Rozprawy doktorskie na temat „Orai1”

Ein durch „Ca2+ Release Activated Ca2+ (CRAC)“-Kanal vermittelter Ca2+-Einstrom ist unverzichtbar für die vollständige Aktivierung von T-Zellen und eine produktive Immunantwort. Im Jahr 2006 führte die Entdeckung des transmembranen Proteins Orai1, einer porenbildenden Untereinheit des CRAC-Kanals, zu einem besseren Verständnis dieses Signalweges. Eine Mutation in Orai1 hat durch die Aufhebung der CRAC-Kanal Funktion eine schwere kombinierte Immundefizienz (SCID) zur Folge (Feske, S. et al. 2006). Die im Rahmen dieser Arbeit präsentierten Experimente hatten die nähere Erforschung der Rolle von Orai1 in Bezug auf die Aktivierung und Entwicklung von Lymphozyten sowie auf die pathogenetische Bedeutung für humane Immundefektsyndrome zum Ziel. So konnte hier durch das Sequenzieren genomischer DNA mehrerer SCID-Patienten eine neue Mutation in Orai1 aufgedeckt werden. Mithilfe intrazellulärer Durchflusszytometrie und Real-Time-PCR gelang es, die Expression von Orai1 auf humanen und murinen Immunzellen, einschließlich T- und B-Lymphozyten, nachzuweisen. Darüber hinaus wurden Orai1 „knock-in“ Mäuse analysiert, welche transgen für eine bei zwei SCID-Patienten gefundene Mutation (R91W) (Feske, S. et al. 2006) sind. Dadurch war es möglich die Funktion von Orai1 und die des CRAC-Kanal vermittelten Ca2+-Einstroms für die Entwicklung und Aktivierung von Lymphozyten zu analysieren. Diese transgenen Mäuse stellen das zu diesem Zeitpunkt erste Tiermodell dar, mit dessen Hilfe die Rolle von CRAC-Kanälen in vivo studiert werden kann.

Calmodulin (CaM) plays an important role in calcium (Ca²⁺)-dependent signal transduction. Ca²⁺ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca²⁺-dependent inactivation (CDI) process in store-operated Ca²⁺ entry (SOCE), by interacting with the N-terminus of the hexameric plasma membrane Ca²⁺ channel Orai1. To understand the relationship between Ca²⁺-induced hydrophobicity of CaM and the CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) were used as an informative probe to better understand the functionality of each EF-hand. ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca²⁺ binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM, as depicted by ANS fluorescence and binding affinity. Such a conclusion is consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai1. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = − 5.02 ± 0.13 kcal/mol) and binding affinity (K_a = 8.92 ± 1.03 x 105 M⁻¹). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ?H than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. Circular Dichroism studies suggested differences in binding most likely resulted from changes in chimera three-dimensional structure rather than secondary structure, as the extent of ?-helical content from apo-, Ca²⁺-, and Orai-CMBD-bound proteins remained similar. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s⁻¹ by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76, indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the unique, open binding mode suggested by already published structural studies.

L'altération de glycosylation est l'une des caractéristiques du cancer du sein. Ainsi le défaut de glycosylation affecte différentes protéines glycosylées responsables des différents processus cancéreux. Les canaux SOC (Store operated channels) constituent la voie majeure de l'entrée du calcium dans les cellules et sont impliqués dans la prolifération, la migration et la survie des cellules cancéreuses du sein. CO-OCS est un nouvel inhibiteur de la glycosylation avec plus de sélectivité vis-à-vis des α-glucosidases, et montre des activités anticancéreuses des cellules cancéreuses du sein, sans affecter les cellules mammaires normales. L'objectif de ma thèse est d'étudier les mécanismes moléculaires par lesquels CO-OCS induit ses effets anti-tumoraux. CO-OCS inhibe la migration des cellules cancéreuses à fort potentiel métastatique. Cet effet anti-migratoire est dû à une réduction de l'expression de la β1-intégrine, de Stim1, et de l'activation des voies de signalisation FAK et ERK1/2 par CO-OCS. Dans les cellules cancéreuses peu invasives, CO-OCS diminue la prolifération et augmente la mortalité de ces cellules en affectant l'expression de 3 protéines : Stim1 et Orai1 : protéines N-glycosylées au niveau du réticulum endoplasmique (RE), et GRP78, protéine de stress du RE. Ainsi en supprimant complétement l'expression de Stim1, CO-OCS réduit la prolifération en accumulant les cellules dans les phases G1 et G2/M du cycle cellulaire. Alors que la réduction de l'expression de GRP78 et d'Orai1 par le CO-OCS augmente respectivement l'apoptose et la nécrose. Par ailleurs, l'invalidation de Stim1 atténue l'effet apoptotique induit par CO-OCS. CO-OCS réduit aussi le contenu calcique du RE. Cette réduction du calcium réticulaire est due à une fuite de calcium par le Translocon. En effet, l'Anisomycine, inhibiteur du Translocon, restore de contenu calcique réticulaire et antagonise l'apoptose induite par le CO-OCS. En conclusion, CO-OCS induit une accumulation de protéines mal-repliées dans le RE induisant ainsi un stress réticulaire. Trois cibles du CO-OCS ont été identifiées : l'expression de Stim1 favorise la prolifération tandis que celle d'Orai1 et de GRP78 protègent respectivement les cellules de l'apoptose et de la nécrose induites par CO-OCS. De plus, en diminuant l'expression de GRP78, CO-OCS induit une fuite du calcium du RE par le Translocon
Alteration in glycosylation pattern is one of the hallmarks of breast cancer. The levels and the abnormal expressions of glycan were found in breast cancer patients. Glycosylation defect can affect different glycosylated proteins which are implicated in cancerogenesis. Changes in intracellular Ca2+ levels can regulate different cellular processes. SOC channels are implicated in breast cancer proliferation, migration and survival. CO-OCS is a new glycosylation inhibitor with more selectivity toward theα- glucosidases exhibited anti-cancer activities in breast cancer cells without affecting the normal mammary cells. The objective of my thesis is investigating the related molecular mechanisms by which CO-OCS induced its anti-tumour effects.CO-OCS impaired breast cancer migration through decrease β1-integrin expression and the activation of FAK and ERK1/2 signalling pathways. CO-OCS also induced anti-migratory effect via Stim1 protein expression down-regulation leading to inhibition of SOCE. Additionally, CO-OCS affected the expression of both Orai1 and Stim1 proteins leading to anti-proliferative effects and cell cycle arrest in G1 and G2/M phase respectively. Moreover, CO-OCS affected the expression of Stim1 at the protein level without affecting its transcript level. GRP78 implicated in CO-OCS apoptotic death. The expression of Stim1 regulated the apoptosis induced by CO-OCS via modulating GRP78 expression. Orai1 down-regulation promoted CO-OCS necrotic effect. CO-OCS induced ER- calcium depletion due to increase in ER calcium leak via the Translocon; Anisomycin (Translocon inhibitor) decreased the apoptosis induced by CO-OCS. In conclusion, these results show that in breast cancer, by targeting Stim1, Orai1 and GRP78, CO-OCS reduced cell proliferation and induced apoptosis and necrosis cell death. Stim1 favours CO-OCS apoptotic effect while Orai1 protected from necrosis induced by CO-OCS. The inhibition of Translocon decreased CO-OCS apoptotic cell death via restoring the ER calcium homeostasis

Alors que l’entrée SOC (store-operated Ca2+ entry) portée par les canaux calciques TRPCs (transient receptor potential canonical) et Orai1 est essentielle dans les cellules non-excitables, son rôle physiologique dans les cardiomyocytes adultes reste à élucider. Néanmoins, il est largement admis qu’une entrée SOC exacerbée dépendante des canaux TRPCs et de la protéine régulatrice STIM1 participe à la pathogenèse de l’hypertrophie et de l’insuffisance cardiaque (IC) par induction de voies pro-hypertrophiques telles que la CaMKII (Ca2+/calmoduline-dépendante kinase II ) et la calcineurine (CaN)/NFAT (Nuclear factor of activated T-cells). Au contraire, une inhibition fonctionnelle ou une extinction génique des canaux TRPCs et de la protéine STIM1 serait cardioprotectrice contre le stress hypertrophique. Cependant, le rôle physiopathologique des canaux calciques Orai1 dans le cœur reste, à ce jour, méconnu et débattu puisque son extinction in vitro présente un effet bénéfique contre l’hypertrophie des cardiomyocytes alors que son extinction in vivo présente des effets délétères avec le développement d’une cardiomyopathie dilatée. De plus amples investigations quant au rôle d’Orai1 dans la physiopathologie cardiaque apparaissent donc primordiales. De ce fait, les objectifs de ma thèse sont d’explorer le rôle de la signalisation calcique dépendante d’Orai1 dans le cœur dans des conditions physiologiques et pathologiques grâce à un modèle de souris transgéniques exprimant un mutant non fonctionnel d’Orai1, spécifiquement dans le cœur (dn-Orai1R91W/tTa) et un inhibiteur pharmacologique sélectif, le JPIII. Tout d’abord, nous montrons que les souris dn-Orai1R91W/tTa présentent une fonction cardiaque normale et une homéostasie calcique impliquée dans le couplage excitation-contraction conservée suggérant qu’Orai1 n’a pas de rôle majeur dans le coeur adulte en condition physiologique. Cependant, nous avons démontré une augmentation de l’expression et de l’activité d’Orai1 dans un modèle murin d’hypertrophie cardiaque induite par surcharge de pression, qui serait délétère pour la fonction ventriculaire. Au contraire, l’inhibition fonctionnelle d’Orai1 par manipulation génétique ou par l’outil pharmacologique (JPIII) semble protéger le coeur des dysfonctions ventriculaires au cours de l’hypertrophie. Cet effet bénéfique passerait par une restauration de l’homéostasie calcique et notamment par un maintien de l’expression de la pompe ATPase SERCA2a. Nous avons également mis en évidence que la voie de l’aldostérone/récepteurs aux minéralocorticoïdes modulait l’expression des canaux TRPC1, -C4, -C5 et notamment Orai1 via la protéine SGK1 (Serum and Glucocorticoid-regulated Kinase 1) dans les cardiomyocytes ventriculaires de rat nouveaux-nés. L’activation de cette voie de signalisation pourrait être à l’origine de la surexpression des canaux TRPCs/Orai1 retrouvée au cours de l’hypertrophie cardiaque. Ces travaux décrivent donc Orai1 comme une cible thérapeutique potentielle dans le traitement de l’hypertrophie cardiaque et de l’IC
While the SOCE (store-operated Ca2+ entry), carried by TRPCs (transient receptor potential canonical) and Orai1 channels, is essential in non-excitable cells, its physiological role in adult cardiomyocytes remains elusive. Nevertheless, it is well established that exacerbated TRPCs/STIM1-dependent Ca2+ entry participates in the pathogenesis of hypertrophy and heart failure (HF) via the induction of pro-hypertrophic signaling pathways, such as CaMKII (Ca2+/calmodulin-kinase II) and calcineurin (CaN)/ NFAT (nuclear factor of activated T-cells). By contrast, functional inhibition or gene silencing of TRPCs and STIM1 is cardioprotective against hypertrophic insults. As for Orai1 Ca2+ channels, their pathophysiological roles in the heart remain unknown and under debate, since in vitro Orai1 silencing has a beneficial effect against cardiomyocyte hypertrophy, whereas in vivo silencing has deleterious effects with the development of dilated cardiomyopathy. Further investigations are necessary to determine the pathophysiological role of Orai1 in the heart. My thesis objectives are to explore the role of Orai1-dependent Ca2+ signaling in the heart under physiological and pathological conditions using a transgenic mouse model expressing a non functional mutant of Orai1, specifically in the heart (dn-Orai1R91W/tTa) and a selective pharmacological inhibitor, JPIII. First, we showed that dn-Orai1R91W/tTa mice have normal cardiac function and conserved Ca2+ homeostasis involved in the excitation-contraction coupling suggesting that Orai1 is not instrumental in regulating cardiac function under physiological conditions. However, we demonstrated an increased Orai1 expression and activity in a mouse model of cardiac hypertrophy induced by pressure overload, which is a maladaptive alteration involved in pathological ventricular dysfunction. By contrast, functional inhibition of Orai1 by genetic manipulation or by the pharmacological tool (JPIII) protects the heart from ventricular dysfunction after pressure overload-induced cardiac hypertrophy. This beneficial effect is related to a restoration of Ca2+ homeostasis and more specifically, is due to preserved ATPase SERCA2a pump expression. We also showed that the aldosterone/mineralocorticoid receptor signaling pathway modulates the expression of TRPC1, -C4, -C5 channels and also the Orai1 channels expression via the SGK1 (Serum and Glucocorticoid-regulated Kinase 1) protein, in neonatal rat ventricular cardiomyocytes. The activation of this signaling pathway could be the cause of the TRPCs/Orai1 channels overexpression found during cardiac hypertrophy. In conclusion, our studies highlighted that Orai1 Ca2+ channels could constitute potential therapeutic target in the treatment of cardiac hypertrophy and HF

Nous avons montré un rôle d’un canal K+, le canal SK3, dans la migration des cellules cancéreuses de sein MDA-MB-435s et le développement de métastases ostéolytiques du cancer du sein. Lors de l’ostéolyse, la [Ca²+]ext augmente dans le microenvironnement osseux. Nous avons voulu déterminer si cette élévation de [Ca²+]ext, pouvait moduler l’expression et l’activité du canal SK3. Nous avons montré que l’augmentation de la [Ca²+]ext: i) favorise l’expression du canal SK3. Cet effet fait intervenir le récepteur au calcium (CaSR), qui en diminuant la [AMPc]int réduit l’activité de la PKA et lève ainsi son inhibition de la transcription du gène KCNN3 (codant pour SK3) ; ii) favorise la migration cellulaire dépendante du canal SK3, mécanisme impliquant également le CaSR ; iii) active le canal SK3 qui, par ailleurs, voit son activité réduite par l’élévation d’AMPc intracellulaire. De plus, l’augmentation d’AMPc délocalise un canal calcique partenaire de SK3, le canal Orai1, et diminue l’entrée constitutive de Ca²+ et la migration dépendantes du canal SK3. En conclusion, nos résultats montrent que l’expression et l’activité de SK3 sont régulées par l’AMPc et le Ca2+ extracellulaire. Ceci permet d’envisager une nouvelle stratégie thérapeutique ciblant l’AMPc pour le traitement des métastases osseuses du cancer du sein
We showed that a K+ channel, SK3 channel, is a mediator of MDA-MB-435s breast cancer cells migration and of osteolytic bone metastasis development of breast cancer. Since [Ca²+]out rises during osteolysis, in bone microenvironment, we study if this [Ca²+]out elevation could modulate SK3 expression and activity. We show that [Ca²+]out elevation: i) increases SK3 expression threw CaSR activation which, in turn, decreases [cAMP]int and PKA activation, leading to loss of its inhibitory effect on KCNN3 transcription; ii) increases SK3-dependent migration threw CaSR activation; iii) increases SK3 channel activity that is in addition, decreased by [cAMP]int elevation. Furthermore, cAMP elevation moves the Ca2+ channel Orai1 (SK3 partner) outside of lipid rafts and reduces the SK3 dependent-constitutive Ca²+ entry and cell migration. Our results show that both SK3 expression and activity are regulated by cAMP and extracellular Ca²+. These results underscore an innovative opportunity to use therapeutic approaches targeting cAMP for the treatment of breast cancer bone metastasis

