Macrophages can be activated by a variety of extracellular signals to polarize to either the M1 (inflammatory and antimicrobial) or to the M2 (wound repair and inflammation resolution) phenotype. Expression of arginase 1 in macrophages is a key marker of the M2 phenotype. Arginase 1 expression is induced by interleukin 4 (IL-4), a cytokine secreted by Th2 helper cells. All-trans retinoic acid (ATRA) is a product of metabolism of dietary retinol (vitamin A). In a manner analogous to hormones, ATRA binds to nuclear receptors in cells and influences gene expression and cell physiology. ATRA is important in the resolution of inflammation systemically and on the cellular level, however it has not been linked to M2 activation or arginase 1 expression. Testing the hypothesis that ATRA can induce arginase 1 in macrophages either directly or indirectly, it was found that ATRA alone cannot cause murine bone marrow-derived macrophages (BMDM) to activate in the M2 phenotype (as indicated by arginase 1 expression), however it can dramatically potentiate induction of arginase 1 expression and activity by IL-4. This is the first observation positively linking ATRA to arginase 1. Lipopolysaccharide (LPS), is a conserved structural component of the outer membrane of Gram negative bacteria, and a potent pyrogen. In metabolic endotoxemia, LPS concentration in the blood is slightly elevated, and over the long term this contributes to diverse inflammatory diseases such as atherosclerosis, obesity, and diabetes. LPS promotes the M1 phenotype and suppresses the M2 phenotype, but its contribution at low doses such as those found in metabolic endotoxemia are not well studied. In order to investigate mechanisms of LPS suppression at low doses, mice deficient in IRAK1 and tollip, key mediators or proinflammatory LPS signaling, were used to study IL-4, ATRA, and LPS crosstalk. LPS suppression of arginase 1 was found to be dependent on IRAK1 and tollip, but only at low doses of LPS. / Ph. D.

Systemic lupus erythematosus (SLE) is an autoimmune disease involving the inflammatory damages of multiple organs. Lupus nephritis (LN) as the manifestation in the kidney occurs in more than 50% of SLE patients and is a major cause of morbidity and mortality. Current treatments consist of immunosuppressants that always lead to compromised immune responses with increased risks of infections as the major side effect. To minimize this side effect, it is crucial to develop new treatments that are more natural and specific. Vitamin A, particularly in the form of its functional metabolite, retinoic acid, has shown some beneficial effects against LN in both lupus-prone mouse models and clinical cases. However, a more systemic evaluation of vitamin A treatment in lupus had not been investigated. In our study, we found paradoxical effects of all-trans-retinoic acid (tRA) on lupus-like disease in MRL/lpr lupus-prone mice. Starting at 6 weeks old when the inflammatory environment had been established in MRL/lpr mice, tRA administration reduced immune cell numbers in the secondary lymphoid organs and improved glomerulonephritis. However, circulating autoantibodies and inflammation in renal tubulointerstitium and other organs were increased. The detrimental effects of tRA were not present in MRL control mice, which didn't have an established inflammatory environment at 6 weeks old as shown in MRL/lpr mice, suggesting that the pro-inflammatory effects of tRA are dependent on the pre-existing inflammatory environment. Therefore, to successfully apply vitamin A-based treatment, it is important to avoid the detrimental effects of tRA on lupus by identifying and then specifically eliminating the critical pro-inflammatory immune cell types in lupus. As treatments usually start after the onset of apparent symptoms in patients at the effector stage of autoimmune responses, targeting the inflammatory contributors at this stage appears to be more practical and critical. Among different types of leukocytes, we chose to focus on dendritic cells (DCs), because they are highly diverse and critical in the immune responses as a bridge between the innate and adaptive immune systems. Plasmacytoid DCs (pDCs) as a candidate target have been