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Initial studies in this work explored the role of interleukin-6 (IL6) and leukemia inhibitory factor (LIF) in preimplantation bovine embryos. Neither cytokine affected the total percentage of embryos which developed to the blastocyst stage in vitro. However, supplementation of IL6 increased blastocyst inner cell mass (ICM) cell number without affecting trophectoderm (TE) cell number. Additionally, we found that IL6 activated signal transducer and activator of transcription 3 (STAT3) specifically within ICM cells. LIF, however, did not affect ICM cell number or activate STAT3 in ICM cells, and was not pursued further. This increase in ICM cell number by IL6 was largely comprised of hypoblast (GATA6+:NANOG-) cells, and most IL6-responsive cells in day 9 blastocysts were hypoblast cells (as measured by STAT3 activation). However, some epiblast (NANOG+) cells were also IL6-responsive, and IL6 appeared to initially slow epiblast differentiation. Finally, IL6-treated blastocysts also had increased transcripts of hypoblast/primitive endoderm (PE) markers. These results indicate that IL6 may improve pregnancy retention of IVP embryos by improving yolk sac development, but further work is needed to confirm this theory. Activation of STAT3 by IL6 could be blocked with a chemical Janus kinase 2 (JAK2) inhibitor (AZD1480). JAK2 inhibition from day 5 to 8 resulted in blastocyst ICMs with fewer than 10% the normal cell number, regardless of IL6 supplementation. This indicates that STAT3 is critical for bovine ICM development. Further analysis revealed that inhibition of JAK2/STAT did not prevent ICM formation but disrupted its maintenance. Additionally, we assessed the suitability of zinc sulfate and a bovine embryonic stem cell culture media (TeSR) for improving bovine embryo development in vitro. Zinc sulfate increased day 8 blastocyst total and ICM cell number. Therefore, zinc sulfate appears to improve blastocyst quality. The TeSR medium improved embryo development beyond day 8. In normal synthetic oviduct fluid, blastocysts degenerated after day 8, while blastocysts moved to TeSR had greatly increased cell numbers, and even exhibited PE migration out from the ICM, a phenomenon that has not been reported in vitro. This indicates that extended blastocyst culture is possible with TeSR media. / Doctor of Philosophy / Bovine embryos have been produced in vitro for the purpose of being transferred to recipient cattle to produce a calf since the 1980s. This practice allows cattle breeders to increase the number of offspring from their best females each year, and also allows for more rapid progress in generational genetic improvement. However, only approximately 10% of bovine oocytes survive and produce a calf. This poor efficiency of bovine in vitro embryo production negatively impacts the procedure's widespread use. A significant portion of these embryo losses are likely a result of inadequate in vitro culture conditions, particularly of the embryo culture media, the fluid in which embryos are grown. This media is often called "synthetic oviduct fluid," or SOF, because it is designed to mimic the fluid present in the cow's oviduct, where the embryo would normally reside. However, SOF is much simpler in nature than actual cow oviduct fluid, and this leads to reduced embryonic survival of in vitro produced embryos. Unfortunately, we know very little of what molecules control and promote bovine embryo development. Therefore, one major goal of bovine embryo research is to identify these factors and add them to SOF. The goal of this work was to examine the ability of three molecules, interleukin-6 (IL6), leukemia inhibitory factor (LIF), and zinc sulfate, to increase the number and quality of blastocysts produced through in vitro culture techniques. Additionally, I tested the replacement of SOF with a complex cell culture media, known as TeSR. This medium is more complex than SOF, and therefore should better promote embryo development. This work revealed that IL6, but not LIF, improves in vitro produced (IVP) bovine blastocyst quality. Unfortunately, neither IL6 nor LIF affected the percentage of embryos which survived to the blastocyst stage. However, IL6, but not LIF, increased the number of cells in the inner cell mass (ICM) of the blastocysts. ICM cells are the portion of the embryo which will produce the future calf. IVP bovine embryos are known to have fewer cells than normal, in vivo derived, blastocysts, and this issue is believed to cause some embryonic death after embryo transfer. Therefore, treatment with IL6 may increase the percentage of embryos which will survive after transfer and produce a calf. We also found the addition of zinc sulfate to SOF to benefit embryo quality. None of the concentrations of zinc significantly improved the percentage of embryos which survived to the blastocyst stage, but 2 µM zinc did increase ICM cell number. Like IL6, this may improve embryo survival after transfer. The use of the TeSR media as a replacement for SOF had some benefits. Unfortunately, this media is unusable for producing embryos for transfer to recipients, as we discovered early embryos could not survive in the media. However, blastocyst-stage embryos thrived in it, and could be cultured in vitro for a longer period of time as a result. Therefore, this media will be a useful tool for studying bovine embryo development in vitro, however it is unlikely to benefit calf production. In summary, this work provides evidence that zinc sulfate and IL6 are beneficial additions to SOF. However, future work is needed to determine if embryos produced with these factors are more able to produce a calf. Additionally, we discovered that TeSR is a superior extended blastocyst culture medium.

