Dissertations / Theses on the topic 'IP3Rs'

L'apoptose, ou « mort cellulaire programmée », joue un rôle clé dans de nombreux processus biologiques. Les protéines de la famille Bcl-2, dont l'expression est souvent altérée dans les cellules tumorales, sont les principaux régulateurs de l'apoptose. Parmi cette famille, la fonction exacte du répresseur apoptotique Nrh, aussi appelé BCL2L10 ou Bcl-B, reste à ce jour mal comprise. Bien que son expression ne soit pas détectable dans la plupart des tissus sains, on retrouve des niveaux élevés de Nrh corrélés à un mauvais pronostique dans les cancers du sein et de la prostate. Nous avons mis au jour un nouveau mécanisme selon lequel Nrz, l'orthologue de Nrh chez le poisson zèbre, interagit avec le domaine de liaison du ligand IP3 du canal calcique IP3R1. Il s'est avéré que la régulation négative des flux calciques par Nrz est critique lors du développement embryonnaire du poisson zèbre. Grâce à ces nouvelles données, nous avons cherché à comprendre la fonction de Nrh chez l'Homme, dans un contexte pathologique. Nous avons montré que Nrh interagit via son domaine BH4 avec le domaine de liaison du ligand du récepteur IP3R1 humain pour réguler l'homéostasie calcique et la mort cellulaire. Cette interaction définit Nrh comme la seule protéine de la famille Bcl-2 à réguler négativement la mort cellulaire exclusivement au niveau du réticulum endoplasmique. Pour aller plus loin, nous avons montré que la dissociation du complexe Nrh/IP3Rs sensibilise des cellules tumorales mammaires à l'action d'agents chimiothérapeutiques. Pour finir, nos résultats apportent une explication moléculaire sur la contribution de Nrh dans la résistance aux thérapies anti-tumorales
Apoptosis, also called “Programmed Cell Death”, plays a key role in many biological processes and pathologies. The B-cell lymphoma 2 (Bcl-2) proteins, whose expression is often altered in tumor cells, are the main regulators of apoptosis.Among this family, the actual physiological function of the human apoptosis inhibitor Nrh, also referred to as BCL2L10 or Bcl-B, remains elusive. Although in most healthy tissues the Nrh protein is nearly undetectable, clinical studies have shown that Nrh expression is correlated with poor prognosis in breast and prostate carcinomas. We have shed light on a novel mechanism by which Nrz, the zebrafish ortholog of Nrh, was found to interact with the Ligand Binding Domain (LBD) of the Inositol-1,4,5-triphosphate receptor (IP3R) type-I Ca2+ channel. Indeed, the regulation of IP3Rs-mediated Ca2+ signaling by Nrz was shown to be critical during zebrafish embryogenesis. We used the knowledge gained with the zebrafish model to investigate Nrh function in cancer. We showed that Nrh interacts with the LBD of IP3Rs via its BH4 (Bcl-2 Homology 4) domain, which is critical to regulate intracellular Ca2+ trafficking and cell death. Actually, this interaction seems to be unique among the Bcl-2 family, and sets Nrh as the only Bcl-2 homolog to negatively regulate apoptosis by acting exclusively at the Endoplasmic Reticulum. Furthermore, we showed that disruption of the Nrh/IP3Rs complex primes Nrh-dependent cells to apoptotic cell death and enhances chemotherapy efficiency in breast cancer cell lines.Lastly our results bring a new insight to the role of Nrh regarding chemotherapy resistance

Competition and intellectual property rights (IPRs) are both necessary for a market to work efficiently and to promote consumer welfare. The tension between them is only apparent. Properly applied, intellectual property rules define a legal framework which allows undertakings to profit from their inventions. This in turn encourages competition among firms and enhances dynamic efficiency, to the benefit of consumer welfare. From this perspective, IPRs and competition generate a fruitful symbiosis. Standard setting represents one of the fields where the interaction between competition law and IPRs clearly comes to light. The collaborative goal of standard setting organizations (SSOs) is to adopt and promote standards that either do not conflict with anyone’s right or, if they do, are developed under condition that patents are licensed under defined terms. On the one hand, patents are important to promote innovation, as they confer exclusive rights to the inventors. On the other, standards are paramount for enhancing the interoperability of products, expanding network externalities, and facilitating the dissemination of knowledge. Conflicts between IP and competition laws may arise in case IPRs owners in standardization contexts overexploit the rights they have been granted. This may lead to the hold-up problem, which represents both a private and public concern. How to strike, then, the optimal balance between IPRs and industry standards? By answering the question, this work aims at filling a gap in the academic literature, which does not appear so far to have attempted an in-depth assessment of the right equilibrium between investment incentives and competition goals in standard setting. Any abuse of market power may harm significantly consumer well-being. At the same time, any form of control of market power should preserve the incentives of firms to invest in the market. The crucial aim, hence, is to define the optimal balance in order to avoid risks of significant losses in consumer and societal welfare.