Le cancer de la prostate (CaP) est le cancer le plus fréquent et le troisième plus mortel chez l’homme en Europe. Les cellules souches cancéreuses (CSC) représentent une sous population de cellules cancéreuses possédant des propriétés de cellules souches qui les rendent résistantes aux thérapies et hautement tumorigènes. Les CSCs sont ainsi associées aux phénomènes de dormance tumorale, puis de rechute suite à leur réactivation. Les mécanismes régulant la transition dormance/prolifération constituent donc une question centrale dans la prise en charge du cancer. L’importance des protéines Orai dans le CaP a déjà été montrée dans de précédentes études, via leur implication dans les canaux de type SOC (store-operated calcium channel) et ARC (arachidonic acid-regulated channel). Cependant, le rôle du canal Orai1 dans la prolifération du CaP, ou son éventuelle implication dans la physiologie des CSC, restaient inconnus. Parallèlement, pour répondre aux limitations de son ciblage direct, nous avons cherché à identifier ses protéines partenaires. Nous nous sommes ainsi intéressés au récepteur Sigma 1 (S1R), une protéine chaperonne dont l’expression augmente dans le CaP, et qui possède de nombreux modulateurs pharmacologiques utilisés en clinique. Ce travail avait donc un double objectif : étudier le rôle d’Orai1 dans le CaP et les CSC prostatiques, et caractériser fonctionnellement le rôle du S1R en tant que nouveau partenaire du canal Orai1. Ces travaux ont tout d’abord permis de mettre en évidence l’importance d’Orai1 dans le contrôle de la transition entre la quiescence et la prolifération des CSCs prostatiques via la voie NFAT. De plus, ces résultats ont été confirmés dans les CSCs de mélanome, montrant que le rôle d’Orai1 serait généralisable au-delà du modèle prostatique. Nous avons également montré que le S1R interagit directement avec Orai1 et module positivement son activité, impactant ainsi la prolifération des cellules cancéreuses prostatiques. Enfin, nous avons mis en évidence la régulation de l’expression de ces protéines par les androgènes, ce qui est d’importance cruciale dans l’évolution du CaP. Nos résultats ont donc permis l’identification d’un acteur central du contrôle de la prolifération du CaP (Orai1), et la caractérisation d’une nouvelle protéine partenaire du canal Orai1 dans le CaP : le S1R. Ces travaux montrent que le S1R et Orai1 pourraient constituer de nouveaux marqueurs intéressants, ainsi que de potentielles nouvelles cibles thérapeutiques
Prostate cancer (PCa) is the most frequent and the third deadliest cancer in men in Europe. Cancer stem cells (CSC) are a rare subset of cancer cells possessing stem cell properties leading to a high resistance to therapy and an enhanced tumorigenicity. As a result, CSCs have been linked to tumor dormancy and relapse upon reactivation. Thus, the mechanisms regulating CSC dormancy/activation transition are of critical importance in PCa. Previous studies showed the importance of Orai proteins in PCa, through their roles in SOC (store-operated channel) and ARC (arachidonic acid-regulated calcium channel) channels. But the role of Orai1 in PCa proliferation and CSC physiology remained to be studied. Moreover, in order to bypass current targeting limitations for Orai1, we aimed to identify a partner protein able to regulate Orai1 in PCa. For this purpose, we focused on the Sigma 1 receptor (S1R), a chaperone protein capable of ion channel regulation. Interestingly, S1R expression is increased in PCa and this protein can bind many pharmacological compounds currently used for other clinical applications. This work thus aimed to first study the role of Orai1 in PCa and CSC physiology, and then characterize the role of S1R as a new regulator of Orai1 in PCa. Our results first show that Orai1 is a key regulator of CSC transition between quiescence and proliferation via the NFAT pathway. Moreover, this role is not limited to PCa, since these results were also confirmed in melanoma CSCs. We also show here that the S1R directly interacts with Orai1 and increases its activity, thus modulating PCa cell proliferation. Finally, we characterized the regulation of Orai1 and S1R expression by androgens, which is highly significant during PCa development. Our results therefore allowed the identification of a key regulator of PCa proliferation (Orai1), and propose an alternative method for its targeting via the identification of its partner protein (S1R). These results could lead to the development of new markers and innovative therapeutic strategies

Os distúrbios na regulação da concentração de cálcio (Ca2+) citoplasmático contribuem para a patogênese da hipertensão arterial. Evidências sugerem que as moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+, enquanto as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo avaliamos a participação de STIM1/Orai1 na regulação das concentrações de Ca2+ citoplasmático e na ativação da contração vascular em aortas de ratos hipertensos. Nossos resultados sugerem que a ativação de STIM1/Orai1 pode representar um novo mecanismo que modula alterações vasculares nos níveis de Ca2+ intracelular na hipertensão arterial e que contribui para as diferenças sexuais de reatividade vascular em animais hipertensos.
Disturbance in the regulation of cytoplasmic calcium (Ca2+) concentration contributes to the pathogenesis of hypertension. Evidences suggest that the stromal interaction molecule (STIM) acts as a sensor of intracellular Ca2+ stores, whereas Orai proteins are the subunits that form CRAC channels. In this study, we evaluated the role of STIM1/Orai1 in the regulation of cytoplasmic Ca2+ concentrations and in the activation of contraction in aortas from hypertensive rats. We also studied how the differential activation of this pathway contributes to sex differences observed between hypertensive rats, as well as the protective effects of the female sex hormones in the vasculature. Our results suggest that activation of STIM1/Orai1 may represent a new mechanism that modulates intracellular Ca2+ concentration during hypertension and contributes to sex differences in the vascular reactivity of hypertensive animals.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-translacional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: a enzima OGT é responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina, enquanto a OGA catalisa a remoção de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvo de O-GlcNAc e o aumento da expressão de proteínas modificadas por O-GlcNAc promove aumento da reatividade vascular para estímulos contráteis. Um dos mecanismos de extrema importância no controle do tônus vascular está ligado à regulação da concentração de cálcio (Ca2+) intracelular, onde destacamos a participação do sistema STIM1/Orai1. As moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+ e as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo investigamos a hipótese de que o aumento dos níveis vasculares de proteínas glicosiladas aumenta a resposta contrátil em aorta de ratos, por mecanismos relacionados ao controle da concentração intracelular de Ca2+.Em nossos experimentos, utilizamos aortas torácicas de ratos incubadas com PugNAc (inibidor seletivo da OGA, ), por 24h. Utilizando protocolo experimental que permite avaliar contrações induzidas pelo influxo de Ca2+ e liberação de Ca2+ intracelular, demonstramos que a incubação com PugNAc aumentou a resposta contrátil à PE bem como a contração durante o período de influxo de Ca2+, induzida pela reintrodução de solução fisiológica contendo Ca2+ (1,56 mM). O bloqueio dos canais CRAC com 2-APB (100 ) e gadolíneo (Gd3+, 100 ) diminuiu significativamente as contrações induzidas pelo influxo de Ca2+ em aortas incubadas com PugNAc. Além disso, estas aortas apresentaram aumento da expressão protéica de STIM1, o que resultaria em maior influxo de Ca2+. A contração induzida por cafeína (20 mM) e serotonina (10 ), a qual reflete a capacidade funcional do retículo sarcoplasmático (RS) em captar Ca2+, foi maior em aortas incubadas com PugNAc. O papel da Ca2+-ATPase (SERCA) foi avaliado com a utilização de tapsigargina, bloqueador da SERCA. O efeito da tapsigargina foi semelhante em artérias incubadas com PugNAc e veículo, apesar do aumento de expressão proteica da SERCA em aortas incubadas com PugNAc. Como a proteína cinase C (PKC) é ativada por aumentos de Ca2+ intracelular, determinamos se a atividade de proteínas alvo da PKC estavam aumentadas. A incubação com PugNAc aumentou a expressão das formas fosforiladas da CPI-17, MYPT-1 e MLC. Em conjunto, estes resultados sugerem que a ativação de STIM1/Orai1, aumento da liberação de Ca2+ intracelular e ativação da via de sinalização da PKC podem representar mecanismos que modulam as alterações vasculares em resposta ao aumento de proteínas glicosiladas por O-GlcNAc.
Glycosylation with O-linked -N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic post-translational modification. The process of O-GlcNAc is controlled by two enzymes: the OGT enzyme catalyses the addition of N-acetyl-glucosamine to the hydroxyl group of serine and threonine residues of a target protein, while OGA catalyzes the cleavage of O-GlcNAc from post-translationally-modified proteins. Proteins with an important role in vascular function are targets of O-GlcNAc and increased levels of proteins modified by O-GlcNAc increase vascular reactivity to contractile stimuli. The regulation of intracellular calcium (Ca2+) concentration, including the activation of STIM1/Orai1, is key in the control of vascular tone. The stromal interaction molecules (STIM) act as sensors of intracellular Ca2+ stores whereas the Orai proteins represent subunits of the Ca2+ release-activated Ca2+ channels (CRAC). We hypothesized that increased levels of vascular O-GlcNAc proteins augment vascular contractile responses by altering mechanisms that regulate the intracellular Ca2+. Rat thoracic aortas were incubated with PugNAc (OGA selective inhibitor, ) for 24h. Using an experimental protocol that evaluates contractions induced by Ca2+ influx and release, we demonstrated that incubation with PugNAc increases contractile responses to phenylephrine (PE) as well as the contraction induced by Ca2+ influx, after depletion of intracellular Ca2+ stores. The CRAC channel blockers, 2-APB (100 ) and gadolinium (Gd3+, 100 ), significantly reduced the contractions induced by Ca2+ influx in aortas incubated with PugNAc. Furthermore, these aortas showed increased STIM1 protein expression, which could result in increased influx of Ca2+ and, in turn, increase vascular contraction. The contraction induced by the release of intracellular Ca2+ stores, stimulated by caffeine (20 mM) and serotonin (10 ), was increased in aortas incubated with PugNAc. The Ca2+-ATPase (SERCA) inhibitor thapsigargin produced similar effects in arteries incubated with PugNAc or vehicle, despite the increased SERCA protein expression in aortas incubated with PugNAc. Since PKC is activated by increases in intracellular Ca2+ and arteries incubated with PugNAc show activation of PKC, we determined whether the activity of proteins that are targets of PKC was increased in PugNAc-treated aortas. Incubation with PugNAc increased the expression of phosphorylated forms of CPI-17, MYPT-1 and MLC. Together, these results suggest that activation of STIM1/Orai1, increased release of intracellular Ca2+ and PKC activation may represent mechanisms that modulate vascular responses upon increased O-GlcNAc proteins.

La Leucémie Myéloïde Chronique (LMC) est une maladie clonale caractérisée par la présence du chromosome Philadelphie codant pour Bcr-Abl, une tyrosine kinase constitutivement active responsable de la leucémogenèse. Bien que très efficaces, les inhibiteurs de tyrosine kinase (ITKs) restent cependant inactifs sur les cellules souches leucémiques. Ce travail de thèse montre que la signalisation calcique, connue pour réguler de nombreux processus dans les cellules saines et cancéreuses, est importante dans la signalisation cellulaire au décours de la LMC. Le rôle des entrées calciques dépendantes des stocks (SOCEs) médiées par STIM1 (STromal Interaction Molecule 1) et les canaux Orai1 et TRPC1 ainsi que des entrées calciques induites par la thrombine a été étudié dans la leucémogenèse. Nous avons observé une diminution de ces entrées dans les cellules exprimant Bcr-Abl pouvant être expliquée par le changement de stœchiométrie Orai1/STIM1. Ceci entraîne la diminution de l'activation de NFAT (Nuclear Factor of Activated T-cells) ainsi que des conséquences sur la prolifération et la migration cellulaire mais pas sur l'apoptose. De plus, les SOCEs sont restaurées dans les cellules cancéreuses après traitement à l'Imatinib, le principal ITK. Nous proposons alors que l'expression de Bcr-Abl joue un rôle sur l'homéostasie calcique en entraînant une dérégulation générale des fonctions cellulaires dans les cellules leucémiques notamment via la voie PKC (Protein Kinase C). Ainsi, ces résultats montrent une dérégulation des entrées calciques dans les cellules exprimant Bcr-Abl, suggérant que la signalisation calcique puisse être une cible thérapeutique en parallèle avec les ITKs
Chronic Myeloid Leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia chromosome encoding for Bcr-Abl, a constitutively active tyrosine kinase responsible for leukemogenesis. Although Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the therapy of Ph+ leukemia, the complete eradication of CML is limited by the emergence of resistance in hematopoietic stem cells. This thesis proposes that calcium (Ca2+) signaling pathways, known to govern a large number of functions in normal and cancer cells, may be important in CML cell signaling. Therefore, we studied the role of Store Operated-Calcium entry (SOCE) (i.e. STromal Interaction Molecule 1 (STIM1), Orai1 and TRPC1 channels) and thrombin induced Ca2+ entry in leukemogenesis. We found a decrease in both calcium entries in Bcr-Abl-expressing cells compared to normal cells. The reduced SOCE seems related to a change in stoichiometry of Orai1/STIM1. This leads to a reduction of the Nuclear Factor of Activated T-cells (NFAT) translocation and functional consequences on cell proliferation and migration but not on apoptosis. Moreover, we showed that SOCE is restored in malignant cells after treatment with Imatinib, the main TKI. We proposed that Bcr-Abl expression could impact on Ca2+ homeostasis enhancing a general disorganization of cell functions in leukemia cells notably via Protein Kinase C (PKC) pathway. Altogether this work shows a deregulation of Ca2+ entry in Bcr-Abl-expressing cells, suggesting that the Ca2+ signaling pathway could be a therapeutic target in parallel with TKIs

Les lymphomes B non-Hodgkiniens (LNHB) représentent le type d’hémopathie maligne le plus fréquent. Ces pathologies sont traitées par l’association de chimiothérapies conventionnelles et d’immunothérapies dirigées contre le CD20. Bien qu’efficace, 40% des patients résistent ou rechutent après le traitement. Deux raisons peuvent expliquer ces échecs thérapeutiques : 1) l’absence de cibles thérapeutiques impliquées dans plusieurs processus oncogéniques et 2) l’absence de modèles pré-cliniques de LNHB pertinents pour le test de molécules thérapeutiques et la compréhension de la lymphomagenèse. Le calcium est un messager ubiquitaire qui est impliqué dans de nombreux processus cellulaires en condition physiologique et pathologique. La principale voie d’entrée de calcium dans les lymphocytes B est l’entrée capacitive de calcium médiée par Orai1 et STIM1. Ces deux protéines ont été largement décrites pour être impliquées dans les processus tumoraux de nombreux cancers, cependant leurs rôles dans la lymphomagenèse restait à élucider. Nos travaux ont révélé l'implication de la signalisation calcique dans la mort induite par le GA101, un anti CD20 de nouvelle génération actuellement en essai clinique. De plus, nous avons mis en évidence l’implication des protéines Orai1 et STIM1 dans la migration des cellules cancéreuses de LNHB. De manière intéressante, l’implication de ces deux protéines dans la migration cellulaire est calcium indépendante, suggérant donc un nouveau rôle de ces protéines. Enfin, grâce à la technologie des capsules cellulaires nous avons établi un nouveau modèle 3D de lymphome mimant la niche tumorale en incluant des cellules du microenvironnement et de la matrice extracellulaire. Ce modèle semble particulièrement pertinent pour le screening de molécules et la compréhension des mécanismes de la lymphomagenèse. Ce travail de thèse révèle ainsi le ciblage de Orai1 et STIM1 comme potentiellement intéressant dans le traitement du LNHB
B-cell non-Hodgkin lymphomas (BNHL) are the most common hematological malignancies, usually treated with a combination of chemotherapy and anti CD20 immunothérapie. However, 40% of patients are resistant or relapse after treatment. These therapeutic failures could be due to 1) lack of therapeutic targets implicated in several oncogenic processes, 2) lack of relevant preclinical BNHL models for drug screening and lymphomagenesis studies. Calcium is an essential second messenger involved in various cell functions. In B cells, calcium entry is mainly due to Orai1 and STIM1 proteins, both of which have been associated with oncogenesis on solid tumors. However, their role in lymphomagenesis still remains to be elucidated. Our work shows that calcium signaling in BNHL cells participates in cell death induced by GA101, a novel anti-CD20 monoclonal antibody. We also demonstrate that Orai1 and STIM1 play a role in BNHL cell migration. Interestingly, both proteins controlled cell migration in a calcium-independent manner, suggesting a new role for these proteins. Finally, using cellular capsule technology, we established a new BNHL 3D model mimicking tumoral niche by including extracellular matrix and stromal cells. This new model could be used for drug screening and understanding lymphomagenesis. In summary, this work suggests that targeting of Orai1 and STIM1 is promising for BNHL treatment