demonstrated to be crucial for the initiation of lupus development by producing IFNα. However, we demonstrated that although pDCs produced a large amount of IFNα during disease initiation, those from late-stage lupus mice were found to be defective in producing IFNα, suggesting that pDC-targeted treatments should be performed at the initiation stage. This will depend on the progress in early diagnosis in the future. Besides pDCs, we identified a CD11c+ cell population absent at the early-stage but gradually accumulating at the late-stage in the kidneys of lupus mice. These cells have a phenotype of mature monocyte-derived DCs, with particularly high CX3CR1 expression on the surface. Consistent with their pathogenic cytokine profile, in vivo administration of anti-CX3CR1-saporin conjugates to dysfunction these cells in MRL/lpr mice significantly reduced proteinuria scores. Ex vivo activation of renal-infiltrating CD4+ T cells showed increased survival rate, proliferation and IFN-γ production of activated CD4+ T cells when they were cultured with these renal-infiltrating CD11c+ cells. These results suggest that the renal-infiltrating CD11c+ cells are pathogenic and promote inflammation in the kidney at the later effector stage of lupus by interacting with renal-infiltrating CD4+ T cells. In conclusion, although vitamin A showed anti-inflammatory effects on reducing glomerulonephritis, its use in lupus treatment should be guarded due to the other potential pro-inflammatory effects induced by the pre-existing inflammatory environment. IFNα-producing pDCs and CX3CR1highCD11c+ monocyte-derived DCs could be specific therapeutic targets to reduce the established inflammation at the early stage and late stage of LN, respectively. Therefore, it is worthwhile to further investigate the comprehensive effects of combination therapy on lupus, with vitamin A administration and pDCs-specific depletion at the early stage, and CX3CR1highCD11c+ monocyte-derived DCs-specific depletion at the late stage. / Ph. D.

Oxidative stress plays a vital role in the pathogenesis of many chronic and acute inflammatory diseases. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two key mediators that are known to induce cellular and tissue oxidative stress. The generation of ROS and RNS is mediated by innate immune signaling processes. Lipopolysaccharide (LPS), a major inflammatory signal, is known to be a potent inducer of ROS/RNS. Thus, strategies that may block LPS-mediated generation of free radicals may hold promise in treating various inflammatory disease processes. However, the molecular mechanisms underlying LPS-mediated ROS/RNS production are not fully defined. Interleukin-1 Receptor associated kinase (IRAK-1), an intracellular kinase downstream of Toll-like Receptor 4 (TLR4) has been shown to contribute to the inflammatory cascade associated with LPS-TLR4 signaling pathway. However, its role in ROS production has not been defined. Therefore, we tested the hypothesis that IRAK-1 plays an important role in regulating ROS/RNS production. Both in vitro and in vivo studies were conducted to investigate the role of IRAK-1 in modulating free radicals as well as oxidative stress. In vitro studies demonstrate that IRAK-1 is a critical molecule involved in the induction of ROS/RNS. IRAK-1 deletion ablated free radical production following LPS challenge in a variety of cell types including macrophages, fibroblasts and microglia. Mechanistically, we observed that IRAK-1 is required for optimal expression and activity of NADPH oxidase subunits and iNOS. IRAK-1 deletion reduced LPS-triggered p47phox membrane translocation, suppressed NOX-1 expression and protein levels as well as hampered Rac1 activation. On the other hand, IRAK-1 deletion sustained antioxidative enzyme activity and levels in IRAK-1-/- macrophages and fibroblasts. In terms of the in vivo physiological consequences, IRAK-1-/- mice exhibited attenuated lipid peroxidation in vital organs, attenuated histopathological lesions in liver and kidney, and reduced endotoxemia-associated mortality. Taken together, IRAK-1 may, at least in part, serve as an important therapeutic target in the treatment of various inflammatory disease processes. / Ph. D.