Doctor en Bioquímica
La macroautofagia, comúnmente referida como “autofagia” es la principal vía de degradación de proteínas, organelos y material citoplasmático, permitiendo de este modo el reciclaje del material intracelular. Este proceso consiste en el englobamiento de fracciones citosólicas por una estructura multimembranar llamada “autofagosoma”, el cual posteriormente se fusiona con el lisosoma para formar el “autofagolisosoma”. Luego el material comprendido en el autofagolisosoma es degradado por enzimas hidrolíticas. Un estudio mostró que la inhibición de la enzima inositolmonofosfatasa (IMPasa) usando litio y L690.330, inducía una disminución de los niveles basales del IP3 y en consecuencia la generación de autofagia. Nuestros resultados confirmaron estos datos previos, demostrando que el pre tratamiento con mio-inositol revierte la autofagia inducida por litio y L-690.330. Además se demuestra que el pre tratamiento con mio-inositol también revertía la autofagia inducida por privación de nutrientes. IP3 es ligando de su receptor de IP3 (IP3R), el cual es el principal canal de Ca2+ a nivel del retículo endoplásmico. El principal objetivo de esta tesis es evaluar el rol del IP3R en la regulación de la autofagia. Los resultados mostraron que la disminución de los niveles proteicos del IP3R usando siRNA específicos, así como el tratamiento con antagonistas químicos del IP3R, tales como xestosponginas B y C, estimulaban significativamente el aumento en los niveles de autofagia. Además, xestospongina B, así como también la privación de nutrientes, indujo una pérdida en la interacción entre Bcl-2 y Beclin-1, los cuales interactúan en condiciones basales. El tratamiento con xestospongina B no perturbó los niveles de Ca2+, tanto en retículo endoplásmico como en el citosol, concluyendo que la autofagia inducida por xestospongina B es independiente de una fluctuación del Ca2+. Los experimentos de inmunoprecipitación mostraron que Beclin-1 (regulador clave en la inducción de la autofagia) interactúa tanto con IP3R así como con Bcl-2 en condiciones basales, y la interacción de este complejo es atenuado bajo condiciones de privación de nutrientes o por tratamiento con ABT737, el cual es un mimetizador de dominios BH3. Este resultado sugiere la presencia de un complejo proteico en la regulación de la autofagia. El papel del retículo endoplásmico en el desarrollo de la autofagia toma gran significancia debido al reclutamiento de proteínas clave (IP3R, Beclin-1 and Bcl-2). La relación entre autofagia y estrés de retículo no es clara y por lo tanto se evaluó el efecto de agentes inductores de estrés de retículo en la inducción de la autofagia. Los resultados mostraron que tunicamicina, tapsigargina y brefeldina-A (agentes inductores de estrés de retículo) activaron el UPR (respuesta a proteínas mal plegadas) e indujeron autofagia. La disminución de los niveles de proteínas claves en el desarrollo de la autofagia (Atg5, Atg10, Atg12, Vps34 y Beclin-1) usando específicos RNAs interferentes atenuaron la autofagia inducida por agentes inductores de estrés de retículo y xestospongina B. Además, la sobreexpresión de Bcl-2 y Bcl-XL con destinación a retículo endoplásmico atenuó la autofagia inducida por xestospongina B e inhibidores de la IMPasa. Esta tesis muestra novedosos resultados, los cuales dan cuenta de un complejo proteico IP3R/Beclin-1/Bcl-2 en la regulación de la autofagia.
