic suppression confirmed previous knowledge for participation of TNF-α and IFN-γ in the pathophysiology of marrow failure in aplastic anemia. In parallel, the molecular data both from the in vitro model, as well as from patients with aplastic anemia, reinforce the implication of Fas/FasL pathway in the pathogenesis of this disease and propose a probable role for TRAIL/Apo2L in the process.

The works presented in this dissertation provide insights into the mechanisms of chemotherapy-induced cognitive impairment (CICI or “ChemoBrain”) and take steps toward outlining a preventive strategy. CICI is now widely recognized as a complication of cancer chemotherapy experienced by a large percentage of cancer survivors. Approximately fifty percent of existing FDA-approved anti-cancer drugs generate reactive oxygen species (ROS). Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, produces the reactive superoxide radical anion (O2-•) in vivo. Dox treatment results in oxidation of plasma proteins, including ApoA-I, leading to TNF-α-mediated oxidative stress in plasma and brain. TNF-α elevation in brain leads to further central nervous system toxicity including mitochondrial dysfunction, neuronal death, and cognitive impairment. Co-administration of the antioxidant drug, 2-mercaptoethane sulfonate sodium (MESNA), prevents Dox-induced protein oxidation and subsequent TNF-α elevation in plasma without interfering with the cancer-killing ability of Dox. In studies presented in this dissertation, we measured oxidative stress in both brain and plasma of Dox-treated mice both with and without MESNA. MESNA ameliorated Dox-induced oxidative protein damage in plasma, confirming our prior studies, and in a new finding led to decreased oxidative stress in brain. Using novel object recognition (NOR), we demonstrated the Dox administration resulted in memory deficits. Using hydrogen magnetic resonance imaging spectroscopy (H1-MRS) techniques, we demonstrated that Dox administration led to a dramatic decrease in choline(phosphocholine)/creatine (Cho/Cr) ratios in mouse hippocampus. The activities of both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D(PLD) were severely diminished following Dox administration. The activity of PC-PLC was preserved when MESNA was co-administered with Dox. In the absence of TNF-α, MRS-indexed Cho/Cr ratio, PLD activity, and mitochondrial oxygen consumption are preserved in brain, and markers of oxidative stress are reduced. Together with results from our previous studies, these results provide strong evidence that TNF-α is strongly associated, if not responsible for CICI. We also tested the notion that O2-• is responsible for Dox-induced plasma protein oxidation and TNF-α release. O2-• resulted in increased oxidative damage to proteins when added to plasma and increased levels of TNF-α in macrophage culture, providing strong evidence that O2-• is responsible for these Dox-induced toxicities.

The Rho family small GTPases are key regulators of the cytoskeleton, through which they impact and control many vital cellular functions, including growth, vesicle trafficking, intercellular junctions, transepithelial transport, migration, and gene transcription. Activation of Rho GTPases is induced by Guanine Nucleotide Exchange Factors (GEFs). We have previously shown that Tumour Necrosis Factor-α (TNF), plasma membrane depolarization, and immunosuppressive drugs activate RhoA through a specific exchange factor, GEF-H1. However, the question of whether other stimuli, such as hyperosmolarity, that activate RhoA, act through GEF-H1 and whether GEF-H1 activates other RhoGTPases was not known. The overall objective of this research project has been to gain insights into the complex mechanism through which the Rho GTPases, Rac and RhoA, are regulated in tubular cells. Specifically, we wished to explore the role and pathway-specific regulation of GEF-H1 in hyperosmotic stress- and TNF-induced signalling in tubular cells. In order to accomplish our goals, we optimized and used affinity precipitation assays to detect GEF-H1 activation (RhoA(G17A) and Rac(G15A)). We found that 1) GEF-H1 is activated by hyperosmotic stress and mediates the hyperosmolarity-induced RhoA activation, as well as nuclear translocation of the Myocardin-Related Transcription Factor (MRTF); 2) TNF induces activation of both Rac and RhoA through GEF-H1, but via different mechanisms. Epidermal Growth Factor Receptor (EGFR)- and Extracellular signal Regulated Kinase (ERK)-dependent phosphorylation at the Thr678 site of GEF-H1 is a prerequisite for RhoA activation only, while both Rac and RhoA activation require GEF-H1 phosphorylation on Ser885. Interestingly, Rac is required for TNF-induced RhoA activation. Together these findings highlight a role for GEF-H1 as an osmosensitive molecule that regulates cellular reprogramming through MRTF. Importantly, we have also uncovered a novel mechanism explaining hierarchical activation of Rac and RhoA by TNF. Such a mechanism could be key in coordinating GEF function and fine-tuning Rac and RhoA activation.

Oxidative stress and inflammation are important factors involved in the progression of heart failure. An important cytokine produced during myocardial infarction (MI) is tumor necrosis factor alpha (TNF-α). TNF-α may induce oxidative stress, cell damage, apoptosis and cardiac dysfunction. Considering the anti-inflammatory and anti-oxidant properties of extra-virgin olive oil and its major component (80%) oleic acid (OA), and their benefits to the cardiovascular system, we hypothesized that the negative effects of TNF-α in the pathogenesis of heart failure will be mitigated by olive oil consumption. This hypothesis was tested by examining the effects of a special diet supplemented with 10% olive oil, in coronary artery ligated animal model of MI. Corn oil (10%) supplementation was used as a control for matching caloric intake. Animals in the sham and ligated groups fed regular chow, olive oil, and corn oil were studied at 4 and 16 weeks post myocardial infarction (PMI). Mortality, diet consumption, weight gain and conduction system abnormalities were comparable among all ligated groups. Echocardiography showed that MI deteriorated cardiac function, and olive oil restored the function. At 16 weeks PMI, only corn oil fed groups showed significant increase in both total cholesterol and HDL. Corn oil was not able to offer protection to the heart, suggesting that the beneficial effects of olive oil are not due to increased caloric intake or increased HDL. MI increased myocardial TNF-α, oxidative stress, lipid peroxidation, pro-apoptotic protein expression (Bax, cleaved Caspase 3, cleaved PARP, TGFβ, Bnip3), cytochrome C release, MAP kinase activation (p38, JNK) and decreased anti-apoptotic protein Bcl-xL expression at both 4 and 16 weeks PMI, and these changes were modulated by olive oil. In order to further test the central role of TNF-α PMI, we examined the possible miti-gation of TNF-α induced changes by OA in isolated adult rat cardiomyocytes. TNF-α in-creased oxidative stress, cell damage, cell death, and apoptosis, while OA treatment miti-gated these TNF-α induced effects. We concluded that TNF-α is implicated in the progression of heart failure subsequent to MI and that OA in olive oil may prevent this progression, through its anti-oxidant, anti-inflammatory, anti-hypertensive, and inotropic effects.