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2011-2012 / L’hypertension artérielle pulmonaire (PAH) est une vasculopathie obstructive caractérisée par une oblitération du lumen des artères pulmonaires distales et une augmentation des résistances vasculaires menant à une augmentation des pressions pulmonaires (PAP) et une hypertrophie ventriculaire droite (RVH) compensatrice. Aucun médicament n’est capable à ce jour de stopper le processus, et lorsque l’hypertrophie compensatrice devient insuffisante, le ventricule droit (RV) se dilate et défaille. A l’origine de ce processus se trouve une hyper-prolifération, une résistance à l’apoptose et une augmentation de la motilité des cellules musculaires lisses vasculaires de l’artère pulmonaire (PASMCs), les rendant « pseudo-malignes ». Le laboratoire a précédemment démontré que le facteur de transcription NFAT (Nuclear factor of activated T-cells) est en partie impliqué dans ces désordres cellulaires en provoquant une augmentation des concentrations calciques intracellulaires et stimulant la prolifération ; et une hyperpolarisation du potentiel de membrane mitochondriale (ΔΨm) inhibant ainsi l’apoptose dépendante des mitochondries. Dans le chapitre 2 nous avons démontré pour la première fois dans le réseau vasculaire pulmonaire que le facteur de transcription STAT3 (Signal transducer and activator of transcription 3) est activé et impliqué de façon directe dans la régulation de l’expression de NFAT et de façon indirecte dans son activation via l’oncoprotéine serine/thréonine kinase Pim1 (Provirus integration site for Murine Moloney leukemia virus). L’inhibition de Pim1 in vitro et in vivo (modèle de rat injecté à la monocrotaline) est associée à une diminution de l’activité de NFATc2 et à un retour à un phénotype normal. De plus, les souris déficientes pour le gène Pim1 sont résistantes à une induction de la PAH par hypoxie ou monocrotaline. De plus nous avons démontré que l’expression de Pim1 corrèle avec la sévérité de la maladie dans le modèle expérimental et le modèle humain. Nous avons donc souligné l’intérêt de Pim1 comme cible thérapeutique et outil de diagnostic. Dans le chapitre 3, nous avons mis en évidence l’implication de la plateforme signalétique c-Src (sarcoma Schmidt-Ruppin A-2 viral oncogene homolog)/FAK (Focal adhesion kinase) dans la régulation du phénotype « pseudo malin » en partie par activation de STAT3. L’inhibition de FAK in vitro diminue la prolifération des cellules pathologiques, augmente leur sensibilité a l’apoptose et réduit leur motilité. In vivo, l’inhibition de FAK réduit les pressions pulmonaires et le remodelage vasculaire faisant de FAK une cible thérapeutique intéressante. Dans le chapitre 4 nous proposons finalement une autre option thérapeutique par l’utilisation de la dehydroepiandrosterone. Cette hormone naturelle a précédemment été remarquée comme bénéfique dans le traitement de l’hypertension artérielle pulmonaire de par ses propriétés vasodilatatrices. Nous avons montré ici qu’en inhibant STAT3 la DHEA possède également des propriétés antiprolifératives et que son utilisation clinique est prometteuse. Durant mes travaux de doctorat, j’ai pu donc mettre en évidence l’implication majeure de l’axe Src/FAK/STAT3/Pim1 dans la pathogénèse de l’hypertension artérielle pulmonaire. J’ai pu proposer diverses solutions thérapeutiques qui pourraient apporter de nouvelles issues cliniques plus ou moins rapidement. / Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by distal pulmonary arteries lumen obliteration and increased vascular resistances, leading to a rise in pulmonary arterial pressure (PAP) and a compensatory right ventricular hypertrophy. Currently available therapies do not permit to reverse the established process and when the hypertrophy become insufficient, the right ventricle dilates and fails. This phenomenon is due to enhanced proliferation, survival and motility of pulmonary artery smooth muscle cells (PASMCs), which acquire a pseudo malignant phenotype. Our group previously described that the transcription factor NFAT (Nuclear factor of activated T-cells) is involved in these cellular disorders by increasing intracellular calcium level and enhancing proliferation; and by hyperpolarizing the mitochondrial membrane potential and decreasing mitochondrial-dependant apoptosis. In the Chapter 2, we demonstrated for the first time in the pulmonary vasculature, that STAT3 (Signal transducer and activator of transcription 3) regulates directly NFATc2 expression and indirectly NFATc2 activity via the oncoprotein serine/threonine kinase Pim1 (Provirus integration site for Murine Moloney leukemia virus). In vitro and in vivo Pim1 inhibition (in the monocrotaline rat model) is associated with decreased NFATc2 activity and reversion of the malignant phenotype. Moreover, Pim1 deficient mice are resistant to monocrotaline or hypoxia-induced PAH. Finally, we demonstrated