Macroautophagy (herein referred to as “autophagy”) is the major catabolic pathway for entire organelles, long-lived/ aberrant proteins and superfluous portions of the cytosol. It consists of the stepwise engulfment of substrate elements into distinctive multimembraned “autophagosomes”, which after fusion with lysosomes form singlemembraned autophagolysosomes. Into the autophagolysosome, the engulfed material is degradated by lisosomal hidrolytic enzymes, leading the recyclage of intracellular material. A study has suggested that myo-inositol-1,4,5-trisphosphate (IP3) could regulate autophagy because inhibition of inositol monophosphatase (IMPasa) by lithium or L-690.330 stimulates autophagy through the depletion of IP3. Our results have confirmed that the reduction of intracellular IP3 levels by IMPasa inhibitors (lithium and L.690.330) stimulates autophagy, whereas the enhancement of IP3 levels by pre treatment whit mio-inositol inhibits the lithium and L.690.330 effect. Moreover we have demostred that autophagy induced by nutrient privation was also inhibited by treatment with mio-inositol, but the effect of nutrient privation in the intracellular IP3 basal levels was not evaluated. IP3 acts on the IP3 receptor (IP3R), an IP3‑activated Ca2+ channel of the endoplasmic reticulum membrane and consequently we wanted to evaluate de roll of IP3R in the regulation of autophagy. The results obtained in this thesis show that knockdown of the IP3 receptor (IP3R) with specifics small interfering RNAs and pharmacological IP3R antagonist (xestospongin B and C) are a strong stimulus for the induction of autophagy, in addition, xestospongin B (like nutrient starvation) induced loss in the interaction between Beclin-1 and Bcl-2. Moreover, the autophagy promoted by xestospongin B not produced alterations in the steady-state Ca2+ levels in the ER or in the cytosol, therefore the autophagy induced by xestospongin B was Ca2+-independent. Immunoprecipitation assays shown that Beclin- 1 (key protein in the regulation of autophagy) interacts with IP3R and Bcl-2 in basal conditions, and this interaction may be attenuated both by nutrient starvation or ABT737 treatment, which is a mimetic compound of BH3. These results suggest the presence of a protein complex in the regulation of autophagy. The treatment whit ER stressors such as tunicamycin, thapsigargin and brepheldine A induced Unfolded Protein Responses (UPR) and autophagy. The autophagy induced by these agents showed to be IRE1α dependent, but the inhibition of autophagy showed an increase in the cell death, indicating a pro survival function of the autophagy upon endoplasmic reticumum stress conditions. The autophagy induced by treatment with xestospongin B and ER stressors was inhibited by knockdown of Atg5, Atg10, Atg12, Vps34 and Beclin-1, which are keys proteins in the autophagic process. We have also evaluated the roll of Bcl-2 and Bcl-XL in the inhibition of autophgy, and the results showed that Autophagy triggered by IMPasa inhibitors and xestospongin B was inhibited by Bcl-2 and Bcl-XL over expression specifically targeted to ER but not Bcl-2 or Bcl-XL proteins targeted to mitocondria. Altogether, these results suggest that IP3R form a regulator complex with Bcl-2 and Beclin-1, which exerts a major role in the physiological control of autophagy

Orientador: Prof. Dr. Alexandre Hiroaki Kihara
Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2016.