Yes / Thyroid hormone (TH) is essential for adult brain function and its actions include several key roles in the hypothalamus. Although TH controls gene expression via specific TH receptors of the nuclear receptor class, surprisingly few genes have been demonstrated to be directly regulated by TH in the hypothalamus, or the adult brain as a whole. This study explored the rapid induction by TH of retinaldehyde dehydrogenase 1 (Raldh1), encoding a retinoic acid (RA)-synthesizing enzyme, as a gene specifically expressed in hypothalamic tanycytes, cells that mediate a number of actions of TH in the hypothalamus. The resulting increase in RA may then regulate gene expression via the RA receptors, also of the nuclear receptor class. In vivo exposure of the rat to TH led to a significant and rapid increase in hypothalamic Raldh1 within 4 hours. That this may lead to an in vivo increase in RA is suggested by the later induction by TH of the RA-responsive gene Cyp26b1. To explore the actions of RA in the hypothalamus as a potential mediator of TH control of gene regulation, an ex vivo hypothalamic rat slice culture method was developed in which the Raldh1-expressing tanycytes were maintained. These slice cultures confirmed that TH did not act on genes regulating energy balance but could induce Raldh1. RA has the potential to upregulate expression of genes involved in growth and appetite, Ghrh and Agrp. This regulation is acutely sensitive to epigenetic changes, as has been shown for TH action in vivo. These results indicate that sequential triggering of two nuclear receptor signalling systems has the capability to mediate some of the functions of TH in the hypothalamus.

Yes / Animals have evolved diverse seasonal variations in physiology and reproduction to accommodate yearly changes in environmental and climatic conditions. These changes in physiology are initiated by changes in photoperiod (daylength) and are mediated through melatonin, which relays photoperiodic information to the pars tuberalis of the pituitary gland. Melatonin drives thyroid‐stimulating hormone transcription and synthesis in the pars tuberalis, which, in turn, regulates thyroid hormone and retinoic acid synthesis in the tanycytes lining the third ventricle of the hypothalamus. Seasonal variation in central thyroid hormone signalling is conserved among photoperiodic animals. Despite this, different species adopt divergent phenotypes to cope with the same seasonal changes. A common response amongst different species is increased hypothalamic cell proliferation/neurogenesis in short photoperiod. That cell proliferation/neurogenesis may be important for seasonal timing is based on (i) the neurogenic potential of tanycytes; (ii) the fact that they are the locus of striking seasonal morphological changes; and (iii) the similarities to mechanisms involved in de novo neurogenesis of energy balance neurones. We propose that a decrease in hypothalamic thyroid hormone and retinoic acid signalling initiates localised neurodegeneration and apoptosis, which leads to a reduction in appetite and body weight. Neurodegeneration induces compensatory cell proliferation from the neurogenic niche in tanycytes and new cells are born under short photoperiod. Because these cells have the potential to differentiate into a number of different neuronal phenotypes, this could provide a mechanistic basis to explain the seasonal regulation of energy balance, as well as reproduction. This cycle can be achieved without changes in thyroid hormone/retinoic acid and explains recent data obtained from seasonal animals held in natural conditions. However, thyroid/retinoic acid signalling is required to synchronise the cycles of apoptosis, proliferation and differentiation. Thus, hypothalamic neurogenesis provides a framework to explain diverse photoperiodic responses. / MRC. Grant Number: MR/P012205/1 - Scottish Government - BBSRC. Grant Number: BB/K001043/1 - Physiological Society

L’acide rétinoïque (AR) agit via des récepteurs nucléaires (RAR) qui sont des facteurs de transcription inductibles par le ligand. Il active aussi des cascades de kinases qui ciblent les RAR et modulent leur activité transcriptionnelle. Cependant, l’ensemble des protéines phosphorylées en réponse à l’AR de même que les conséquences des dérégulations du « kinome » sur les effets l’AR et le fonctionnement des RAR demeurent mal connus. J’ai comparé les effets de l’AR sur le phosphoprotéome de deux lignées de cellules de cancer du sein : MCF7, qui est sensible à l’AR, et BT474, qui surexprime le récepteur a activité tyrosine kinase erbB-2 et est résistante à l’AR. De nombreuses différences ont été observées avec des répercussions sur l’expression des gènes de même que sur la phosphorylation, le recrutement aux promoteurs des gènes cibles et la dégradation de RAR alpha par le protéasome. J’ai aussi montré que la dégradation de RAR alpha met en jeu TRIM24 qui contrôle sa déubiquitination. / Retinoic acid (RA) acts by binding to specific nuclear receptors (RARs), which are ligand-dependant transcription factors. RA also has non-genomic effects and activates kinase cascades that target RARs and modulate their transcriptional activity. However, the proteins that are phosphorylated in response to RA remain to be identified. The consequences of dysregulations of the "kinome" on the non-genomic effects of RA and on RAR function also require further investigation. I compared the effect of RA on the phosphoproteome of two breast cancer cell lines: MCF7, which is RA-sensitive, and BT474, a RA-resistant cell line that overexpresses the receptor tyrosine kinase erbB-2. Multiple differences were observed with consequences on gene expression as well as on phosphorylation, recruitment on target genes promoters and RARalpha degradation by the proteasome. In the context or RARalpha degradation, I showed the involvement of TRIM24 which controls RARα deubiquitination.