Alzheimer'™s disease (AD) is the most common form of dementia among elderly population. More than twenty years ago the so-called amyloid hypothesis was formulated based on the major histopathological hallmarks of AD, among which the amyloid plaques are the most known and studied. This hypothesis was prompted by the discovery of three genes that, whereas mutated, are associated with the familial forms of the disease (FAD). One of these genes encodes for the amyloid precursor protein (APP), a single-pass type I transmembrane protein that undergoes sequential cleavages operated by the secretase family of enzymes. The last and key secretase, called gamma-secretase, is composed of four proteins, among which we found either presenilin 1 (PS1) or presenilin 2 (PS2), encoded by other two genes (PSEN1/PSEN2) that are responsible for FAD pathogenesis. Autosomic dominant mutations in either APP, PSEN1 or PSEN2 cause accelerated Abeta deposition due to an increased Abeta42/Abeta40 ratio. While the vast majority of AD cases are sporadic, FAD patients bearing PS2 mutations show a clinical course much similar to that of sporadic patients. By many groups it was found that PSs are capable of perturbing cellular Ca2+ homeostasis, and, particularly, our group demonstrated that PS2, either bearing FAD-linked mutations or wild-type (WT), lowers endoplasmic reticulum (ER) and Golgi apparatus Ca2+ content, interacts with SERCA pump, dampening its function, and tethers ER and mitochondria; all of these pleiotropic effects are independent of its gamma-secretase activity. Recently another group identified PS2 as a regulator of the ER Ca2+ content, together with Orai2, a plasma membrane channel implicated in the Store-Operated Ca2+ Entry (SOCE). This latter phenomenon is impaired in AD, and specifically it is down-tuned in mutant PS-bearing cells. Taken together this body of information offered an interesting background to study the interplay between ER Ca2+ levels, SOCE defects and APP processing/Abeta production. Taking advantage of the PS2-based AD mouse models available in our laboratory, namely the homozygous single transgenic (TG) line expressing the FAD-linked mutant PS2-N141I (line PS2.30H) and the homozygous double transgenic (2TG) line expressing PS2-N141I together with the Swedish double mutant APP-K670M/N671L (line B6.152H), we could investigate the expression pattern of Orai2 in the nervous tissue. Western blot analyses on cortices and hippocampi revealed that Orai2 was overexpressed in cortices from TG and 2TG mice, when compared to C57BL/6 (WT) mice. This overexpression was mainly due to the neuronal contribution since it was even higher in cortical neuronal cultures and in situ Orai2 was found only in neurons, as assayed by immunohistochemical analysis of brain slices. Orai2 up-regulation, that is the condition found in TG and 2TG neurons, is capable of perturbing cellular Ca2+ homeostasis. Particularly, when overexpressed it caused a significant decrease in IP3-induced ER Ca2+ release in both H4-APPswe and HEK29T cells; these results are consistent with a decreased ER Ca2+ level, as measured with the ER-targeted probe G-CEPIA1er. In addition to this, Orai2 revealed to be a less efficient mediator of SOCE than Orai1, since it dampened SOCE when overexpressed alone and it produced a much smaller SOCE when overexpressed with STIM1 as compared with Orai1 plus STIM1 overexpression. Conversely to our expectations, Orai2 downregulation had a noticeable effect neither on IP3-induced ER Ca2+ release nor on total store Ca2+ content; it however improved Ca2+ entry upon store depletion. As far as subcellular localization goes, Orai2 overexpression did not increase the fraction of protein present in the ER and it appeared that most of the protein was found at the early endosomal level, as revealed by immunofluorescence staining of various subcellular compartments. This holds true moving to cortical neurons, where Orai2 was preferentially found in Rab5-EEA1 positive endosomes in primary cultures from WT mice, with a dramatic accumulation at this level in neurons from 2TG mice, possibly reflecting the increased early-endosome compartment that characterizes the AD phenotype. Orai2 localization is, however, dynamic, meaning that it moves in and out of endosomes when properly stimulating neurons with compounds able to induce neuronal activity or to stimulate SOCE. This behaviour is anyway different among the three genotypes, with TG neurons showing a greater tendency to retrieve Orai2 in endosomes upon cell stimulation, and 2TG neurons being unable to properly tune their endosome pool, possibly because of its higher accumulation level. Whether these changes involve also Orai1 has still to be evaluated and it will give us a better picture of this unknown phenomenon. Finally, evidence is provided that a down-tuning of SOCE is associated with increased levels of secreted Abeta42, as measured by ELISA performed on conditioned media from mutant APP-expressing cells such as CHO-7PA2 and H4-APPswe.
La malattia di Alzheimer (AD) costituisce la forma di demenza più comune nella popolazione anziana. Ormai più di vent'anni fa è stata formulata la cosiddetta ipotesi della cascata amiloide, la quale si basa sulle placche amiloidi, uno dei principali marker di AD, tra i più conosciuti e studiati. La formulazione di quest'™ipotesi fu permessa dalla scoperta di tre gene i quali, allorché mutati, sono associati con la forma familiare di AD (FAD). Uno di questi geni codifica per la proteina precursore dell'™amiloide (APP), la quale è una proteina di tipo I a singolo dominio transmembrana che viene tagliata in maniera sequenziale dagli enzimi della famiglia delle secretasi. L'™ultima secretasi a tagliare APP, nonché la più importante nell'™AD, è chiamata gamma-secretasi ed è composta da quattro proteine, che comprendono o la presenilina 1 (PS1) o la presenilina 2 (PS2), codificate dagli altri due geni (PSEN1/PSEN2) responsabili della patogenesi di FAD. Mutazioni autosomiche dominanti in APP, PSEN1 o PSEN2 causano una deposizione più veloce di Abeta dovuta ad un aumentato rapporto Abeta42/Abeta40. La maggior parte dei casi di AD, tuttavia, è sporadica; i pazienti FAD con mutazioni in PS2 mostrano un decorso clinico della malattia molto più simile a quello dei pazienti sporadici. Molti gruppi di ricerca hanno dimostrato la capacità delle PSs di perturbare l'™omeostasi cellulare del Ca2+, e in particolare il nostro gruppo ha dimostrato che PS2, sia nella forma mutata associata a FAD che nella forma wild-type (WT), abbassa il contenuto di Ca2+ del reticolo endoplasmatico (ER) e dell'apparato di Golgi. Inoltre essa interagisce con la pompa SERCA, diminuendone il funzionamento, e modula la vicinanza fra ER e mitocondri; tutte queste funzioni pleiotropiche sono indipendenti dalla sua attività gamma-secretasica. Recentemente un altro gruppo ha identificato PS2 come un regolatore del contenuto di Ca2+ del ER, insieme ad Orai2, il quale è un canale della membrana plasmatica implicato nell'™entrata di Ca2+ dipendente dallo svuotamento del ER (SOCE). Quest'™ultimo fenomeno è alterato nell'AD, e in particolare è ridotto nelle cellule che esprimono PS mutate. Questa serie di informazioni rappresenta una base interessante per lo studio dell'™interazione fra contenuto di Ca2+ del ER, diminuzione di SOCE e metabolismo di APP/produzione di Abeta. Grazie ai modelli murini di AD basati su PS2 presenti in laboratorio, specificatamente il modello singolo transgenico (TG), esprimente il mutante FAD PS2-N141I (linea PS2.30H) e il modello doppio transgenico (2TG), esprimente PS2-N141I insieme al mutante Swedish APP-K670M/N671L (linea B6.152H), abbiamo potuto investigare il pattern di espressione di Orai2 nel tessuto nervoso. Nei Western blot di cortecce ed ippocampi abbiamo notato come Orai2 sia sovra-espressa nella corteccia dei topi TG e 2TG se confrontati con topi di controllo C57BL/6 (WT). Quest'aumento di espressione è dovuto principalmente ad un contributo neuronale, sia in quanto è maggiore in colture neuronali pure, sia perché Orai2 è presente solamente nei neuroni in situ, come rivelato dall'analisi immunoistochimica di fettine di cervello. L'™aumentata espressione di Orai2, così come si ritrova nei neuroni TG e 2TG, è in grado di alterare l'™omeostasi cellulare del Ca2+. In particolare, quando sovra-espressa, Orai2 causa una riduzione significativa del rilascio di Ca2+ indotto da IP3, sia in cellule H4-APPswe che HEK293T; questi risultati sono in accordo con una riduzione del contenuto di Ca2+ del ER, condizione osservata utilizzando la sonda G-CEPIA1er, direzionata al ER. Inoltre, Orai2 si è rivelato essere un mediatore di SOCE meno efficiente di Orai1, infatti diminuisce SOCE quando espresso da solo e, se co-espresso con STIM1, dà vita ad un SOCE meno ampio di quello prodotto da Orai1 co-espresso con STIM1. Contrariamente a quanto atteso la riduzione di Orai2 non produce alcuna diminuzione né del rilascio di Ca2+ indotto da IP3, né del contenuto totale di Ca2+ dei depositi intracellulari; essa tuttavia produce un lieve aumento dell'™ingresso di Ca2+ causato dalla deplezione dei depositi. Per quanto riguarda la localizzazione subcellulare, la sovra-espressione di Orai2 non aumenta la frazione di questa proteina presente nel ER, la maggior parte di Orai2 è infatti presente a livello degli 'early endosomes', come dimostrato marcando numerosi compartimenti subcellulari in immunofluorescenza. Ciò resta vero anche nei neuroni corticali in coltura. Nei neuroni WT, Orai2 si trova preferenzialmente negli endosomi positivi per Rab5 ed EEA1, a questo livello la localizzazione aumenta nei neuroni 2TG, un accumulo causato probabilmente dall™aumento degli 'œearly endosomes' tipico del fenotipo AD. Nonostante sia presente a livello endosomiale, la localizzazione di Orai2 è dinamica, e cioè cambia fra dentro e fuori dagli endosomi a seconda dello stimolo usato per aumentare l'™attività neuronale o per indurre SOCE. Questo dinamismo appare diverso fra i tre genotipi, dove i neuroni TG mostrano una maggior tendenza ad accumulare Orai2 negli endosomi dopo stimolazione della cellula, mentre i neuroni 2TG risultano incapaci di modulare questo aspetto, probabilmente perché in queste cellule l'™accumulo di endosomi è prossimo alla saturazione. Resta da valutare se questi cambi di localizzazione interessano anche Orai1, un'™informazione che ci permetterà di avere un'™idea più precisa di questo fenomeno sconosciuto. Infine vi è evidenza che la diminuzione di SOCE è associata ad un aumento dei livelli di Abeta42 secreta, così come misurato tramite saggio ELISA sui terreni condizionati provenienti da cellule che esprimono una forma mutata di APP, quali le CHO-7PA2 e le H4-APPswe.

Les personnes en surcharge pondérale semblent préférer une alimentation riche en graisse. Face à l'épidémie d'obésité qui touche nos Sociétés tant urbaines que rurales, élucider les mécanismes de la détection des lipides alimentaires devient un enjeu majeur. Il avait précédemment été admis que la glycoprotéine CD36 exprimée dans les papilles caliciformes de souris, est impliquée dans la perception oro-gustative des lipides alimentaires. Dans ce travail, nous avons montré que l'acide linoléique (LA), en activant les phospholipases A2, sPLA2, cPLA2 et iPLA2 via CD36, produit de l'acide arachidonique (AA) et la lyso-phosphatidylcholine (lyso-PC). LA déclenche un influx calcique dans les cellules CD36-positives et induit la production du facteur CIF (Calcium Influx Factor). CIF, AA et lyso-PC exercent différentes actions sur l'ouverture des canaux SOC (Stored Operated Calcium Channel) constitués de protéines Orai et contrôlés par STIM1. Stim1 est un senseur calcique situé sur la membrane du réticulum endoplasmique activé par la déplétion du calcium intracellulaire. Nous avons utilisé la technologie siRNA et des modèles de souris transgéniques pour montrer que CIF et lyso-PC activent des canaux calciques homodimériques composés de protéines Orai1 tandis qu’AA active des canaux hétéro-pentamériques composés d’Orai1 et Orai3. Nous avons également montré que STIM1 régule la production de CIF dans les cellules stimulées par la thapsigargine et l’acide linoléique ainsi que l'ouverture de deux types de canaux calciques. Par ailleurs les souris au phénotype Stim1-/- perdent la préférence spontanée pour les lipides observé chez les animaux de type sauvage. D’un autre côté les cellules CD36-positive de souris Stim1-/- sont incapables de libérer la sérotonine dans l'environnement extracellulaire. Nos résultats suggèrent que des acides gras à longue chaine (AGLC) induisent la signalisation calcique régie par STIM1 via CD36. La perception oro-gustative des lipides alimentaires détermine la préférence spontanée pour les lipides observée chez les mammifères
The lipid-binding glycoprotein CD36, expressed by circumvallate papillae (CVP) of the mouse tongue, has been shown to be implicated in oro-gustatory perception of dietary lipids. We demonstrate that linoleic acid (LA) by activating sPLA2, cPLA2 and iPLA2 via CD36, produced arachidonic acid (AA) and lyso-phosphatidylcholine (Lyso-PC) which triggered Ca2+ influx in CD36-positive taste bud cells (TBC), purified from mouse CVP. LA induced the production of Ca2+ influx factor (CIF). CIF, AA and Lyso-PC exerted different actions on the opening of store-operated Ca2+ (SOC) channels, constituted of Orai proteins and regulated by STIM1, a sensor of Ca2+ depletion in the endoplasmic reticulum. We used siRNA technology and transgenic mice models and observed that CIF and Lyso-PC opened Orai1 channels whereas AA-opened Ca2+ channels were composed of Orai1/Orai3. STIM1 was found to regulate LA-induced CIF production and opening of both kinds of Ca2+ channels. Furthermore, Stim1–/– mice lost the spontaneous preference for fat, observed in wild-type animals. The CD36-positive TBC from Stim1–/– mice also failed to release serotonin into extracellular environment. Our results suggest that fatty acid-induced Ca2+ signaling, regulated by STIM1 via CD36, might be implicated in oro-gustatory perception of dietary lipids and the spontaneous preference for fat