Anóxia é uma das maiores causas de morbidade e mortalidade neonatal, especialmente em neonatos pré-maturos, constituindo um importante problema de saúde pública devido às sequelas neurológicas permanentes em pacientes. A privação de oxigênio dispara uma série de cascatas, culminando em morte celular em regiões cerebrais mais vulneráveis, como o hipocampo. Neste processo de morte celular causada pela privação de oxigênio, o cálcio citosólico possui um papel crucial. Receptores intracelulares de inositol 1,4,5-trifosfato (IP3Rs) são importantes reguladores de níveis de deste cálcio, no entanto, não se sabe sobre sua função na anóxia. O objetivo deste estudo é analisar se os IP3Rs do tipo 1 (IP3R1) participam no processo de morte no hipocampo de ratos após a anóxia neonatal. A análise quantitativa de real-time PCR revelou uma diminuição da expressão gênica de IP3R1 24 horas após a anóxia neonatal. Na análise da distribuição de células IP3R1-positivas foi observada uma densidade de IP3R1 na região de CA1 em ambos os grupos, porém, não se observou diferença entre os grupos controle e anóxia. Interessantemente os animais anóxia apresentaram uma alta colocalização de IP3R1 e marcador de núcleo (DAPI), sugerindo que a anóxia causa uma translocação de IP3R1 para o núcleo nas células hipocampais. Além disso, o padrão de marcação mostrou diferentes tamanhos de clusters dos receptores, indicando uma organização diferente entre os grupos. Foi injetado 2-APB, um bloqueador de IP3R1, ou veículo, no hipocampo de forma bilateral após a anóxia. Foi utilizado metodologias de marcação de células degeneradas e foi visto que no grupo 2APB houve uma diminuição do número de células FJC-positivas e TUNEL-positivas em comparação ao grupo veículo anóxia. Porém, não foi observado nenhuma diferença de marcação entre os grupos na imunofluorescência de caspase-3 ativada. Não foi detectada nenhuma diferença entre os grupos no teste de labirinto de Barnes. No teste de campo aberto, observou-se que o grupo 2APB apresentam maiores níveis de ansiedade. Desta forma, este estudo pode contribuir com novas perspectivas na investigação de mecanismos de neurodegeneração ativadas pela privação de oxigênio.
Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR analysis revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. Distribution analysis of IP3R-positive cells was performed and we observed higher IP3R1 pixels quantity in CA1 of both groups; however, we were not able to observe alterations between control and anoxia animals. Interestingly, we observed that anoxia animals present a higher colocalization of IP3R1 and nucleus marker (DAPI), suggesting that neonatal anoxia may cause IP3R1 translocation to the nucleus in hippocampal cells. Furthermore, puncta-labelling pattern showed different cluster sizes, larger in control group, indicating different organization between groups. We injected 2-APB, an IP3R1 blocker, or vehicle in hippocampus bilaterally after anoxia. Labelling techniques of degenerate cells was performed and we observed that 2APB group decrease the number of FJC-positive cells compared to vehicle anoxia group. In contrast, TUNEL labelling and active caspase-3 immunofluorescence showed no difference between groups. Barnes maze test showed no differences between 2APB group and anoxia vehicle group. On the other hand, the open field test showed that 2APB group presents higher anxiety levels than vehicle group. In this way, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation.