L'acide rétinoïque (AR) régule la myogénèse embryonnaire. Dans le cadre de ce projet de thèse, nous avons d'une part utilisé l'AR pour moduler la myogénèse embryonnaire, dans la perspective d'étudier les conséquences de cette modulation sur le potentiel ultérieur de croissance et identifier les mécanismes moléculaires mobilisés.D'autre part, nous avons étudié la fonction de deux gènes régulés par l'AR et susceptibles de participer au contrôle de la myogénèse embryonnaire.La première partie du travail a été réalisée sur les modèles truite et poisson-zèbre. Nous avons montré que chez la truite comme chez le poisson zèbre, une incubation dans l'AR entrainait une activation de l'expression de Fgf8et de la différenciation des fibres musculaires rapides. Toutefois, chez la truite, nous n'avons pas pu mettre en évidence de régulation des MRF, indiquant qu'une autre voie est utilisée pour activer la myogénèse chez cette espèce.Dans la seconde partie de ce travail, la fonction de deux gènes régulés par l'AR et exprimés dans le mésoderme a été étudiée chez le poisson-zèbre. Le gène vertnin est exprimé essentiellement dans le tailbud. Quand il est inactivé par injection d'un oligo nucléotide morpholino antisens, on observe une altération de la formation des somites (mais pas de modification apparente du processus de segmentation) et une altération de l'intégrité des fibres lentes. Les fibres lentes sont en effet irrégulièrement espacées et les espaces au niveau des myoseptes verticaux peuvent être anormalement larges et les jonctions myotendineuses mal formées. Le gène arrestine β2aest exprimé dans les somites néo-formés puis également dans le mésoderme présomitique et le tailbud. Son inactivation par injection d'OM antisens entraine l'apparition du phénotype U-type et une altération de la morphologie des fibres lentes avec des fibres qui se détachent des jonctions myotendineuses. / Retinoic acid (RA) regulates embryonic myogenesis. During this thesis project, we first used RA to modulate embryonic myogenesis in order to study consequences of this modulation on the future potential for growth and to identify the underlying molecular mechanisms. Second part deals with the characterisation of the function of two genes regulated by the RA which may be involved in the control of embryonic myogenesis.The first part of the work was performed on the trout and zebrafish models. We have shown that in trout as in zebrafish, incubation in RA produced an activation of Fgf8 expression and differentiation of fast muscle fibers.However in trout, we did not observed regulation of MRF expression indicating that an alternative pathway isused to activate myogenesis in this species.In the second part of this work, the function of two genes regulated by the RA and expressed in the mesodermwas studied in zebrafish. The vertnin gene is expressed primarily in the tailbud. When it is inactivated by injection of antisense morpholino oligonucleotide, there is an alteration in the somites morphogenesis (but no apparent change in the process of segmentation) and impairment of the integrity of the slow muscle fibers. Slowfibers are indeed irregularly spaced and the vertical myosepta can be abnormally large. In addition myotendinous junctions display some abnormal branches. The arrestin β 2a gene is expressed in last formed somites and then also in the presomitic mesoderm and the tailbud. Its inactivation by injection of antisense MO leads to the appearance of the U-type phenotype and alteration of the slow muscle fibers morphology which detach frommyotendinous junctions