L'altération de glycosylation est l'une des caractéristiques du cancer du sein. Ainsi le défaut de glycosylation affecte différentes protéines glycosylées responsables des différents processus cancéreux. Les canaux SOC (Store operated channels) constituent la voie majeure de l'entrée du calcium dans les cellules et sont impliqués dans la prolifération, la migration et la survie des cellules cancéreuses du sein. CO-OCS est un nouvel inhibiteur de la glycosylation avec plus de sélectivité vis-à-vis des α-glucosidases, et montre des activités anticancéreuses des cellules cancéreuses du sein, sans affecter les cellules mammaires normales. L'objectif de ma thèse est d'étudier les mécanismes moléculaires par lesquels CO-OCS induit ses effets anti-tumoraux. CO-OCS inhibe la migration des cellules cancéreuses à fort potentiel métastatique. Cet effet anti-migratoire est dû à une réduction de l'expression de la β1-intégrine, de Stim1, et de l'activation des voies de signalisation FAK et ERK1/2 par CO-OCS. Dans les cellules cancéreuses peu invasives, CO-OCS diminue la prolifération et augmente la mortalité de ces cellules en affectant l'expression de 3 protéines : Stim1 et Orai1 : protéines N-glycosylées au niveau du réticulum endoplasmique (RE), et GRP78, protéine de stress du RE. Ainsi en supprimant complétement l'expression de Stim1, CO-OCS réduit la prolifération en accumulant les cellules dans les phases G1 et G2/M du cycle cellulaire. Alors que la réduction de l'expression de GRP78 et d'Orai1 par le CO-OCS augmente respectivement l'apoptose et la nécrose. Par ailleurs, l'invalidation de Stim1 atténue l'effet apoptotique induit par CO-OCS. CO-OCS réduit aussi le contenu calcique du RE. Cette réduction du calcium réticulaire est due à une fuite de calcium par le Translocon. En effet, l'Anisomycine, inhibiteur du Translocon, restore de contenu calcique réticulaire et antagonise l'apoptose induite par le CO-OCS. En conclusion, CO-OCS induit une accumulation de protéines mal-repliées dans le RE induisant ainsi un stress réticulaire. Trois cibles du CO-OCS ont été identifiées : l'expression de Stim1 favorise la prolifération tandis que celle d'Orai1 et de GRP78 protègent respectivement les cellules de l'apoptose et de la nécrose induites par CO-OCS. De plus, en diminuant l'expression de GRP78, CO-OCS induit une fuite du calcium du RE par le Translocon
Alteration in glycosylation pattern is one of the hallmarks of breast cancer. The levels and the abnormal expressions of glycan were found in breast cancer patients. Glycosylation defect can affect different glycosylated proteins which are implicated in cancerogenesis. Changes in intracellular Ca2+ levels can regulate different cellular processes. SOC channels are implicated in breast cancer proliferation, migration and survival. CO-OCS is a new glycosylation inhibitor with more selectivity toward theα- glucosidases exhibited anti-cancer activities in breast cancer cells without affecting the normal mammary cells. The objective of my thesis is investigating the related molecular mechanisms by which CO-OCS induced its anti-tumour effects.CO-OCS impaired breast cancer migration through decrease β1-integrin expression and the activation of FAK and ERK1/2 signalling pathways. CO-OCS also induced anti-migratory effect via Stim1 protein expression down-regulation leading to inhibition of SOCE. Additionally, CO-OCS affected the expression of both Orai1 and Stim1 proteins leading to anti-proliferative effects and cell cycle arrest in G1 and G2/M phase respectively. Moreover, CO-OCS affected the expression of Stim1 at the protein level without affecting its transcript level. GRP78 implicated in CO-OCS apoptotic death. The expression of Stim1 regulated the apoptosis induced by CO-OCS via modulating GRP78 expression. Orai1 down-regulation promoted CO-OCS necrotic effect. CO-OCS induced ER- calcium depletion due to increase in ER calcium leak via the Translocon; Anisomycin (Translocon inhibitor) decreased the apoptosis induced by CO-OCS. In conclusion, these results show that in breast cancer, by targeting Stim1, Orai1 and GRP78, CO-OCS reduced cell proliferation and induced apoptosis and necrosis cell death. Stim1 favours CO-OCS apoptotic effect while Orai1 protected from necrosis induced by CO-OCS. The inhibition of Translocon decreased CO-OCS apoptotic cell death via restoring the ER calcium homeostasis

La myopathie à agrégats tubulaires (TAM) est une maladie génétique qui se caractérise par la présence d’agrégats tubulaires dans les biopsies musculaires de patients. Notre équipe a identifié pour la première fois des mutations dans STIM1 comme étant à l’origine de cette maladie. STIM1 (stromal interaction molecule 1) est le senseur calcique du réticulum sarco/endoplasmique (RE/RS). En effet, en cas de diminution du calcium (Ca2+) dans le RE/RS, STIM1 se déplie, oligomérise et migre à proximité de la membrane plasmique (MP) pour activer le canal calcique ORAI1 et permettre le remplissage des stocks. Ce mécanisme est le «store-operated Ca2+ entry» (SOCE). D’autres équipes ont rapporté une mutation dans STIM1 (p.R304W) conduisant à une TAM associée à d’autres symptômes, ou encore syndrome de Stormorken. Ainsi, ce travail a eu pour but d’étudier et de comparer l’impact des mutations TAM et Stormorken à différents niveaux du SOCE. Nous avons ainsi montré que les mutations TAM et Stormorken conduisent à une augmentation de l’expression de STIM1, à la formation de clusters constitutifs de STIM1 à proximité de la MP, ainsi qu’au recrutement du canal ORAI1 et à l’activation de la voie du NFAT, dépendante du Ca2+
Tubular aggregate myopathy (TAM) is a genetic disorder characterized by tubular aggregates in muscle biopsies of patients. Our team identified for the first time mutations in STIM1 as causative of this disease. STIM1 (stromal interaction molecule 1) is the main calcium (Ca2+) sensor of the endo/sarcoplasmic reticulum (ER/SR). Following Ca2+ depletion of the ER/SR, STIM1 unfolds, oligomerizes and migrates close to the plasma membrane (PM) to activate the Ca2+ channel ORAI1, leading to Ca2+ entry. This mechanism is the «store-operated Ca2+ entry» (SOCE). Several teams report a mutation in STIM1 (p.R304W) leading to TAM associated with other symptoms, described as Stormorken syndrome. Therefore, this work aims to assess and compare the impact of TAM and Stormorken mutations at different stages of the SOCE pathway. We show that TAM and Stormorken mutations lead to an increase expression of the protein, a constitutive STIM1 clustering near the PM, to ORAI1 constitutive recruitment and to the activation of a Ca2+ -dependent pathway: the NFAT pathway

Cholestasis is an important liver pathology. During cholestasis bile acids accumulate in the bile canaliculus affecting hepatocyte viability. The actions of bile acids require changes in the release of Ca2+ from intracellular stores and in Ca2+ entry. The target(s) of the Ca2+ entry pathway affected by bile acids is, however, not known. The overall objective of the work described in this thesis was to elucidate the target(s) and mechanism(s) of bile acids-induced modulation of hepatocytes calcium homeostasis. First, it was shown that a 12 h pre-incubation with cholestatic bile acids (to mimic cholestasis conditions) induced the inhibition of Ca2+ entry through store-operated Ca2+ channels (SOCs), while the addition of choleretic bile acids to the incubation medium caused the reversible activation of Ca2+ entry through SOCs. Moreover, it was shown that incubation of liver cells with choleretic bile acids counteracts the inhibition of Ca2+ entry caused by pre-incubation with cholestatic bile acids. Thus, it was concluded that SOCs are the target of bile acids action in liver cells. Surprisingly, despite the effect of choleretic bile acids in activating SOCs, the Ca2+ dye fura-2 failed to detect choleretic bile acid-induced Ca2+ release from intracellular stores in the absence of extracellular Ca2+. However, under the same conditions, when the sub-plasma membrane Ca2+ levels were measured using FFP-18 Ca2+ dye, choleretic bile acid induced a transient increase in FFP-18 fluorescence. This evidence suggested that choleretic bile acids-induced activation of Ca2+ entry through SOCs, involving the release of Ca2+ from a region of the endoplasmic reticulum (ER) located in the vicinity of the plasma membrane.

La fibrose pancréatique est la principale caractéristique de deux pathologies majeures du pancréas, la pancréatite chronique et le cancer du pancréas. Le développement de ce dense tissu conjonctif est essentiellement dû à l'activation de cellules stellaires pancréatiques (CSP). Les CSPs activées sont caractérisées, entre autres, par un potentiel prolifératif élevé, une sécrétion abondant de diverses cytokines, ainsi que par une expression importante de α-actine musculaire lisse (αSMA). Ces processus cellulaires sont connus pour être, parmi d'autres, contrôlés par le Ca2+ intracellulaire, agissant comme un second messager universel. Plusieurs types de canaux calciques membranaires assurent l’entrée du Ca2+ dans les cellules non-excitable notamment les canaux Orai1 et TRPC1. Si le rôle de ces canaux est largement étudié dans les cellules cancéreuses, le rôle du Ca2+ en général et de ces deux canaux en particulier dans les mécanismes moléculaires et cellulaires des CSP activées reste méconnu. Dans ce travail de thèse, nous avons étudié le rôle des canaux Orai1 et TRPC1 dans la prolifération et la sécrétion de cytokines des CSPs activées. En effet, ces derniers sont deux processus majeures, caractéristiques de leur état d'activation, participant à l'aggravation de la fibrose pancréatique. Nous avons démontré que les canaux Orai1 et TRPC1 sont exprimés et fonctionnels dans les CSP humaines activées. Orai1 est activé par la déplétion en Ca2+ du réticulum endoplasmique alors que TRPC1 agit comme un canal Ca2+ mécano-sensible, car il est activé à la suite d'une stimulation par une pression extracellulaire élevée, permettant une augmentation de l'influx Ca2+ dans les CSPs. Orai1 et TRPC1, favorisent tous les deux, la prolifération des CSPs en régulant la progression du cycle cellulaire en phase G1, et aussi la sécrétion des cytokines, à travers des voies de signalisation différentes. Nous avons rapporté que Orai1 module la prolifération cellulaire ainsi que la sécrétion de TGFβ1 via la voie de signalisation AKT, tandis que TRPC1 régule la prolifération et la sécrétion d'IL-6 par une voie ERK1/2-dependante and une voie SMAD2-dependante. L'un des importants résultats de ce travail est l'identification d'une boucle autocrine positive induite par le TGFβ1 secrété, qui stimule la prolifération médiée par la voie Orai1/AKT-dépendante via l'augmentation de l'expression d'Orai1 et de l'entrée SOCE médiée par Orai1. En outre, nous avons démontré que TRPC1 peut interagir et former un complexe protéique avec l'αSMA et la forme phosphorylée de SMAD2. En effet, TRPC1 est impliqué dans l'activation de la voie SMAD2 par sa propre liaison au facteur SMAD2, participant ainsi à la régulation de l'expression de l'αSMA. L'ensemble de nos résultats ont mis en évidence le rôle essentiel des canaux Orai1 et TRPC1 dans les processus d'activation de CSPs humaines. Ces données suggèrent qu'ils pourraient devenir des potentielles cibles thérapeutiques pour le traitement de la fibrose pancréatique, et par conséquent de la pancréatites chronique et/ou du cancer du pancréas
Excessive pancreatic fibrosis is the central pathological feature of two major pancreatic diseases, chronic pancreatitis and pancreatic cancer. The key effectors driving this dense fibrotic tissue development are the well characterized now activated pancreatic stellate cells (PSCs). Activated PSCs are typified, among others, by elevated proliferative ability and abundant secretion of various cytokines, as well as increased αSMA (α-smooth muscle actin) expression. These cellular processes are known to be triggered by Ca2+, acting as an intracellular second messenger. Therefore, several plasma membrane calcium channels allow the Ca2+ entry in non-excitable cells including store-operated calcium entry (SOCE) and stretch activated channels. However, up until now, the role of Ca2+ and these Ca2+ channels in the molecular and cellular mechanisms of PSC’s activation remains poorly known. In the current thesis work, we aimed to investigate the role of Orai1 and TRPC1 channels in human activated PSC’s proliferation and cytokine secretion, two main PSC's activation features, participating in pancreatic fibrosis exacerbation. We demonstrated that Orai1 and TRPC1 are functionally expressed, and they are mediating the SOCE and stretch Ca2+ influx respectively, in human activated PSCs. Indeed, TRPC1 lead to an increased Ca2+ influx under pressurization. Both, Orai1 and TRPC1 promote PSC's proliferation by regulating the cell cycle progression in G1 phase, and cytokines secretion, through different signalling pathways. We reported that Orai1 modulates the cell proliferation and TGFβ1 secretion through an AKT-dependent pathway, whereas TRPC1 regulates PSC's proliferation and IL-6 secretion via ERK1/2 and SMAD2-dependent pathways. Interestingly, we showed that Orai1-mediated TGFβ1 secretion induces an autocrine positive feedback loop, leading to the stimulation of Orai1/AKT-dependent proliferation via the increase of Orai1 expression and Orai1-mediated SOCE. Another important finding in this work, was the identification of a protein network between TRPC1, αSMA and the phosphorylated active form of SMAD2. We found that TRPC1 is involved in the activation of SMAD2 via its binding to SMAD2, participating to the regulation of αSMA expression. Taken together, our data highlighted the crucial role of Orai1 and TRPC1 channels in the activation of human PSCs, suggesting that they could become potential therapeutic targets for pancreatic fibrosis treatment, and thereby for chronic pancreatitis and pancreatic cancer

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.

Les personnes en surcharge pondérale semblent préférer une alimentation riche en graisse. Face à l'épidémie d'obésité qui touche nos Sociétés tant urbaines que rurales, élucider les mécanismes de la détection des lipides alimentaires devient un enjeu majeur. Il avait précédemment été admis que la glycoprotéine CD36 exprimée dans les papilles caliciformes de souris, est impliquée dans la perception oro-gustative des lipides alimentaires. Dans ce travail, nous avons montré que l'acide linoléique (LA), en activant les phospholipases A2, sPLA2, cPLA2 et iPLA2 via CD36, produit de l'acide arachidonique (AA) et la lyso-phosphatidylcholine (lyso-PC). LA déclenche un influx calcique dans les cellules CD36-positives et induit la production du facteur CIF (Calcium Influx Factor). CIF, AA et lyso-PC exercent différentes actions sur l'ouverture des canaux SOC (Stored Operated Calcium Channel) constitués de protéines Orai et contrôlés par STIM1. Stim1 est un senseur calcique situé sur la membrane du réticulum endoplasmique activé par la déplétion du calcium intracellulaire. Nous avons utilisé la technologie siRNA et des modèles de souris transgéniques pour montrer que CIF et lyso-PC activent des canaux calciques homodimériques composés de protéines Orai1 tandis qu'AA active des canaux hétéro-pentamériques composés d'Orai1 et Orai3. Nous avons également montré que STIM1 régule la production de CIF dans les cellules stimulées par la thapsigargine et l'acide linoléique ainsi que l'ouverture de deux types de canaux calciques. Par ailleurs les souris au phénotype Stim1-/- perdent la préférence spontanée pour les lipides observé chez les animaux de type sauvage. D'un autre côté les cellules CD36-positive de souris Stim1-/- sont incapables de libérer la sérotonine dans l'environnement extracellulaire. Nos résultats suggèrent que des acides gras à longue chaine (AGLC) induisent la signalisation calcique régie par STIM1 via CD36. La perception oro-gustative des lipides alimentaires détermine la préférence spontanée pour les lipides observée chez les mammifères

Dans ce travail, nous démontrons que STIM1, un senseur calcique activé par la déplétion du Ca2+ intracellulaire du réticulum endoplasmique, est indispensable pour la signalisation calcique et la préférence oro-sensorielle du gras. Nous observons que l'acide linoléique (LA), en activant les phospholipases A2 via CD36, produit de l'acide arachidonique (AA) et de la lyso-phosphatidylcholine (lyso-PC). Cette activation déclenche un influx calcique dans les cellules CD36-positives, et induit la production du facteur CIF (Ca2+ Influx Factor). CIF, AA et lyso-PC exercent différentes actions sur l'ouverture des canaux SOC (Stored Operated Calcium Channel) constitués de protéines Orai et contrôlés par STIM1. Par ailleurs, les souris au phénotype Stim1-/- perdent la préférence spontanée pour les lipides et la libération de la sérotonine à partir des cellules gustatives dans le milieu extracellulaire chez les animaux sauvages. Nous demontrons aussi que la signalisation calcique médiée via CD36 est doublement modulée lors de l'obésité. L'augmentation de la [Ca2+]i dans les cellules gustatives observée chez le Psammomys obesus, un modèle d'obésité nutritionelle, est fortement diminuée chez les souris rendues obèses par un regime hyperlipidique. Nous avons constaté également que l'interaction de LA avec le CD36 induit l'activation des MAP Kinases de la voie MEK1/2/ERK1/2/Elk-1 qui est non seulement à l'origine de l'activation des aires cérébrales telles que le NTS, le noyau arqué, l'hippocampe mais aussi indispensable pour la préférence spontanée pour les lipides alimentaires. Nos résultats suggèrent pour la prémière fois, que la voie ERK1/2 des MAPK et la signalisation calcique lipidique controlée par STIM1 sont impliquées dans la perception oro-gustative des lipides