L’homéostasie calcique est régulée par de nombreux canaux ioniques, parmi lesquels des canaux intracellulaires perméables au Ca2+, comme l’IP3R. Récemment, la protéine Bcl-2 a été montré comme régulant l’activité de ce canal ionique. Cependant, les acteurs moléculaires précis de cette interaction ne sont pas très bien établis. Ici nous montrons grâce à une nouvelle technique que l’IP3R est inhibé par le domaine BH4 de Bcl-2 et que ce domaine est nécessaire et suffisant pour inhiber son activité. De plus, la liaison de l’ABT-199 dans la poche hydrophobe de Bcl-2 conduit à un changement de structure du domaine BH4. Le niveau d’expression des différentes isoformes d’IP3R ainsi que des protéines Bcl-2 et Bcl-xL ont été étudié dans différentes lignées cancéreuses prostatiques. De manière intéressante, l’expression du récepteur à l’IP3 de type 3 (IP3R3) est augmentée en fonction de l’agressivité des lignées cancéreuses prostatiques. De plus, nous pouvons observer un effet important de l’IP3R3 sur la migration et l’invasion des lignées humaines de cancer de la prostate. Globalement, ces données montrent que l’IP3R3 participe à l’augmentation du potentiel métastatique des cellules cancéreuses prostatiques. Par conséquent, l’IP3R3 peut être un marqueur diagnostic intéressant ainsi qu’une cible thérapeutique, notamment pour les stades avancés de cancer de la prostate
Calcium homeostasis is regulated by various ion channels, among which intracellular Ca2+-permeable channels, such as IP3R. Lately, Bcl-2 protein have been shown to regulate this ion channel activity. However, the study of the functional properties of IP3R in interaction with Bcl-2 is not a straightforward procedure and the molecular players implicated in that interaction are still not well established. Here, we show with the use of a new electrophysiological method, that the IP3R is inhibited by Bcl-2 via its BH4 domain and that the BH4 domain of Bcl-2 can inhibit by itself the single channel activity of the IP3R. Moreover, the binding of the ABT-199 in the hydrophobic groove of Bcl-2 leads to a tail-flip structural change in BH4 domain. We also studied the expression level of different IP3R isoforms as well as Bcl-2 and Bcl-xL protein in different prostate cancer cell lines. Interestingly, IP3R type 3 (IP3R3) expression is increased according the aggressiveness of prostate cancer cell lines. Indeed, IP3R3 was expressed preferentially in highly aggressive prostate cancer cell lines. Moreover, we can observe an significantly important effect of the IP3R3 on migration and invasion properties of human prostate cancer cell lines. Our study also revealed that IP3R3 was not involved in viability, proliferation. Overall, these data provide evidence on IP3R3 contribution to the increased metastatic potential of human prostate cancer cells. Therefore, IP3R3 could provide new perspective molecular target for the disease suppression, in particular at its advances stages

Au cours du vieillissement, les artères cérébrales subissent des modifications structurelles et fonctionnelles, notamment au niveau des cellules musculaires lisses (CML). La CML a pour rôle de maintenir la réactivité vasculaire via une signalisation calcique qui fait intervenir différents acteurs pouvant ainsi réguler deux phénomènes : la contraction et la relaxation. Ces acteurs rassemblent, au sein d’une même cellule, des canaux (CCVD, RYR, IP3R), des pompes calciques (SERCA, PMCA, NCX, STIM/ORAI) et leurs régulateurs (PLB, FKBP12.6, TRPP2, SARAF, TRIC). La restriction calorique (RC), apparaît comme étant un facteur retardant le vieillissement et ses pathologies. Notre travail s’est donc fortement impliqué dans l’étude de la signalisation calcique de la CML, en se focalisant sur les altérations génomiques et fonctionnelles au cours du vieillissement des artères cérébrales chez la souris C57Bl6/j. Nous avons ainsi pu mettre en évidence une altération de la signalisation calcique qui passe en partie par une modulation des niveaux d’expressions génique et protéique des canaux et pompes calciques impliqués dans ce phénomène, et par une modification fonctionnelle en termes de signaux calciques et de contraction. Après 5 mois de régime RC, il a été mis en évidence un ralentissement des altérations de la signalisation calcique liées au vieillissement et une diminution de l’oxydation des CML