A deterioração oxidativa de alimentos é o fator determinante na qualidade sensorial e nutricional do produto, podendo ocorrer em alimentos complexos tanto na fase contínua como na fase dispersa. Em geral, a formação de radicais e espécies reativas de oxigênio e nitrogênio é o evento primário que ocorre priori ao progresso da oxidação de componentes e estruturas sensíveis e está comumente associado a eventos na fase aquosa. A oxidação de lipídeos é frequentemente investigada em alimentos e em meio biológico separadamente dos eventos no meio aquoso e a proteção antioxidante somente avaliada na fase dispersa. Entretanto, a oxidação de proteínas e eventos na fase contínua vêm despertando considerável interesse e aparentemente antioxidantes eficientes na fase dispersa não protegem de forma eficaz as proteínas e os componentes na fase contínua. Um futuro progresso na proteção antioxidante de alimentos e sistemas biológicos origina de uma abordagem holística dos processos redox envolvidos em ambas as fases dispersa e contínua, bem como o uso combinado de espécies antioxidantes e antiredutoras para um superior efeito antiradical global. Neste sentido, foram preparados três novos compostos antiradicais de dupla função (antioxidante e antiredutor) com adequado balanço hidrofílico-lipofílico e aprimorada propriedade antiradical global. Os antiradicais diferulato de astaxantina, retinoato de quercetina e retinoato de epicatequina se mostraram melhores antioxidantes e antiredutores do que seus precursores isoladamente ou em misturas, apresentando superior eficiência na desativação do oxigênio singlete excitado, captação do radical 1-hidroxietila, menor efeito pró-oxidante em sistemas de oxidação catalisados por íons de ferro, e eficiência na redução do reativo estado triplete da safranina (E1/2 = 1,48 V vs. NHE). / The oxidative deterioration of foods is the determining factor in the sensory and nutritional quality of the product, which may occur in complex food both at the continuous and the dispersed phases. In general, the formation of radicals and reactive oxygen and nitrogen species is the primary event that occurs prior to the progress of oxidation of components and sensitive structures and is commonly associated with events in the aqueous phase. The oxidation of lipids is often investigated in food and biological media separately from the events in the aqueous phase and the measurement of the antioxidant protection is only evaluated in the dispersed phase. However, the oxidation of proteins and the events in the continuous phase have attracted considerable interest and apparently, efficient antioxidants in dispersed phase do not effectively protect proteins and components in the continuous phase. A further progress in the antioxidant protection of food and biological systems arise from a holistic approach of the redox processes involved in both the dispersed and continuous phases as well as the combined use of antioxidant and antireductant species for a superior overall antiradical effect. In this sense three new antiradical compounds with dual function (antioxidant and antireductant) were prepared with proper hydrophilic-lipophilic balance and global antiradical properties. The antiradicals astaxanthin diferulate, epicatechin retinoate, and quercetin retinoate are shown to be better antioxidant and antireductant than their precursors, isolated or in mixture, exhibiting enhanced singlet excited oxygen deactivation, 1-hydroxyethyl radical scavenging, lower pro-oxidative effect in the oxidative system catalysed by iron ions and an efficient reduction of the reactive triplet state of safranine (E1 / 2 = 1.48 V vs. NHE).