Des cellules souches neurales (CSN) persistent dans le cerveau adulte et produisent des neurones et des cellules gliales tout au long de la vie de l’individu. Les CSN suscitent un intérêt considérable pour la médecine régénératrice mais leur utilisation thérapeutique potentielle nécessite au préalable d’approfondir les connaissances sur leurs mécanismes de régulation. Les glioblastomes, quant à eux, sont les tumeurs cérébrales les plus fréquentes chez l’adulte et les plus mortelles. Au sein de ces tumeurs, les cellules souches de glioblastomes (CSG) seraient issues de la transformation maligne des CSN et seraient responsable de l’initiation, de la propagation et de la résistance aux traitements des tumeurs. Des analyses transcriptomiques ont suggéré un rôle majeur de la signalisation calcique au sein des CSN et des CSG. Représentant une des voies principales d’entrée du calcium dans la cellule, les canaux calciques SOC (Store-Operated Channels) régulent de nombreux processus cellulaires, y compris la progression tumorale. L’objectif des travaux de cette thèse est d’évaluer le rôle des SOC dans les CSN et les CSG.Nous avons établi par des approches in vitro et in vivo, que les CSN de souris adulte expriment des SOC fonctionnels et que leur inhibition pharmacologique diminue la prolifération et l’autorenouvellement des CSN, propriété indispensable au maintien de la population souche. La deuxième partie de nos travaux montre que les CSG issues de cultures primaires de patients expriment des SOC dont l’inhibition altère la prolifération et l’autorenouvellement de ces cellules.Ainsi, les résultats obtenus lors de cette thèse mettent en évidence un rôle essentiel des SOC dans la régulation de l’autorenouvellement des CSN et des CSG. Les CSG étant responsables de la résistance aux traitements dans le glioblastome, ces travaux ouvrent des perspectives thérapeutiques ciblant les canaux calciques pour contrer cette pathologie au pronostic sombre
Neural stem cells (NSC) persist in the brain of adult mammals and fuel the brain with new neurons and glial cells all lifelong. Recruited by brain injuries, NSC are considered with great interest by regenerative medicine. However, the development of new therapeutic approaches based on the use of NSC requires an in-depth knowledge of the mechanism regulating these cells. Glioblastomas are the most frequent and deadliest form of adult brain tumors. Within the tumor, glioblastoma stem cells (GSC) form a subpopulation of cells that is considered as responsible of tumor initiation, propagation and relapse, as these cells are particularly resistant to anti-tumoral treatments. GSC and NSC share key characteristics and numerous studies suggest that GSC arise from transformed NSC. Transcriptomic analysis of NSC and of GSC revealed an enrichment of calcium signaling transcripts in these two cell populations. Representing a major way of calcium influx into cells, Store-Operated Channels (SOC) are mobilized in response to a wide range of extracellular factors. SOC regulate many cellular processes and are often hijacked in cancer to promote tumor progression.The aim of this thesis is to evaluate potential SOC involvement in NSC and GSC regulation.The first part of this work, relying on in vitro and in vivo studies, demonstrates that NSC from adult mice express functional SOC whose inhibition by pharmacological agents reduces NSC proliferation and self-renewal. In the second part of this thesis, we demonstrate that GSC from primary cultures derived from patients express SOC, as do NSC, and that SOC inhibition reduces GSC ability to proliferate and self-renew.Accordingly, the results of this thesis demonstrate that SOC regulate NSC and GSC self-renewal, a property that is essential to maintain stem cells pool. As GSC are responsible for glioblastomas treatment resistance, our studies point to a potential new therapeutic way, via calcium channels, against this deadly pathology

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
Durante longos anos, os antagonistas da vitamina K e as heparinas foram os únicos anticoagulantes disponíveis. Apesar de eficazes na prevenção/tratamento das doenças tromboembólicas, apresentam numerosas limitações. No sentido de ultrapassar estas limitações, têm vindo a ser desenvolvidos novos fármacos, que ao contrário dos anteriores atuam num único fator da coagulação específico. Após vários estudos de eficácia e segurança, o dabigatrano etexilato (inibidor direto da trombina), o rivaroxabano e o apixabano (inibidores diretos do fator Xa) foram aprovados para prevenção de acidente vascular cerebral (AVC) e do tromboembolismo venoso em pacientes submetidos a artroplastia eletiva da anca ou joelho, para reduzir o risco de AVC e embolismo sistémico em pacientes com fibrilhação auricular não-valvular e também como tratamento em pacientes com trombembolismo venoso agudo. Estes novos anticoagulantes orais além de serem farmacologicamente previsíveis, não sofrem interações significativas com alimentos, nem com outros fármacos, não necessitam de monitorização laboratorial regular e são de administração oral. Os resultados dos estudos demonstraram que são pelo menos tão eficazes como a varfarina mas mais seguros, uma vez que apresentam um risco de hemorragias major inferior. No entanto, muito ainda está por explorar, sendo necessário prosseguir com as investigações nesta área, conhecendo melhor os efeitos a longo prazo e garantindo uma melhor eficácia e segurança para os pacientes.
For many years, vitamin K antagonists and heparins were the only available anticoagulants. Although effective in the prevention/treatment of thromboembolic diseases, they have numerous limitations. In order to overcome these drawbacks, new drugs that act on a single specific coagulation factor have been developed. After several studies on efficacy and safety, dabigatran etexilate (direct thrombin inhibitor), rivaroxaban and apixaban (factor Xa inhibitors) have been approved for prevention of stroke and venous thromboembolism in patients undergoing elective arthroplasty of hip or knee, to reduce the risk of stroke and systemic embolism in patients with non-valvular atrial fibrillation and in the treatment of patients with acute venous thromboembolism. These new oral anticoagulants are pharmacologically predictable, do not suffer from interaction with other drugs or with food, do not require regular laboratory monitoring and are orally active. The results of the studies showed that are at least as effective as warfarin but safer, since the risk of major bleeding is shorter. However, much remains to be explored, it is necessary to proceed with the investigations in this area, ensuring better efficacy and safety for patients.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Introduction: oral breathing (RO) can influence tongue movements patterns and this can cause persistent alterations in speech, chewing and swallowing throughout life. Studies show that the use of acoustic analysis allows us to infer tongue position during the production of speech. This can be helpful in the processes of diagnosis and speech therapy. Objectives: to present acoustic data of the formant patterns (frequency and intensity of the first three formants F1, F2 and F3, and the bandwidth of the first formant F1) of Brazilian Portuguese (PB) oral vowels as produced by individuals diagnosed with mouth breathing (RO) and individuals presenting no respiratory alterations (RN). Methodology: the corpus consisted of words, inserted in carrier sentences, containing all of the seven oral vowels in stressed position and semi-spontaneous speech. It was recorded by two groups of subjects (RO and RN). These two groups were further divided into two age groups (7-10 years and 10 years and one month to 12 years). Results: statistical differences between RO s and RN s vowel formants (frequency and intensity) have been found. The comparison between the vowel formant structures revealed that the F1 frequencies of the open vowels produced by the RO group were lower than those produced by the RN group, but the F2 frequencies were found to be lower in anterior vowels. These data can be interpreted as related to diminished tongue movement and jaw openness. The comparison among age groups (7 years -10 years and 10 years and 1 month 12 years) within RO and RN groups showed that all formant frequencies (F1, F2 and F3) of the vowels produced by the 7 -10 age group were higher than the older group. This finding can be interpreted as correlated to laryngeal and craniofacial growth. The intensity values of the vowels produced by the RO group were found to be higher than the ones produced by the RN group. This finding can to be correlated with the higher degrees of laryngeal tension (hyperfunction) which characterized RO s voice qualities and characteristics of oral cavity size due to high and narrowed palate area. Conclusion: comparison between the formant structures of the vowels produced by the RO and the RN groups were found to differ in terms of formant frequency (F1, F2 and F3) and intensity (I1, I2 e I3)
Introdução: a ocorrência de respiração oral (RO) interfere negativamente no posicionamento da língua, podendo causar alterações persistentes de fala, mastigação e deglutição por várias etapas da vida. Estudos mostram que o instrumental acústico propicia a condição de inferir o posicionamento do dorso da língua durante a produção de fala, o que poderia auxiliar os processos diagnóstico e terapêutico da RO. Objetivo: apresentar dados acústicos comparativos do padrão de formantes (frequência, intensidade dos três primeiros formantes F1, F2 e F3 e banda do primeiro formante) das vogais orais do português brasileiro (PB) de amostras de fala de indivíduos com diagnóstico de respiração oral (RO), em comparação a indivíduos sem alterações respiratórias (RN). Metodologia: 8 falantes com respiração oral e 8 sem alterações respiratórias com idades entre 07 a 10 anos e 10 anos e 01 mês a 12 anos foram submetidos à sessão de gravação de amostras de fala contendo trechos de fala semi-espontânea e sentenças-veículo com as sete vogais orais do PB inseridas. As gravações foram analisadas por meio do software de livre acesso Praat, para extração de medidas acústicas referentes à freqüência, intensidade e banda de formantes. As medidas acústicas foram submetidas à análise estatística por meio do teste T. Resultados: A caracterização do padrão formântico das vogais orais do grupo RO em relação ao RN revelou diferenças estatísticas em termos dos parâmetros de frequência e intensidade. No caso da frequência, F1 revelou-se rebaixado nas vogais abertas e semi abertas do grupo RO, enquanto F2 revelou-se rebaixado nas vogais anteriores do grupo RO. Tais dados sugerem diminuição da movimentação de língua (no eixo da altura e do deslocamento ântero-posterior) e da abertura da mandíbula. Quando foram comparadas as subfaixas etárias (07 a 10 anos e 10 anos e 01 mês a 12 anos) para cada um dos grupos (RO e RN) detectou-se aumento de todas as freqüências formânticas (F1, F2 e F3) na primeira subfaixa etária, revelando que houve ampliação da extensão total do trato vocal na etapa seguinte, provavelmente pelo processo de crescimento craniofacial e laríngeo. As medidas de intensidades formânticas (I1, I2 e I3) apresentaram-se aumentadas no grupo RO em relação ao RN. Tal achado foi relacionado a efeitos de ajustes de tensão laríngea (hiperfunção) e de modificações na dimensão da cavidade oral consequentes ao quadro de respiração oral, tais como palato duro alto e estreitado. Conclusão: os falantes respiradores orais apresentaram alterações na estrutura formântica das vogais orais do PB, em comparação aos dados de falantes sem alteração da respiração, com destaque para as medidas de freqüência (F1, F2 e F3) e de intensidade (I1, I2 e I3)

A pílula é uma forma eficaz no controlo da fertilidade e desde o início da sua comercialização foi sujeita a vários estudos para melhorar as suas características e aplicações. Porém, existem ainda questões por esclarecer no que se refere ao seu efeito sobre o cancro da mama, no humor, libido e a garantia da sua eficácia em mulheres obesas. As pílulas ou contraceptivos orais (CO) são, também, muito associadas a mitos cuja evidência científica provou não serem verdade: efeitos na fertilidade, no aumento de peso, necessidade de realização de pausas periódicas para “descanso do corpo”, entre outros. A manifestação dos seus efeitos secundários (reais ou associados a mitos) e outras desvantagens ao nível da administração (toma diária, de preferência sensivelmente à mesma hora do dia) leva a que os CO estejam associados a uma taxa de descontinuação elevada ou compliance inadequada e, consequentemente, ao comprometimento da sua eficácia. Dado que os conhecimentos e atitudes em relação aos CO podem nem sempre reflectir as tendências no seu uso, apesar de serem um bom indicador das percepções do público sobre este método, foram aplicados 205 questionários a uma amostra aleatória de utilizadoras de pílulas adquiridas em farmácias de uma região da Beira Interior, para estudar as noções e a dinâmica associadas à sua utilização: tipo de CO usado, troca e descontinuação do seu uso e contribuição do médico/farmacêutico para esta dinâmica. Após utilização de SPSS, verificou-se que, de uma maneira geral, existem noções correctas sobre os CO, excepto a noção relativa à alteração do desejo sexual. Verificou-se desconhecimento nalguns aspectos como a infertilidade, o risco de cancro da mama e o facto de as adolescentes fumadoras poderem usar CO. Um maior nível de escolaridade ou o uso há mais tempo de CO não garante que exista um maior conhecimento sobre CO. As mulheres entrevistadas usavam essencialmente CO de 3ª ou 4ª geração, apresentando uma elevada taxa de descontinuação devido a períodos de tempo sem necessidade de contracepção (principalmente gravidez), enquanto as trocas ocorridas se deveram principalmente a efeitos secundários. Foram verificadas outras situações de contra-indicação para uso de CO, particularmente, a sua utilização por mulheres obesas e fumadoras com mais de 35 anos. As fontes de informação principais a que recorrem as utilizadoras de CO são o próprio folheto informativo e o médico. Para além da necessidade em desmistificar outras noções sobre os CO, é essencial investigar com maior profundidade a dinâmica de utilização de CO e detectar quais os principais aspectos associados aos erros de compliance durante o uso de CO.
The pill is an effective way to control fertility and several studies have been conducted since its marketing in order to improve their characteristics and applications. However, there are still issues to clarify with regard to its effect on breast cancer, mood, libido, and about its efficacy in obese women. Pills or oral contraceptives (OC) are also associated with myths that have been proven not to be true by scientific evidence, such as effects on fertility, weight gain, the necessity of performing periodic breaks for "rest of the body", among others. Manifestation of side effects (real or associated with myths), and some disadvantages related to the administration (daily dose at the same time) relate OC to a high rate of discontinuation or poor compliance, consequently, compromise their effectiveness. Knowledge and attitudes towards OC does not always reflect trends in their use. However, due to the fact that they are a good indicator of public perceptions about this method, 205 questionnaires were applied to a random sample of pill users acquired at pharmacies of a region of Beira Interior, to study the concepts and dynamics associated with their use: type of OC use, exchange and discontinuation of its use and contribution of the doctor/pharmacist to this dynamic. By using the SPSS statistical software, it was found that, in general, there are correct notions about the OC, except for the modification of sexual desire. Results revealed lack of knowledge about some aspects, such as infertility due the pill use, risk of breast cancer and the fact that adolescent smokers may use OC. A higher educational level or use of pill in a longer time period doesn’t means that there is grater knowledge about this contraception method Women who were asked were taking mainly 3rd or 4th generation OC and it was found a high rate of discontinuation due to periods of time with no need of contraception (especially in pregnancy), while exchanges that occurred were mainly due to side effects. Contraindications for use of OC were observed, particularly its use by obese women and smokers with more than 35 years. The main sources of information to which users of OC seek are leaflet that comes with the pill and the doctor. Apart from need to demystify other notions about OC, it’s essential investigate in greater depth the dynamics of using CO and detect main issues associated with compliance errors when using OC.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
IntroduÃÃo: CÃlulas-tronco cancerÃgenas constituem uma subpopulaÃÃo de cÃlulas neoplÃsicas que apresentam propriedades fenotÃpicas de diferenciaÃÃo, renovaÃÃo celular e proliferaÃÃo semelhantes Ãs cÃlulas-tronco normais, sendo responsÃveis pela manutenÃÃo tumoral. Objetivo: investigar a imunoexpressÃo de CD133, marcador de cÃlulas-tronco cancerÃgenas, em displasias epiteliais orais e em carcinomas epidermoides orais. Material e MÃtodo: a amostra se constituiu de 15 casos de CEO e 15 casos de DEO, sendo realizada a imuno-histoquÃmica pela tÃcnica da estreptoavidina-biotina, utilizando o anticorpo anti-CD133 (GTX60471, GeneTexÂ, San Antonio, TX, USA), com diluiÃÃo de 1:650 e recuperaÃÃo antigÃnica com citrato PH 6. A anÃlise quantitativa foi realizada por meio da contagem percentual de cÃlulas com imunomarcaÃÃo positiva em cinco campos, no aumento de 400X, utilizando o programa Image J. Os resultados foram obtidos e comparados entre grupos por meio dos testes t de Student e ANOVA multifatorial seguido do pÃs-teste de Bonferroni, tomando como base os nÃveis de significÃncia de 5%. Resultados: a avaliaÃÃo imuno-histoquÃmica evidenciou marcaÃÃo positiva em todos os casos da amostra (100% dos casos). No grupo de DEO, observou-se que 77,6Â16.0 das cÃlulas epiteliais exibiam imunoexpressÃo positiva para CD133 e, no grupo de CEO, verificou-se que 82.6Â7.2 das cÃlulas epiteliais exibiam imunoexpressÃo positiva para CD133; contudo, nÃo houve diferenÃa estatisticamente significativa entre os grupos estudados (p=0.283). Ademais, observou-se que, com relaÃÃo a sexo, localizaÃÃo anatÃmica e grau de displasia, a marcaÃÃo positiva ocorreu da seguinte forma: sexo masculino (DEO: 76.4Â10.9 e CEO: 82.9Â6.3) (p=0.526) e feminino (DEO: 78.0Â17.9 e CEO: 82.1Â8.9) (p=0.588); lÃngua (DEO: 69.6Â23.2 e CEO: 83.5Â9.3) (p=0.217), mucosa jugal (DEO: 84.8Â14.7 e CEO: 79.0Â5.7) (p=0.618) e palato (DEO: 74.5Â6.7 e CEO: 86.8Â10.3); DEO leve (78.0Â18.4), DEO moderada (72.7Â11.4) e DEO severa (80.1Â1.8) (p=0.899). Todavia, nÃo houve diferenÃa estatisticamente significativa entre os grupos estudados. ConclusÃo: sugere-se que a presenÃa dessa subpopulaÃÃo celular pode nÃo ser imprescindÃvel para a determinaÃÃo do fenÃtipo maligno