During aging, cerebral arteries undergo structural and functional changes, particularly in smooth muscle cells (SMC). SMC is responsible for maintaining vascular reactivity via calcium signaling involving different actors and can regulate two phenomena: contraction and relaxation. These actors regroup channels (CCVD, RYR, IP3R) calcium pumps (SERCA, PMCA, NCX, STIM / ORAI) and their regulators (PLB, FKBP12.6, TRPP2, SARAF, TRIC). Caloric restriction (CR) appears as a factor in delaying aging and its pathologies. Our work is strongly involved in the study of calcium signaling in SMC, focusing on genomic and functional alterations during aging of cerebral arteries in mice C57BL6/J. We were able to demonstrate an altered calcium signaling, which is partly through modulation of gene and protein expression levels of calcium channels and pumps involved in this phenomenon, and a functional change in terms of calcium signals and contraction. After 5 months under RC, it was highlighted a slow calcium signaling alterations associated with aging and a decrease of SMC oxidation by SAMP8

Ca2+ is a universal intracellular messenger that regulates many cellular responses. Most cells express inositol 1,4,5-trisphosphate receptors (IP3R) that mediate Ca2+ release from the endoplasmic reticulum (ER) when they bind IP3 produced after activation of cell-surface receptors. Vertebrate genomes encode three closely related subtypes of IP3R (IP3R1-3). High-resolution optical analyses have revealed a hierarchy of IP3-evoked Ca2+ signals that are thought to arise from the co-regulation of IP3Rs by IP3 and Ca2+. The smallest events ('blips') report the opening of single IP3Rs, Ca2+ 'puffs' report the almost simultaneous opening of a few clustered IP3Rs, and as stimulus intensities increase further Ca2+ signals propagate regeneratively as Ca2+ waves. The aim of this study was to establish whether all three IP3R subtypes can generate Ca2+ puffs. I first used a haploid cell line (HAP1 cells) to generate, using CRISPR/Cas9, a line lacking all endogenous IP3Rs. However, for analyses of Ca2+ puffs, I used HEK cells that had been engineered, using CRISPR/Cas9 to disrupt endogenous genes, to express single IP3R subtypes. Local Ca2+ signals evoked by flash-photolysis of caged- IP3 were recorded using Cal520 and total internal reflection fluorescence (TIRF) microscopy in human embryonic kidney (HEK) cells. The Flika algorithm was used, and validated, for automated detection of Ca2+ puffs and to measure their properties. IP3 evoked Ca2+ puffs in wild-type HEK cells and in cells expressing single IP3R subtypes. In wild-type cells, the Ca2+ signals invariably propagated regeneratively to give global increases in cytosolic [Ca2+]. This occurred less frequently in cells expressing single IP3R subtypes, commensurate with their lower overall levels of IP3R expression. The properties of the Ca2+ puffs, including their rise and decay times, durations, the size of the unitary fluorescence steps as channels closed channel during the falling phase, and the estimated number of active IP3Rs in each Ca2+ puff, were broadly similar in each of the four cell lines. The latter observation suggests that despite lower overall levels of IP3R expression (~30%) in cells with single subtypes relative to WT cells, there is a mechanism that ensures formation of similarly sized IP3R clusters. The only significant differences between cell lines were the slower kinetics of the Ca2+ puffs evoked by IP3R2, which may suggest dissociation of IP3 from its receptor contributes to the termination of Ca2+ puffs. My results demonstrate, for the first time, that all three IP3R subtypes can generate Ca2+ puffs. I conclude that Ca2+ puffs are fundamental building blocks of all IP3-evoked Ca2+ signals.