that Pim1 expression correlates with disease progression both in animal and human model. Thus, we underlined Pim1 as a potent therapeutic target and an interesting diagnosis tool. In the chapter 3, we showed that the signaling hub c-Src (sarcoma Schmidt-Ruppin A-2 viral oncogene homolog)/FAK (Focal adhesion kinase) is implicated in the regulation of the PASMCs pseudo malignant phenotype, in part by activating STAT3. FAK inhibition in vitro decreases PASMCs proliferation, survival and motility. In vivo, FAK inhibition is associated with decreased PAP and decreased vascular remodeling, making FAK as an interesting therapeutic target. In the chapter 4, we suggest dehydroepiandrosterone (DHEA) as another therapeutical option. This natural hormone is known to be beneficial in PAH through their vasodilating properties. We showed here that by inhibiting STAT3 activation, DHEA also has anti-proliferating properties. Therefore, clinical use of DHEA for PAH can be promising. During my PhD studies, I showed the critical implication of the Src/FAK/STAT3/Pim1 in PAH pathogenesis. I contributed to increase the knowledge on PAH pathogenesis and suggested some therapeutical solutions that can be useful to improve patient outcome.

Sepsis é uma disfunção de órgãos causada por uma resposta desregulada do hospedeiro em decorrência de uma infecção e que eventualmente leva a morte. A identificação de moléculas que minimizem este processo pode fornecer alvos terapêuticos para prevenir a falência de órgãos durante a sepse. O supressor de sinalização de citocinas 1 (SOCS1) é conhecido por regular negativamente a sinalização de receptores de citocinas e de receptores do tipo Toll (TLRs). No entanto, os alvos celulares e mecanismos moleculares envolvidos nas ações de SOCS1 durante a sepse são desconhecidos. Para determinar o papel de SOCS1 durante a sepse polimicrobiana, camundongos C57BL/6 foram tratados com um peptídeo inibidor do domínio KIR (kinase inhibitor region) do SOCS1 (iKIR) e submetidos à CLP (ligação e perfusão do ceco). O tratamento com iKIR aumentou a mortalidade, a carga bacteriana e a produção de citocinas inflamatórias induzida pela CLP. Além disso, observou-se que animais deficientes de SOCS1 nas células mielóides (SOCS1?myel) também tiveram aumento na carga bacteriana e na produção de citocinas proinflamatórias, quando comparados com camundongos SOCS1fl. O aumento na susceptibilidade a sepse foi acompanhado pelo aumento da via glicolítica nas células peritoneias e no pulmão desses animais. Assim, foi observado aumento da produção de ácido láctico e da expressão de enzimas glicolíticas como hexoquinase-1 (Hk1), lactato desidrogenase A (Ldha) e o transportador de glicose 1 (Glut-1) em camundongos sépticos tratados com iKIR ou SOCS1?myel. A expressão desses genes da via glicolítica foi dependente da via de ativação STAT3/HIF-1?. O tratamento com 2-deoxiglicose (inibidor da via glicolítica) diminuiu a susceptibilidade à sepse em camundongos tratados com iKIR. Estes resultados indicam um papel até agora desconhecido de SOCS1, como um regulador de reprogramação metabólica que reduz a resposta inflamatória exacerbada e o dano de órgãos durante a sepse. / Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Identification of pleiotropic molecular brakes might provide therapeutic targets to prevent organ failure during sepsis. Suppressor of cytokine signaling 1 (SOCS1) is known to negatively regulate signaling by cytokine and Tolllike receptors (TLRs). However, the cellular targets and molecular mechanisms involved in SOCS1 actions during sepsis are unknown. To address this in a cecal ligation puncture (CLP) model of sepsis, we treated C57BL/6 mice with an antagonist peptide (iKIR) that blocks the kinase inhibitory region (KIR) domain of SOCS1 and prevents its actions. iKIR treatment increased mortality, bacterial burden and inflammatory cytokine production induced after CLP. We also found that myeloid cell-specific SOCS1 deletion (SOCS1?myel) rendered mice more susceptible to sepsis, shown by higher bacterial loads and inflammatory cytokines than SOCS1fl littermate control mice. O aumento na susceptibilidade a sepse foi acompanhado pelo aumento da via glicolítica nas células peritoneias e pulmão desses animais. These effects were accompanied by increase of glycolysis function in peritoneal cells and lung of SOCS1?myel. Thus, it was observed increased expression of the glycolytic enzymes, hexoquinase-1 (Hk1), lactate dehydrogenase A (Ldha), and glucose transporter 1 (Glut-1) in iKIR-treated or SOCS1?myel septic mice. These events were dependent on the activation of STAT3/HIF-1? pathway. Blocking glycolysis with 2-deoxyglucose ameliorated the increased susceptibility to sepsis in iKIR-treated CLP mice. Together, we unveiled a heretofore unknown role of SOCS1 as a regulator of metabolic reprograming that reduces overwhelming inflammatory response and organ damage during sepsis.