LUNA, Ealber Carvalho Macedo. Expressão imuno-histoquímica de cd133 em displasias epiteliais orais e carcinomas epidermoides orais. 2015. 61 f. Dissertação (Mestrado em Odontologia) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2015.
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C ancer stem cells are a subpopulation of neoplastic cells, which have properties phenotypic differentiation, cell renewal and proliferation similar to normal stem cells are r esponsible for tumor support. Objective: To investigate the immunoreactivity of CD133, a marker of cancer stem cells in oral epithelial dysplasia and oral squamous cell carcinoma . Methods: 15 cases were selected as CEO and 15 cases of DEO, being held by immunohistochemistry by the streptavidin - biotin technique, usi ng anti - CD133 antibody (GTX60471, GeneTex®, San Antonio, TX, USA) with dilution of 1: 650 and antigen retrieval with citrate pH 6. the quantitative analysis was performed using the percentage of cells with positive immunostaining count in 5 fields at 4 00X magnification using the Imag e program J. the results were obtained and compared between groups through the Student t test and ANOVA followed by Bonferroni multifactorial post - test, based on the levels of significance of 5%. Results : Immunohistochemical eva luation showed positive staining in all cases the sample (100% of cases). In DEO group, it was observed that 77.6 ± 16.0 epithelial cells exhibited positive immunostaining for CD133 and CEO group was found that 82.6 ± 7.2 epithelial cells exhibited positiv e immunostaining for CD133; however, there was no statistically significant difference between groups (p = 0.283). Moreover, it was observed that, with respect to gender, anatomical location and degree of dysplasia, the positive staining was as follows: ma le (DEO: 76.4 ± 10.9 and CEO: 82.9 ± 6.3) (p = 0.526) and female ( DEO: 78.0 ± 17.9 and CEO: 82.1 ± 8.9) (p = 0588); tongue (DEO: 69.6 ± 23.2 and CEO: 83.5 ± 9.3) (p = 0.217), buccal mucosa (DEO: 84.8 ± 14.7 and CEO: 79.0 ± 5.7) (p = 0.618) and palate (DEO : 74.5 ± 6 .7 and CEO : 86.8 ± 10.3); mild DEO (78.0 ± 18.4), moderate DEO (72.7 ± 11.4) and DEO severe (80.1 ± 1.8) (p = 0899). However, there was no statistically significant difference between groups . Conclusion it is suggested that the presence of this cell subpopulation may not be essential for determining the malignant phenotype .
Células-tronco cancerígenas constituem uma subpopulação de células neoplásicas que apresentam propriedades fenotípicas de diferenciação, renovação celular e proliferação semelhantes às células-tronco normais, sendo responsáveis pela manutenção tumoral. Objetivo: investigar a imunoexpressão de CD133, marcador de células-tronco cancerígenas, em displasias epiteliais orais e em carcinomas epidermoides orais. Material e Método: a amostra se constituiu de 15 casos de CEO e 15 casos de DEO, sendo realizada a imuno-histoquímica pela técnica da estreptoavidina-biotina, utilizando o anticorpo anti-CD133 (GTX60471, GeneTex®, San Antonio, TX, USA), com diluição de 1:650 e recuperação antigênica com citrato PH 6. A análise quantitativa foi realizada por meio da contagem percentual de células com imunomarcação positiva em cinco campos, no aumento de 400X, utilizando o programa Image J. Os resultados foram obtidos e comparados entre grupos por meio dos testes t de Student e ANOVA multifatorial seguido do pós-teste de Bonferroni, tomando como base os níveis de significância de 5%. Resultados: a avaliação imuno-histoquímica evidenciou marcação positiva em todos os casos da amostra (100% dos casos). No grupo de DEO, observou-se que 77,6±16.0 das células epiteliais exibiam imunoexpressão positiva para CD133 e, no grupo de CEO, verificou-se que 82.6±7.2 das células epiteliais exibiam imunoexpressão positiva para CD133; contudo, não houve diferença estatisticamente significativa entre os grupos estudados (p=0.283). Ademais, observou-se que, com relação a sexo, localização anatômica e grau de displasia, a marcação positiva ocorreu da seguinte forma: sexo masculino (DEO: 76.4±10.9 e CEO: 82.9±6.3) (p=0.526) e feminino (DEO: 78.0±17.9 e CEO: 82.1±8.9) (p=0.588); língua (DEO: 69.6±23.2 e CEO: 83.5±9.3) (p=0.217), mucosa jugal (DEO: 84.8±14.7 e CEO: 79.0±5.7) (p=0.618) e palato (DEO: 74.5±6.7 e CEO: 86.8±10.3); DEO leve (78.0±18.4), DEO moderada (72.7±11.4) e DEO severa (80.1±1.8) (p=0.899). Todavia, não houve diferença estatisticamente significativa entre os grupos estudados. Conclusão: sugere-se que a presença dessa subpopulação celular pode não ser imprescindível para a determinação do fenótipo maligno

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
El actual trabajo tiene como objetivo el estudio de la representación y la memoria de narrativas verbales acerca del Boto y del Curupira en las comunidades del interior de la ciudad de Santarém, en Pará. Se discuten, en general las líneas, la cuestión de la literatura verbal, las clases de este tipo de literatura, más necesariamente leyendas y mitos, así como algunos conceptos de narrativas, bajo punto de vista de los teóricos del tema. Después, haremos una discusión acercada de los elementos teatrales del narrador en el momento donde cuenta sus historias; también el estudio enfatizará el papel de la memoria como resultado de las experiencias diarias y la influenza del lugar en las prácticas cotidianas de los contadores. Consideraciones, do mismo modo, serán hechos relativas a las personajes tanto en el romance cuanto en el teatro para, y al final, una análisis de las personajes Boto e Curupira presentadas por los contadores en sus narrativas.
O presente trabalho tem como objetivo o estudo da performance e da memória a partir de narrativas orais sobre o Boto e o Curupira contadas em comunidades do interior do município de Santarém, estado do Pará. Discutem-se, em linhas gerais, a questão da literatura oral, os gêneros desse tipo de literatura, mais precisamente lendas e mitos, assim como alguns conceitos de narrativas, sob o ponto de vista de teóricos do assunto. Em seguida, aborda-se a questão dos elementos performáticos de que se vale o contador no momento em que conta seus causos; também destacam-se o papel da memória enquanto resultado do entrelaçamento das experiências cotidianas e a importância do lugar nas práticas cotidianas dos contadores. Apresentam-se considerações sobre a personagem no romance e no teatro para, em seguida, a análise das personagens Boto e Curupira a partir das informações apresentadas pelos contadores em suas narrativas.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
Os doentes transplantados são pacientes que cada vez mais nos surgem no consultório dentário e tal deve-se aos grandes avanços que se tem desenvolvido nesta última década quer nas técnicas cirúrgicas, quer nas terapêuticas medicamentosas usadas para evitar uma eventual rejeição do transplante. E é devido ao aumento da sua afluência, mas também por ser um tema pouco referido e no qual ainda é necessário realizar mais estudos, que neste trabalho se pretende retratar quais as lesões orais associadas aos transplantes realizados mais comummente, podendo eles ser do tipo cardíaco, hepático, renal ou de células hematopoiéticas. Tem ainda como objetivo descrever quais as manifestações clínicas das diferentes lesões, quais os sintomas que o paciente apresenta e como as tratar, mas também quais os melhores métodos para ajudar na prevenção destas mesmas. Contudo existe uma carência de protocolos definidos, por isso esta monografia pretende também sugerir alguns, com base em diferentes e variadas propostas feitas por vários autores ao longo desta última década. Este trabalho resume-se à ideia de que é necessário intervir na saúde oral dos pacientes com transplantes, não só para lhes melhorar o dia-a-dia e diminuir as suas comorbilidades, mas também para prevenir e evitar que se iniciem tais transtornos. Transplant patients are patients who increasingly emerge in the dental office and this is due to the great progress that has been developed over the last decade both in surgical techniques and in drug therapies used to prevent a possible rejection of the transplant. It is due to their increased affluence - and with this being a rarely mentioned issue still needing further studies - that this work is intended to portray what oral lesions associated with transplants performed more commonly, them being from the heart, liver, kidney or hematopoietic cells. It is also my goal to describe the clinical manifestations of different lesions and the symptoms of the patient and and how to treat them, but also what are the best methods to help prevent them. However, there is a lack of defined protocols, so this monograph also aims to suggest some, based on different proposals made by several authors over the last decade. This work comes down to the idea that it is necessary to interfere in the oral health of patients with transplants, not only for their everyday lives and decrease their comorbidities, but also to prevent and avoid the start of such disorders.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
Introdução: As anomalias congénitas abrangem alterações de estrutura, função e metabolismo da criança. Resultantes de alterações físicas e /ou mentais, podendo estar presentes logo no nascimento ou manifestar-se mais tardiamente. Estima-se que 7,6 milhões de crianças ao nascimento sejam portadoras de uma anomalia congénita. Qualquer alteração no desenvolvimento embrionário pode originar anomalias congénitas que podem variar desde pequenas assimetrias até defeitos com maiores comprometimentos estéticos e funcionais. Objetivo: O objetivo do presente trabalho é dar a conhecer a existência de algumas anomalias congénitas com grande envolvimento da cavidade oral provocando más oclusões devido a erupções dentárias alteradas e alterações no crescimento dos ossos da face. Algumas dessas anomalias são: Fenda Lábio-Palatina, Disostose Cleidocraniana, Disostose Craniofacial entre outras. Materiais e Métodos: Pesquisou-se em duas bases de dados a literatura relevante quanto à temática proposta, com limite temporal dos últimos dez anos e com as seguintes palavras-chaves: anomalias congénitas com manifestações orais, fenda Lábiopalatina, Disostose Cleidocraniana, Disostose Crâniofacial, Disostose Mandíbulo- Facial, Torcicolo, Sífilis Congénita, Toxoplasmose Congénita, Paralisia Cerebral, Síndrome Incontinência Pigmentar, Paralisia Facial, Displasia Ectodérmica, Síndrome de Rieger, Síndrome de Pierre-Robin, Síndrome de Hallerman-Streiff. Os artigos foram selecionados segundo o seu rigor científico e interesse para o tema. Conclusões: A etiologia das anomalias congénitas é multifatorial sendo idêntica em quase todas as anomalias. As anomalias craniofaciais têm um número significativo dentro das anomalias congénitas sendo por isso muito diversificadas e complexas. A fenda Lábio-palatina é das anomalias craniofaciais mais frequente e conclui-se que as manifestações orais são idênticas em cada anomalia congénita estudada. Introduction: Congenital anomalies include changes in structure, function and metabolism of the child. Resulting from physical and / or mental abnormalities that can be presented at birth or may manifest themselves later. It is estimated that 7.6 million children are born with a congenital anomaly. Any change in embryonic development can cause birth defects which may vary from small asymmetries to defects with higher aesthetic and functional impairment. Objective: This work intends to shed some light on the existence of some congenital anomalies with great involvement on the oral cavity causing malocclusion due to altered dental eruptions and changes in facial bones' growth. Some of these anomalies are: Cleft Lip and Palate, cleidocranial dysostosis, Craniofacial dysostosis among others. Materials and Methods: Two databases on relevant literature regarding the proposed theme were searched. The search time span was comprised in the last ten years and keywords like congenital anomalies with oral manifestations, cleft lip and palate, cleidocranial dysostosis, craniofacial dysostosis, mandibular-facial dysostosis, Torticollis, Congenital Syphilis, Congenital Toxoplasmosis, Cerebral Palsy, Incontinence Pigmentosa Syndrome, Facial Paralysis, Ectodermal Dysplasia, Rieger Syndrome, Pierre -Robin syndrome, Hallerman-Streiff were used. Articles were selected according to their scientific rigor and relevance to the theme. Conclusions: The etiology of congenital abnormalities is multifactorial, being identical in almost all anomalies. Craniofacial anomalies are predominant within congenital anomalies, being diverse and complex. The cleft lip and palate are the most common craniofacial anomalies. Thus, it was concluded that oral manifestations are identical in each study congenital anomaly.

Mestrado em Estudos Portugueses
Nesta dissertação aborda-se o tema da ditongação do Português, no que respeita aos seus ditongos orais decrescentes e crescentes. A motivação para este trabalho tem na base um interesse em exercer algo prático no âmbito da fonética experimental; a constatação de lacunas nesta área de estudo e o confronto no que respeita a teorias relativas aos ditongos decrescentes, sendo estes, normalmente, como os verdadeiros ditongos do Português a ser considerados. Este estudo tem como objectivo a verificação deste tipo de sequências vocálicas enquanto ditongos dignos desta designação. Para tal, procedemos à gravação de um corpus construído especificamente para esse fim e à análise dos seus dados relativamente à sua duração e valores formânticos, nomeadamente de F1 e F2. Podemos concluir, pela análise efectuada, que os ditongos crescentes podem também ser considerados ditongos do Português, embora possuidores de uma natureza diferente dos seus pares decrescentes. ABSTRACT: In this dissertation we approach the subject of diphthongs in Portuguese in what concerns its oral falling and rising diphthongs. The basis motivation to this work is related to a strong interest on the experimental phonetics, to verifiable lacks in this area and also to several theories that should be challenged about the unique assumption on the falling diphthongs. This study has its aim on the verification of these vocalic sequences as diphthongs worthy of that name. For that, we proceed to the recording of a corpus so built, and to the analyses of its data in what concerns its duration and formant values, say F1 and F2. We can conclude, by the made analyses, that the falling diphthongs can also be considered Portuguese diphthongs, although they have a different nature from its raising pairs.