L'apoptose est un processus cellulaire fondamental pour l'homéostasie tissulaire. Ce type de mort cellulaire est sous le contrôle des protéines de la famille Bcl-2 qui régulent la perméabilité de la membrane externe de la mitochondrie. Cependant, au-delà de leur rôle dans le contrôle de l'apoptose, les protéines de la famille Bcl-2 peuvent intervenir dans d'autres processus tels que le cycle cellulaire ou le métabolisme. Au sein du laboratoire, nous nous intéressons tout particulièrement aux rôles non-apoptotiques des protéines Bcl-2 au cours du développement embryonnaire. Grâce à l'utilisation du poisson zèbre, nous avons pu montrer que les protéines de la famille Bcl-2 contrôlent différents processus au cours du développement grâce à leur capacité à réguler l'homéostasie calcique. En effet, nous avons montré que la protéine anti-apoptotique Nrz participe au remodelage du cytosquelette d'actine au cours de l'épibolie en régulant la concentration de calcium cytosolique par son interaction avec le récepteur à l'IP3 (IP3R). Nous avons de plus pu montrer que Nrz diminue la sortie de calcium du réticulum endoplasmique en inhibant la fixation de l'IP3 sur son récepteur. Nous avons également identifié un nouveau membre pro-apoptotique de la famille Bcl-2, Bclwav, spécifiquement exprimée chez les poissons et le xénope. Cette protéine participe à la régulation de l'homéostasie calcique mitochondriale en interagissant avec VDAC. Nous avons de plus montré que cette activité est essentielle pour les mouvements de convergence et d'extension au cours du développement embryonnaire précoce du poisson zèbre
Apoptosis is a key cellular process for tissue homeostasis. Apoptotic cell death is under control of Bcl-2 family proteins which regulate outer mitochondrial membrane permeability. However, beyond their role in apoptosis, Bcl-2 family proteins are also involved in other cellular processes such as cell cycle or metabolism. In our laboratory we are interested in non-apoptotic functions of Bcl-2 family proteins in embryonic development. Using zebrafish model we have shown that Bcl-2 proteins control different processes during early development thanks to their ability to regulate calcium homeostasis. Indeed, we have shown that the anti-apoptotic protein Nrz participates in actin cytoskeleton remodeling during epiboly by regulating cytosolic calcium concentration via an interaction with the IP3 receptor (IP3R). We have also demonstrated that Nrz decreases calcium release from the endoplasmic reticulum by inhibiting IP3 fixation on its receptor. We have furthermore identified a new pro-apoptotic member of Bcl-2 family, Bcl-wav which is expressed only in fish and frogs. This protein regulates mitochondrial calcium homeostasis by interacting with VDAC. We have moreover shown that this activity is essential for convergence and extension movements during early zebrafish development

Trata-se de pesquisa empírica cujo objeto são os indicadores do Índice Paulista de Responsabilidade Social (IPRS). À luz deste índice, se analisa educação e desenvolvimento nos municípios do noroeste paulista, buscando compreender que fatores levam aos bons resultados nos indicadores de escolaridade, obtidos por um grupo de municípios com baixos níveis de riqueza, denominados de grupo 3 do IPRS. O trabalho busca, por meio de um olhar crítico da composição das variáveis do IPRS, analisar a capacidade destas expressarem a realidade do ambiente educacional dos municípios e, ao mesmo tempo, serem úteis na definição de políticas visando fortalecer as dinâmicas de desenvolvimento dos mesmos. Com isso objetiva contribuir com o debate sobre a formulação de indicadores educacionais mais adequados à análise do desenvolvimento na atualidade. Parte-se da constatação da relevância da educação nas atuais dinâmicas de desenvolvimento, não mais compreendido como sinônimo de crescimento econômico, ressaltando a inclusão da dimensão educacional nos instrumentos de mensuração do desenvolvimento humano. São discutidos os conceitos de desenvolvimento como liberdade e de capital social como parâmetros para se compreender o surgimento de uma nova geração de indicadores de desenvolvimento dentre os quais o índice paulista. Estes conceitos são abordados com base nas referências teóricas fornecidas por Sen, Putnam, Bourdieu e Coleman, objetivando analisar a capacidade do IPRS em incorporar estes conceitos nas suas dimensões. Paralelamente a isso se apresenta e analisa os indicadores do IPRS no estado de São Paulo e nos municípios da região noroeste, buscando compreender o que leva a emergência de um grupo expressivo de municípios pobres em termos de riqueza mas com indicadores sociais satisfatórios. Para tanto são utilizados os dados secundários do banco de dados do IPRS da Fundação Seade. Com isso se constata dois aspectos: a relevância da dimensão escolaridade nos bons resultados sociais obtidos por estes municípios e a insuficiência dos indicadores educacionais do índice paulista, especialmente no que se refere a incorporar outras variáveis quantitativas relacionadas ao ambiente educacional disponível quanto variáveis referentes à qualidade dos processos educativos desenvolvidos. As seguintes considerações finais são apresentadas: o IPRS é um indicador que avança na sua estruturação e capacidade de retratar a realidade do desenvolvimento dos municípios, fugindo da padronização dos indicadores sintéticos, contudo, ainda apresenta limitações no que tange a capacidade de analisar o ambiente educacional e a qualidade da educação ofertada e, conseqüentemente, contribuir de maneira efetiva como instrumento de monitoramento e avaliação das políticas que visam fortalecer as dinâmicas de desenvolvimento dos municípios paulistas.