Nos últimos anos, nós propusemos um modelo de duas etapas para a padronização antero-posterior do coração. Ácido retinóico (AR) produzido pela enzima RALDH2 induz o destino sino atrial nos precursores cardíacos posteriores. Subsequentemente, estes precursores adquirem a capacidade de expressar RALDH2, formando uma onda caudo-rostral desta enzima. A nossa hipótese é que esta onda surgiu nos para padronizar as células precursoras da bomba circulatória ancestral em regiões de influxo e efluxo, resultando na origem das câmaras cardíacas. Para testar se a onda cauro-rostral é ancestral nos vertebrados, nós mapeamos a expressão de RALDH2 em relação ao campo cardíaco em anfíbios, vertebrados basais e no cordado invertebrado anfioxo. Nossos dados sugerem que o modelo de duas etapas está presente em anfíbios e peixes. Clonagem do gene RALDH em lampréias indica presença de AR no campo cardíaco. Em anfioxo, a caracterização do padrão de expressão do ortólogo da RALDH2 revela ausência da onda caudo-rostral. Nossos resultados sugerem que a onda caudo-rostral de RALDH2 foi cooptada nos vertebrados para padronizar o campo cardíaco no eixo AP, o que corrobora a hipotése de que este mecanismo foi importante na origem evolutiva das câmaras cardíacas. / In the last years, we have proposed a 2-step model for the establishment of cardiac chamber identities. Retinoic acid (RA) produced by its synthetic enzyme RALDH2, induces an atrial fate in posterior cardiac precursors of amniote embryos. Subsequently, a RALDH2 caudorostral wave engulfs posterior precursors. Our hypothesis is that this wave evolved in vertebrates to pattern an ancestral circulatory pump into AP fields, which were later fashioned into cardiac chambers. To test whether the wave is an ancestral or derived feature of amniotes, we mapped expression of RALDH2 in relation to the cardiac field in amphibians, basal vertebrates and the amphioxus. Our data suggests RA signaling patterns amphibian and piscine hearts. Cloning of RALDH in lampreys shows that RA synthesis takes place in the heart field. In the amphioxus, cloning of RALDH reveals a vertebrate-like expression pattern, although the RALDH2 wave is absent. Our results support the hypothesis that the caudorostral wave of RALDH2 was coopted to pattern the vertebrate cardiac field. This supports the hypothesis that the caudorostral wave of RALDH2 was an important player in the evolutionary origin of the cardiac chambers.

O mastocitoma é o tumor cutâneo mais comum dos cães, representando 7% a 21% dos tumores da pele e tecidos moles, 11% a 27% dos tumores malignos cutâneos nessa espécie. Eles possuem uma grande variedade de aparência e comportamento, o qual o torna um desafio seu tratamento. Os retinóides são uma promessa na luta contra o câncer. Entretanto, há poucos estudos sobre os efeitos dos retinóides em neoplasias caninas. O presente trabalho teve como objetivo caracterizar a cultura primária de mastocitomas caninos assim como investigar a quimiosensibilidade deste tumor ao ácido retinóico todo-trans (ATRA). A cultura primária de mastocitomas caninos foi realizada em co-cultivo com fibroblastos, que demonstrou uma interação favorável entre mastócitos e fibroblastos, com uma sobrevida média de 30 dias. A quimiosensibilidade dos mastocitomas caninos ao ATRA não mostrou diferenças entre os graus de mastocitomas, ou seja, tanto um mastocitoma grau II ou III respondem igualmente ao ATRA nas doses estudadas. Foi constatado também que o mastocitoma é mais sensível na concentração 10-4M de ATRA (p < 0,002). Existe também um efeito já nas primeiras 24h, mas esse não se altera em 48h, entretanto se intensifica após 72h. Podemos inferir, então, que a maior quimiosensibilidade de mastocitomas caninos ao ATRA se dá após 72h de exposição na dose de 10-4M. Podemos concluir que o ATRA apresenta efeitos sobre as células de mastocitomas caninos e pode ser usado como potencial adjuvante no tratamento desta neoplasia. / Mast cell tumor (MCT) is one of the most frequent neoplasms that affect the skin and soft tissue of the dog, representing about 7% a 21% of all skin tumors and 11% a 27% of malignant skin tumors in this specie. They present a great variety of appearance and behavior, which becomes a challenge to the treatment. The retinoids are well recognized as promising antitumor agents. However, there have only been a few reports about the effect of retinoids in canine cancers. The aim of this study was to characterize the primary mast cell tumor culture and to investigate the chemosensitivity of this tumor to all trans retinoic acid (ATRA). The primary cell culture of MCT was performed as co-cultive with fibroblasts, showing a positive interaction between mast cells and fibroblasts, with a lifetime of 30 days. The chemosensitivity of MCT to ATRA showed no difference between grade II or III, thus either a MCT grade II or grade III has the same response with ATRA at the doses studied. It has been shown that the MCT is more sensible at the dose 10-4M (p < 0,002). There is also an effect on first 24h untill 48h, changing after 72h. According to these results, it is possible to state that the great chemosensitivity of MCT to ATRA is after 72h of exposition at 10-4M. We can conclude that ATRA may be a potential adjunctive chemotherapeutic agent for the treatment of canine mast cell tumor.