This study aimed to validate an instrument designed to assess the oral motor skills in children at 9 months of age, as well as to assess the influence of oral behaviors and breastfeeding on them. Six judges with oralfacial motricity experience and 125 children belonging to the 4th Regional Health of Rio Grande do Sul State participated in this study. For the content analysis, the instrument was sent to judges in order to verify the agreement about its relevance and pertinence. In a second moment, the instrument was applied in children. A questionnaire answered by parents investigated the presence of oral behaviors in this population. The results showed satisfactory content validity and reliability; however, the factorial validity indicated a need for revision since five of the eleven items not shown to be contributing to assess the oral motor skills in children at this age. Regarding the influence of oral behaviors and breastfeeding, the pacifier negatively affected the abilities of suction while breastfeeding positively affected them. It was also found that although most children did not present ability to use the cup, the behavior of putting objects in mouth has promoted the development of such ability. We concluded that the instrument proposed can be use to evaluate the oral motor skills in children at 9 months of age. The oral motor skills developed well when breastfeeding was present.
Este trabalho teve como objetivo validar um instrumento elaborado para avaliar as habilidade orais de crianças, aos 9 meses de idade, bem com verificar a influência de hábitos orais e do aleitamento materno sobre as mesmas. O estudo contou com a participação de 6 juízes, com formação e experiência na área da Motricidade Orofacial, e de 125 crianças com cerca de 9 meses de idade, pertencentes à região da 4ª Coordenadoria Regional de Saúde do Rio Grande do Sul. Para a análise de conteúdo, o instrumento foi enviado aos juízes com o objetivo de verificar a concordância sobre a pertinência e validade dos itens propostos. Em um segundo momento, o instrumento foi aplicado às crianças participantes. Através de questionário aplicado aos pais foi investigada a presença de hábitos orais na população estudada. O instrumento elaborado apresentou validade de conteúdo e de fidedignidade satisfatórias, no entanto, a análise fatorial indicou a necessidade de reformulação do instrumento, uma vez que cinco dos onze itens avaliados não acrescentaram contribuição para avaliar as habilidades orais de crianças aos 9 meses de idade. Em relação à influência de hábitos orais e do aleitamento materno, verificou-se que o uso da chupeta influenciou negativamente as habilidades de sucção. Já o aleitamento materno favoreceu o desenvolvimento das habilidades orais na sucção, no grupo estudado. Verificou-se também que, embora a maioria das crianças não apresentasse habilidade para usar o copo, o hábito de introduzir objetos na boca favoreceu o desenvolvimento de tal habilidade. Conclui-se que o instrumento elaborado pode ser utilizado na avaliação das habilidades orais de crianças, aos 9 meses de idade. As habilidades orais desenvolveram-se de forma mais adequada na presença do aleitamento materno.

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Os carcinomas de células escamosas orais (CCEOs) são tumores malignos da cavidade oral que abrangem aproximadamente 50% de todos os cânceres de cabeça e pescoço, e são a sexta causa de morte por câncer em todo o mundo. Acredita-se que 16-62% destes carcinomas se desenvolvam de lesões potencialmente malignas (OPMLs) conhecidas como leucoplasias orais. Embora se saiba que os CCEOs possam se desenvolver a partir de leucoplasias, a avaliação clínica e histológica existente possue um valor prognóstico limitado para predizer quais dessas OPMLs progredirão a carcinoma. Neste contexto, a busca por marcadores genéticos associados ao prognóstico de leucoplasias é importante, uma vez que estes seriam utilizados como ferramentas mais robustas para a predição da transformação maligna das leucoplasias orais. Apesar de alguns estudos mostrarem alterações genéticas envolvidas na progressão do câncer oral, genes capazes de predizer a transformação maligna de leucoplasias à carcinoma permanecem desconhecidos. A aplicação de análises genômicas em ampla escala, como as utilizadas este estudo, possui potencial para superar este problema, uma vez que permite a identificação de genes/vias específicas envolvidos nesta doença. Além disso, a inclusão de amostras sequênciais de leucoplasias que progrediram e CCEOs de um mesmo paciente, permite uma comparação mais acurada de modificações genéticas que ocorrem nestas lesões durante a progressão oral. Para tal, aplicamos a análise de expressão de microRNAs e a análise do número de cópias do DNA para a identificação de alterações genéticas associadas com a progressão do câncer oral. Este estudo foi o primiero a identificar uma assinatura de miRNAs (miR-146a, miR- 181b, miR-184, miR-21, miR-345, miR-518b, miR-520g, miR-649, miR-196a e miR-206) foi identificada como associada com a progressão ao câncer oral...
Not Available

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
Os distúrbios alimentares são um problema de saúde a nível mundial. Com a sua prevalência a aumentar, torna-se cada vez mais importante a sua integração nas várias áreas de saúde, incluindo a Medicina Dentária. Alguns fatores sociais como a pressão sobre manter o corpo em forma e obter um estilo de vida saudável podem contribuir para a ocorrência destas desordens. O género feminino é o mais afetado, no entanto o número de casos do género masculino tem vindo a aumentar nos últimos anos. Com a ocorrência de distúrbios alimentares, todo corpo humano é afetado sofrendo alterações a vários níveis e áreas. A cavidade oral é uma dessas áreas. O âmbito desta dissertação é explorar de que forma a saúde oral pode ser prejudicada, observando como distintas patologias que nela ocorrem se relacionam com os distintos distúrbios alimentares. O objetivo desta revisão bibliográfica é apresentar conceitos, características e sintomas dos distúrbios alimentares e associar as suas manifestações na cavidade oral. Patologias orais como erosão, cárie dentária, xerostomia, bruxismo, hipertrofia das glândulas salivares e lesões nos tecidos moles, destacam-se em pacientes que sofram de perturbações alimentares. Todas elas serão abordadas nesta revisão bibliográfica. Para a elaboração desta tese, foi efetuada uma pesquisa nas bases de dados, “PubMed/Medline”, “Science Direct” e Chohrane library, utilizando como palavras-chave: eating disorders, anorexia, obesity, bulimia, tooth erosion, oral health, mouth diseases. vi O papel do médico dentista é indispensável para estes pacientes. Detetar as lesões numa fase inicial, criar estratégias preventivas para o tratamento de novas lesões e controlar a progressão das lesões existentes, fazem parte das várias funções que o dentista tem. Sendo este um problema de saúde tão grave e abrangente é necessário que tenha a devida atenção dos profissionais de saúde. Com uma equipa multidisciplinar de várias áreas da saúde, incluindo a medicina dentária, é possível não só auxiliar este pacientes, bem como trata-los e trazer-lhes melhor qualidade de vida.
Eating disorders are a worldwide health issue. With its prevalence increasing, it becomes more and more important to integrate them in the various areas of health, including dentistry. Some social factors such as pressure on keeping the body in shape and getting a healthy lifestyle can contribute to the occurrence of these disorders. The female gender is most affected, however the number of male gender cases has been increasing in recent years. With the occurrence of eating disorders, all of the human body is affected, suffering alterations in different levels and areas. The oral cavity is one of these areas. The aim of this dissertation is to figure out how oral health can be affected, by watching the various diseases that can happen on it, and how they relate to eating disorders. The purpose of this literature review is to present concepts, characteristics and symptoms of eating disorders and associate them with its manifestations in the oral cavity. Oral pathologies such as erosion, dental cavities, xerostomia, bruxism, enlarged salivary gland and soft tissue injuries, can occur in patients suffering from eating disorders. All of them will be addressed in this literature review. To elaborate this thesis ,was performed a research in databases such as " PubMed / Medline ," " Science Direct " and Chohrane library , using as keywords : eating disorders , anorexia, obesity , bulimia, tooth erosion , oral health , mouth diseases. The role of the dentist is essential for these patients. Detecting lesions at an early stage, create preventive strategies for the treatment of new lesions and monitoring the progression of existing lesions, are part of several functions that the dentist has. This is such a serious and increasing health issue that it is necessary that the health professionals pay attention to it. With a multidisciplinary team of several health care areas, including dentistry, we can not only assist these patients and treat them but also bring them better quality of life.

Resumo: Os carcinomas de células escamosas orais (CCEOs) são tumores malignos da cavidade oral que abrangem aproximadamente 50% de todos os cânceres de cabeça e pescoço, e são a sexta causa de morte por câncer em todo o mundo. Acredita-se que 16-62% destes carcinomas se desenvolvam de lesões potencialmente malignas (OPMLs) conhecidas como leucoplasias orais. Embora se saiba que os CCEOs possam se desenvolver a partir de leucoplasias, a avaliação clínica e histológica existente possue um valor prognóstico limitado para predizer quais dessas OPMLs progredirão a carcinoma. Neste contexto, a busca por marcadores genéticos associados ao prognóstico de leucoplasias é importante, uma vez que estes seriam utilizados como ferramentas mais robustas para a predição da transformação maligna das leucoplasias orais. Apesar de alguns estudos mostrarem alterações genéticas envolvidas na progressão do câncer oral, genes capazes de predizer a transformação maligna de leucoplasias à carcinoma permanecem desconhecidos. A aplicação de análises genômicas em ampla escala, como as utilizadas este estudo, possui potencial para superar este problema, uma vez que permite a identificação de genes/vias específicas envolvidos nesta doença. Além disso, a inclusão de amostras sequênciais de leucoplasias que progrediram e CCEOs de um mesmo paciente, permite uma comparação mais acurada de modificações genéticas que ocorrem nestas lesões durante a progressão oral. Para tal, aplicamos a análise de expressão de microRNAs e a análise do número de cópias do DNA para a identificação de alterações genéticas associadas com a progressão do câncer oral. Este estudo foi o primiero a identificar uma assinatura de miRNAs (miR-146a, miR- 181b, miR-184, miR-21, miR-345, miR-518b, miR-520g, miR-649, miR-196a e miR-206) foi identificada como associada com a progressão ao câncer oral... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Not Available
Orientador: Patricia Pintor dos Reis
Coorientador: Suzanne Kamel-Reid
Banca: Ilce Mara Syllus Cólus
Banca: Cláudia Ap. Rainho
Banca: Wilson Araújo da Silva
Banca: Clarice Sampaio Alho
Doutor

Background: Cardiovascular disease is often characterised by functional and structural changes in the blood vessel wall, usually associated with endothelial dysfunction. Therefore, interest in targeting the dysfunctional endothelial cell has heightened. Ca2+ signalling is crucial to endothelial cell physiology as it drives a number of intracellular signalling pathways. Store-operated Ca2+ entry (SOCE) mediated by Orai1 channels is of particular interest as it provides a major Ca2+ influx pathway in endothelial cells, driving cell migration and proliferation, ultimately contributing to endothelial repair and integrity, angiogenesis and wound healing events. The novel hypothesis is that modulation of Orai1 channels will have important physiological effects on endothelial cell function. Methods and Results: A novel series of Orai1 inhibitors has been identified in this thesis with improved pharmacological and physicochemical properties compared to existing SOCE inhibitors. Optimisation of compounds’ structure-activity relationships (SAR) via Ca2+ measurement assays using human umbilical vein endothelial cells (HUVECs) has revealed important insights into functionally important features of SOCE blockers. In conjunction with in silico modelling, a novel binding site in the Orai1 channel located in a small extracellular pocket has been proposed. The generation and subsequent testing of a quaternary ammonium analogue of the parent compound, JPIII supports the hypothesis. The novel SOCE inhibitors suppressed endothelial cell migration and proliferation without affecting cell viability. Reassuringly, the small molecules were well tolerated in vivo, supporting further development and testing of such blockers in animal models of cardiovascular disease. Here, a novel transgenic murine model with the potential for temporal and conditional disruption of Ca2+ permeation in Orai1 channels has been generated and characterised, offering new possibilities for better understanding of the physiological and pathological roles of Orai1 and the therapeutic potential of targeting this channel. Conclusion: Novel SOCE inhibitors have been characterised in vitro, their mechanism of action interrogated and their properties optimised for the next stages of in vivo testing in an animal model of cardiovascular disease.

Orientadora: Profª. Drª. Luciana Reichert Assunção Zanon
Coorientador: Prof. Dr. Fabian Calixto Fraiz
Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Odontologia. Defesa: Curitiba, 2016
Inclui referências : f. 36-39;41-44
Área de concentração
Resumo: A doença celíaca é considerada uma enteropatia imunomediata induzida pela ingestão de glúten na dieta e está relacionada a padrões genéticos com características epidemiológicas diferentes em populações específicas. Esta pesquisa analisou a ocorrência e características de manifestações orais em indivíduos sul brasileiros com a doença celíaca (DC). Estudo transversal envolvendo 40 pacientes com DC assistidos no ambulatório de gastropediatria do Hospital de Clínicas da Universidade Federal do Paraná, Curitiba, Brasil. Os indivíduos foram classificados de acordo com o tipo de DC em clássica, não clássica e assintomática. O grupo controle (n=40) sem o diagnóstico da doença, pareados por idade. Os participantes ou seus responsáveis responderam a um questionário pré-testado, com questões relacionadas ao histórico médico e odontológico, frequência de escovação e de consumo de alimentos com potencial cariogênico. Clinicamente, avaliou-se em dentes decíduos e permanentes a presença de defeitos de desenvolvimentos de esmalte (DDE) segundo a classificação de AINE e cárie dentária segundo critérios da OMS por um examinador calibrado (kappa?0,826). Presença de ulcerações aftosas recorrentes (UAR) e boca seca foi obtida por relato dos pacientes. Os dados foram analisados por meio de testes não paramétricos (?=0,05). A mediana da idade dos participantes foi 16,50 e as idades variaram entre 5 e 34 anos. Pacientes celíacos apresentaram 2,83 vezes a chance de ocorrência de DDE que o grupo controle (P=0,045). Quanto ao relato de boca seca, indivíduos com DC mostraram 9,15 vezes a chance desta alteração que no grupo controle (P=0,002). Não houve diferença entre os grupos para a ocorrência de UAR e experiência de cárie não tratada. Houve uma tendência de maior presença de DDE entre celíacos com a forma clássica da doença (P=0,054). DDE do tipo específico, ou seja, com o envolvimento dos quatro quadrantes dentários, foi significativamente maior em indivíduos com DC (P=0,048). Do total de 1962 dentes permanentes avaliados, 59 apresentaram DDE, sendo 71,8% dos casos em pacientes celíacos (P=0,001), com predominância nos molares permanentes (P=0,009). Conclui-se que DC aumentou a chance de desenvolvimento de DDE e sensação de boca seca. O exame clínico bucal é uma ferramenta importante no diagnóstico e monitoramento de casos de DC. Palavras-chave: doença celíaca, hipoplasia do esmalte, manifestações bucais
Abstract: Celiac disease (CD) is considered a imunomediata enteropathy induced by ingestion of gluten in the diet and is related to genetic patterns with different epidemiological characteristics in specific populations. This research examined the occurrence and characteristics of oral manifestations in Brazilian southern individuals with CD. Crosssectional study involving 40 patients with CD assisted at the Pediatric Gastroenterology Clinic at Clinical Hospital of the Federal University of Paraná, Curitiba, Brazil. The subjects were classified according to the type of DC classical, non-classical and asymptomatic. The control group (n = 40) without the diagnosis of the disease, age-matched. The participants or their guardians answered a tested questionnaire with questions related to medical and dental history, frequency of toothbrushing and consumption of food with cariogenic potential. Clinically evaluated in primary and permanente teeth the presence of dental enamel defects (DED) according to the classification of AINE and dental caries according to WHO criteria by a calibrated examiner (kappa?0,826). The presence of recurrent aphthous ulcerations (RAU) and dry mouth was obtained from the reporting of the patients. Data were analyzed using nonparametric tests (? = 0.05). The median age of participants was 16.50 and the ages ranged from 5 to 34 years. Patients with CD had 2.83 times the chance of occureence of DED than the control group (P = 0.045). As for the dry mouth report, individuals with AD showed 9.15 times the chance of this alteration in the control group (P = 0.002). There was no difference between groups for the occurrence of RAU and untreated caries experience. There was a trend toward greater presence of DED between celiacs with the classic form of the disease (P = 0.054). DED specific type, in other words, with the involvement of the four dental quadrants was significantly higher in patients with CD (P = 0.048). Of the total of 1962 evaluated permanent teeth, 59 presented DED, and in 71.8% of the cases was in patients with CD (P = 0.001), especially in permanent molars (P = 0.009). It is concluded that CD increased the chance of DED and dry mouth. The oral clinical examination is an important tool in the diagnosis and monitoring of cases of CD. Key-words: celiac disease, dental enamel hypoplasia, oral manifestations

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A presente dissertação é um estudo descritivo sobre os Monaikó, identificando-os no conjunto da família Karíb. Foram registradas narrativas orais com informantes Monaikó cujo intuito foi de reconhecer se eles seriam um grupo ou subgrupo dos Makuxi. Por isso, que do ponto de vista etnográfico, as narrativas orais ajudaram tanto a revelar uma identidade própria como também uma variação linguística entre os falantes que se declaram Monaikó. Nessa perspectiva, com o objetivo de saber se existiria realmente uma língua Monaikó ou dialeto, recorreu-se aos vários conceitos referentes à língua, dialeto e variação linguística para subsidiar a análise dos dados. O corpus linguístico constituiu-se de dados da fonologia, da morfologia e morfossintaxe, além de uma lista de itens lexicais. Em termos metodológicos este trabalho selecionou sete comunidades onde vivem os Monaikó: Araçá da Serra, Placa, Contão, Canta Galo, Igarapé do Galo, Pedra Branca e Enseada, todas localizadas na Terra Indígena Raposa Serra do Sol entre os municípios de Pacaraima, Normandia e Uiramutã Roraima, além de 21 informantes que contribuíram com a pesquisa. Dessa forma, constatou-se a existência do dialeto Monaikó, bem como sua população, ainda que de forma preliminar, pois os dados analisados não foram suficientes para comprovar a existência de uma língua. Ficou evidente também que, apesar de os Monaikó já estarem convivendo há muitos anos espalhados entre os Makuxi, eles trazem consigo o desejo de se autoafirmarem como diferentes desses últimos a fim de fortalecer suas identidades.
This dissertation is a descriptive study on Monaikó, identifying them throughout the Carib family. We recorded oral narratives with Monaikó informants whose purpose was to recognize whether they would be a group or subgroup of Makuxi. For this reason, from the ethnographic point of view, the oral narratives helped so much to reveal their own identity but also a linguistic variation among speakers who declare themselves as Monaikó. From this perspective, aiming to know if there was really a language or dialect Monaikó, we appealed to the various concepts related to language, dialect and language variation to help analyze the data. The language corpus consisted of data from phonology, morphology and morphosyntax, and a list of lexical items. In methodological terms this study selected seven communities where Monaikó live: Araçá da Serra, Placa, Contão, Canta Galo, Igarapé do Galo, Pedra Branca and Enseada, all located in Raposa Serra do Sol Indigenous area between the cities of Pacaraima, Normandia and Uiramutã - Roraima, and 21 informants who contributed to the research. Thus, we confirmed the existence of Monaikó dialect as well as its population, albeit preliminary, because the data analyzed were not enough to prove the existence of a language. It also became evident that, although Monaikó are already living together for many years scattered among Makuxi, they bring with them the desire to assert themselves different from Maluxi in order to strengthen their identities.