Sepsis é uma disfunção de órgãos causada por uma resposta desregulada do hospedeiro em decorrência de uma infecção e que eventualmente leva a morte. A identificação de moléculas que minimizem este processo pode fornecer alvos terapêuticos para prevenir a falência de órgãos durante a sepse. O supressor de sinalização de citocinas 1 (SOCS1) é conhecido por regular negativamente a sinalização de receptores de citocinas e de receptores do tipo Toll (TLRs). No entanto, os alvos celulares e mecanismos moleculares envolvidos nas ações de SOCS1 durante a sepse são desconhecidos. Para determinar o papel de SOCS1 durante a sepse polimicrobiana, camundongos C57BL/6 foram tratados com um peptídeo inibidor do domínio KIR (kinase inhibitor region) do SOCS1 (iKIR) e submetidos à CLP (ligação e perfusão do ceco). O tratamento com iKIR aumentou a mortalidade, a carga bacteriana e a produção de citocinas inflamatórias induzida pela CLP. Além disso, observou-se que animais deficientes de SOCS1 nas células mielóides (SOCS1?myel) também tiveram aumento na carga bacteriana e na produção de citocinas proinflamatórias, quando comparados com camundongos SOCS1fl. O aumento na susceptibilidade a sepse foi acompanhado pelo aumento da via glicolítica nas células peritoneias e no pulmão desses animais. Assim, foi observado aumento da produção de ácido láctico e da expressão de enzimas glicolíticas como hexoquinase-1 (Hk1), lactato desidrogenase A (Ldha) e o transportador de glicose 1 (Glut-1) em camundongos sépticos tratados com iKIR ou SOCS1?myel. A expressão desses genes da via glicolítica foi dependente da via de ativação STAT3/HIF-1?. O tratamento com 2-deoxiglicose (inibidor da via glicolítica) diminuiu a susceptibilidade à sepse em camundongos tratados com iKIR. Estes resultados indicam um papel até agora desconhecido de SOCS1, como um regulador de reprogramação metabólica que reduz a resposta inflamatória exacerbada e o dano de órgãos durante a sepse. / Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Identification of pleiotropic molecular brakes might provide therapeutic targets to prevent organ failure during sepsis. Suppressor of cytokine signaling 1 (SOCS1) is known to negatively regulate signaling by cytokine and Tolllike receptors (TLRs). However, the cellular targets and molecular mechanisms involved in SOCS1 actions during sepsis are unknown. To address this in a cecal ligation puncture (CLP) model of sepsis, we treated C57BL/6 mice with an antagonist peptide (iKIR) that blocks the kinase inhibitory region (KIR) domain of SOCS1 and prevents its actions. iKIR treatment increased mortality, bacterial burden and inflammatory cytokine production induced after CLP. We also found that myeloid cell-specific SOCS1 deletion (SOCS1?myel) rendered mice more susceptible to sepsis, shown by higher bacterial loads and inflammatory cytokines than SOCS1fl littermate control mice. O aumento na susceptibilidade a sepse foi acompanhado pelo aumento da via glicolítica nas células peritoneias e pulmão desses animais. These effects were accompanied by increase of glycolysis function in peritoneal cells and lung of SOCS1?myel. Thus, it was observed increased expression of the glycolytic enzymes, hexoquinase-1 (Hk1), lactate dehydrogenase A (Ldha), and glucose transporter 1 (Glut-1) in iKIR-treated or SOCS1?myel septic mice. These events were dependent on the activation of STAT3/HIF-1? pathway. Blocking glycolysis with 2-deoxyglucose ameliorated the increased susceptibility to sepsis in iKIR-treated CLP mice. Together, we unveiled a heretofore unknown role of SOCS1 as a regulator of metabolic reprograming that reduces overwhelming inflammatory response and organ damage during sepsis.