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Investiga o uso de descrições nominais anafóricas em narrativas orais, mais precisamente, a relação existente entre o uso dessas formas e as partes que constituem a estrutura da narrativa. Observamos as funções que as descrições nominais anafóricas exercem em decorrência dessa relação, bem como a orientação argumentativa que tais expressões imprimem no discurso do narrador. Para esse empreendimento, seguimos um percurso teórico no qual discutimos conceitos de narrativa oral, definimos estrutura da narrativa e problematizamos referenciação, anáfora e as estratégias de referenciação com núcleos nominais, entre as quais destacamos as descrições nominais. Para os estudos da narrativa oral e sua estrutura, recorremos, de modo particular, aos estudos de Labov (1972). Quanto à referenciação, anáfora e estratégias de referenciação, seguimos os postulados de Marcuschi (2005; 2007), Koch (1996; 2001; 2004; 2005; 2006; 2008), Mondada e Dubois (2003) e Lima (2004). O corpus deste estudo é constituído por dez narrativas orais gravadas em vídeo e, posteriormente, transcritas. Os informantes são vigilantes noturnos que atuam no centro de Castanhal (PA). Fazemos, a partir de nossa análise, uma classificação das funções que as descrições nominais anafóricas exercem nas diferentes partes da narrativa. Propomos, outrossim, uma classificação dessas formas, levando em consideração seus diferentes graus de argumentatividade.
It investigates the use of nominal anaphoric descriptions in oral narratives, more specifically, the relationship between the use of these strategies and the constituents that form the structure of the narrative. We observe the functions nominal anaphoric descriptions exercise according to that relationship as well as the argumentative orientation that such expressions print on the speech of the narrator. To do so, we follow a theoretical path in which we discussed the concepts of oral narrative, we define the narrative structure and problematize the notions of referenciation, anaphora and referenciation strategies with nominal cores, among which we highlight the nominal descriptions. For studies of oral narrative and its structure, we used, in particular, the studies of Labov (1972). As for referenciation, anaphora and referenciation strategies, we follow the postulates of Marcuschi (2005, 2007), Koch (1996, 2001, 2004, 2005, 2006, 2008), Dubois and Mondada (2003) and Lima (2004). The corpus of this study is composed of ten oral narratives videotaped and later transcribed. Informants are night watchmen who work in the center of Castanhal (PA). Accordind to our analysis, we do a classification of the functions that the nominal anaphoric descriptions exercise in different parts of the narrative. We propose, instead, a classification of these forms, considering their different degrees of argumentativity.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
O presente estudo tem como objectivo, através de uma revisão de literatura, despertar e alertar os médicos dentistas para as possíveis complicações do uso contínuo do piercing oral/perioral. O uso do piercing tem sido realizado, por várias civilizações, com objetivos estéticos, culturais, políticos e religiosos. Na sociedade actual, o seu uso está ligado à adolescência, tendo conotações de cariz sexual, provocação social, rebeldia e estética. Os piercings localizados dentro e em torno da cavidade oral, são comuns na língua, lábios, dentes, bochecha e úvula, podendo provocar inúmeras condições patológicas. As complicações decorrentes da utilização do piercing oral/perioral, podem ser imediatas ou tardias, dando uma atenção especial aos possíveis efeitos nocivos e traumáticos tanto nos tecidos duros como nos tecidos moles da cavidade oral. Após a colocação do piercing oral/perioral, deve ser tido em conta um conjunto de medidas e cuidados de modo a reduzir a probabilidade de complicações decorrentes do seu uso. Convém salientar que o piercing oral favorece a acumulação de restos alimentares e dificulta a higiene oral. A contínua utilização tem inúmeras consequências que podem ir desde fracturas dentárias e infecções até mesmo a condições pré-malignas. Usualmente, pode tornar-se necessário o emprego de terapêutica medicamentosa analgésica, anti-inflamatória ou antibiótica no período pós-colocação. Assim sendo, o médico dentista, tem o dever de alertar e esclarecer o paciente sobre o uso e manutenção do piercing oral explicando as suas devidas complicações. It is the purpose of this study to inform dentists, through a review in medical literature, of the possible complications of the continuous use of the oral/perioral piercing. Different civilizations have been using piercings due to aesthetic, cultural, political and religious motivations. Today, its use is mainly associated with the process of growing up in adolescence, carrying with it sexual connotations, the desire to be socially provocative and also attitudes of rebellion and aesthetic beliefs. Piercings inserted inside and around the mouth are common in the tongue, lips, teeth, cheeks and uvula, being, all of them, likely to cause several pathological conditions. Complications deriving from the use of oral/perioral piercings can be immediate or arrive later in life. Special attention should be given to the possible harmful and traumatic effects either in the soft or in the bone tissues of the mouth. A lot of care must be taken after inserting the oral/perioral piercing so as to reduce the possible complications of its use. It is important to emphasize the fact that the oral piercing favours the heaping of food items and makes oral hygiene more difficult. Its continuous use brings with it several consequences, from tooth fractures and infections to even pre-malignant conditions. The use of analgesic, anti-inflammatory or antibiotic therapy may prove to be necessary immediately after the piercings’ insertion; therefore, the dentist must give the patient all the details about the use and preservation of the oral piercing, making clear that some complications may also take place.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
A doença celíaca (DC) é uma enteropatia comum, caraterizada pela permanente intolerância ao glúten. Tem sido descrito por vários autores a importância das manifestações orais provocadas por esta doença para um diagnóstico precoce, sendo as mais comuns, defeitos de esmalte, baixa incidência de cárie e ulcerações aftosas recorrentes. Objectivo: Neste trabalho pretendeu-se determinar e avaliar a prevalência de defeitos de esmalte, cárie dentária e ulcerações aftosas recorrentes em crianças com diagnóstico prévio de doença celíaca. Métodos: Foi realizado um estudo observacional descritivo da prevalência de defeitos de esmalte, usando a classificação de Aine (1986); da avaliação e registo de cárie dentária de acordo com o Índice cpod/CPOD (OMS, 1998) e um inquérito dirigido aos responsáveis das crianças celíacas. Este estudo decorreu de janeiro a junho de 2012 no Centro Clínico Académico do Hospital de Braga, onde foram observadas 29 crianças/adolescentes com idades compreendidas entre os 4 e os 18 anos (±10,25). Resultados: Observou-se que 42,86% dos inquiridos apresentava defeitos de esmalte típicos da DC na dentição permanente e 21,43% na dentição decídua; o índice de CPOD/cpod igual a zero observado na dentição permanente foi de 60,9% e de 44,4% na dentição decídua; e uma incidência de 25% para a presença de ulcerações aftosas recorrentes antes do início de uma dieta isenta de glúten (DIG) e de 57,1% para a presença de ulcerações aftosas recorrentes para depois do início de uma DIG. Conclusão: O esmalte é um importante marcador biológico das alterações ocorridas durante o período da sua formação, pelo que se torna essencial o conhecimento das alterações provocadas pela DC, por parte do profissional de saúde, nomeadamente do Médico Dentista para se poder fazer um diagnóstico precoce. Celiac disease (CD) is a common enteropathy characterized by permanent intolerance to gluten. It has been described by several authors the importance of oral manifestations caused by this disease for early diagnosis, the most common are enamel defects, the low incidence of caries and recurrent aphthous ulcerations. Objective: This study aimed to determine and avaliate the prevalence of enamel defects, dental caries and recurrent aphthous ulceration in children with a previous diagnosis of celiac disease. Methods: It was conducted a descriptive observational study of the prevalence of enamel defects, using the classification of Aine (1986); assessment and registration of dental caries according to the DMFT (WHO, 1998) and a survey among caretakers of children with celiac disease. This study took place from 2012 January to June into the Academic Medical Center Hospital of Braga, where were observed 29 children/adolescents aged 4 to 18 years (± 10.25). Results: It was observed that 42.86% of respondents had typical-CD enamel defects in the permanent dentition and 21.43% in the primary dentition; the index of DMFT observed in the permanent dentition was 60.9% and 44.4% in primary dentition, and a 25% incidence for the presence of recurrent aphthous ulcers before the onset of a gluten-free diet (GID) and 57.1% for the presence of recurrent aphthous ulcers after starting to a GID. Conclusion: Dental enamel is an important biomarker of changes occurring in the period of their formation, and therefore it is essential the knowledge about changes caused by CD, by the healthcare professional, including the dentist, in order to make an early diagnosis.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
Dados da Organização Mundial de Saúde (OMS) de 2012 demonstram que a incidência de cancro a nível mundial tem vindo a aumentar, e referem cerca de 14 milhões de novos casos por ano. O cancro infantil representa 0,5% a 4,6% de todos os cancros, variando a sua taxa de incidência entre 50 a 200 novos casos, por milhão de crianças, em todo o mundo. Nos países ocidentais, os protocolos terapêuticos antineoplásicos atualmente empregues melhoram de forma significativa as taxas de sobrevivência do cancro, agravando contudo a morbidade nos pacientes. Na cavidade oral, a terapia oncológica é responsável por efeitos adversos agudos e também por complicações a longo prazo. Nas crianças estas manifestações orais são ainda mais prevalentes que nos adultos e contribuem para a morbilidade e até mesmo para a mortalidade destes doentes. Pretendeu-se assim, com este trabalho, realizar uma revisão sistemática de literatura científica, publicada nos últimos 15 anos, acerca das complicações orais do tratamento oncológico em crianças. Bem como, compilar diretrizes de atuação clínica para orientar o médico dentista no atendimento destes doentes. Durante os meses de Janeiro a Maio de 2015, procedeu-se a uma ;pesquisa bibliográfica nas bases de dados PubMed e B-on, sendo consultados também outros bancos de dados como LILACS – BIREME, SciELO, utilizando as seguintes palavras chave: cancer, children, leukemia, limphoma, “oral manifestations”, radiotherapy, chemotherapy; separadas ou associadas pelo operador de pesquisa, booleano AND. Na pesquisa foram empregues os seguintes limites: artigos publicados nos últimos 15 anos, abstract disponível, estudos em humanos e artigos e língua inglesa, portuguesa e espanhola. Desta pesquisa resultou um total de 116 artigos que foram selecionados primeiramente pelos títulos, seguidamente pela leitura dos abstracts e, finalmente, do artigo por inteiro, obtendo-se assim 68 artigos, para revisão. Foram ainda considerados artigos de referência publicados em anos anteriores, livros de texto médicos e publicações portuguesas com dados epidemiológicos sobre cancro infantil em Portugal. Os efeitos colaterais agudos e crónicos, da terapia antineoplásica, mais frequentemente observados na cavidade oral são então: mucosite, hemorragia oral, disgeusia, infeções oportunistas, disfunção das glândulas salivares, cárie dentária, neurotoxicidade, osteorradionecrose, disfunção na articulação temporomandibular e anomalias do desenvolvimento dentário e craniofacial. O médico dentista desempenha portanto um papel fundamental na prevenção diagnóstico e tratamento dessas complicações. Logo, estas crianças deverão ser incluídas, antes mesmo de iniciar a oncoterapia, num rigoroso programa de acompanhamento médico-dentário, que deve prolongar-se mesmo depois de terminado o tratamento do cancro. Idealmente, estes programas deverão ter lugar nas próprias instituições de cuidados de saúde oncológicos, para promover a estreita colaboração do médico dentista com os restantes membros da equipa oncológica, e assim garantir cuidados orais e aconselhamento especializado, que contribuirão para melhorar a sua qualidade de vida e para a diminuir a morbilidade e mortalidade. World Health Organization (WHO) data from 2012 show that cancer`s incidence has been globally increasing, and mentions around 14 million new cases a year. Childhood cancer represents from 0.5% to 4.6% of all cancer, with an incidence rate between 50 to 200 new cases per million children worldwide. In western countries, currently used antineoplastic therapeutic protocols have improved significantly cancer surviving rates, yet increasing, patients’ morbidity. Oncological therapy is responsible for acute adverse effects and long-term complications in the oral cavity. In children, these oral manifestations are even more prevalent than in adults and contribute to their morbidity and mortality. Therefore, this works objective was to conduct a systematic review of scientific literature published in the last 15 years, about the oral complications of oncological treatment in children. Furthermore, it was intended to gather guidelines on clinical procedures to guide the dentist in the care of pediatric oncological patients. During the months of January until March 2015, PubMed and B-on databases were sceened for Portuguese, Spanish and English abstact-free articles, published in the last 15 years, using the words: cancer, children, leukemia, lymphoma, ”oral manifestation”, radiotherapy, chemotherapy; separated or associated by the Boolean operator ”AND”. Other databases such as LILACS – BIREME, and SciELO were also used for the research aplling the same limitations and key words. 116 articles were found. Article selection was accomplished firstly by the title and abstract reading and finally, by full article analysis. A total of 68 articles were reviewed. For better understanding of the theme to develop, reference articles from previous years were also reviewed; Medical textbooks and epidemiological data on pediatric cancers in Portugal were also consulted. The acute and chronical side effects of anti-neoplastic therapy, more frequently observed in the oral cavity are: mucositis, oral bleeding, dysgeusia, opportunistic infections, salivary gland dysfunction, dental caries, neurotoxicity, osteoradionecrosis, temporomandibular joint dysfunction and dental and craniofacial growth anormalities. So, it is unquestionable the dentist`s malor role in the diagnosis, prevention and treatment of these complications. Therefore, before starting the oncological therapy, children should enter a strict dental monitoring program, that must be extended even after completing cancer treatment. Ideally, these dental programs should take place in the oncologic healthcare institutions, in order to promote dentist and oncological team close cooperation and ensure professional oral care and expert advice. Implementing these measures will be a strong asset towars improving pediatric oncological patients` quality of life and in decreasing their morbidity and mortality.