The following study is an empirical research, having as its object the indicators of the Índice Paulista de Responsabilidade Social (São Paulo Index of Social Responsibility, here referred as IPRS). Having this Index as reference, we analyze education and development of the cities located in the Northwest of São Paulo State, aiming to understand which factors lead to the good results in schooling indicators obtained by a group of low income cities, rated in group 3 in the IPRS. The study?s purpose is to analyze (through a detailed and critical look at the variables that compose the IPRS) its capacity of expressing the reality of the educational environment in the cities as well as being useful in the definition of policies that would aim to strengthen its development actions. Considering this, we intend to contribute to the debate on the creation of educational indicators that would be more adequate to the analysis of development nowadays. We take as a premise the relevance of education in the current development actions, not seen as a synonym to economical growth anymore, stressing the inclusion of the educational dimension in the measuring instruments of human development. We address the concepts of development seen as freedom and social capital as a guideline to understand the advent of a new generation of development indicators among which is the São Paulo index. These concepts are approached based on the references of theories given by Sen, Putnam, Bourdieu and Coleman, purposing to analyze the capacity of IPRS to encompass all these concepts within its boundaries. Concurrently, we present and examine the IPRS indicators in São Paulo State and in the cities of the Northwestern region, attempting to understand what leads to the appearance of an expressive number of low income cities with satisfactory social indicators. In order to do that we use the secondary data of the IPRS database from Fundação Seade. Considering this, two aspects are evidenced: the relevance of the schooling dimension in the good social results obtained by these cities and the inadequacy of the educational indicators in the São Paulo index, especially in taking into consideration other quantitative variables related to the educational environment available and to the quality of the educational processes developed. The following final points are then presented: the IPRS is an indicator that moves towards to its organization and capacity of depicting the reality of the cities development, distancing itself from the standardization of synthetic indicators, though still presenting some limitation regarding the ability to analyze the educational environment and the education quality offered, and thus effectively collaborate as an instrument of monitoring and assessment of the policies that aim the strengthening of the development actions for São Paulo cities.

Le cancer du sein est le cancer féminin le plus fréquent et le plus létal chez la femme dans le monde. Malgré l'amélioration du dépistage dans les phases précoces du développement tumoral, il demeure difficile de traiter les phases tardives lorsque les processus métastatiques sont engagés. Le développement métastatique dépend notamment de l'acquisition de capacités migratoires par les cellules épithéliales impliquant un remodelage du cytosquelette, hautement dépendant de la concentration calcique intracellulaire. Alors que les travaux se sont intéressés à l'implication des canaux ioniques membranaires dans les processus de migration, le rôle des récepteurs à l'inositol 1,4,5-trisphosphate (IP₃Rs) reste peu étudié et donc méconnu. Dans un premier temps nous avons montré une augmentation du niveau d'expression d'IP₃R3 avec le niveau du potentiel migratoire de trois lignées cancéreuses mammaires humaines : MCF-7 (les moins migrantes), MDA-MB-231 et MDA-MB-435s (les plus migrantes). D'autre part, nous montrons que la modulation de l'expression d'IP₃R3 module leurs capacités migratoires: elles sont diminuées par l'inhibition d'IP₃R3, alors qu'elles sont augmentées par la surexpression d'IP₃R3 dans les cellules mammaires peu migrantes. De plus, l'inhibition d'IP₃R3 révèle un signal calcique oscillant, alors que sa surexpression induit un signal calcique maintenu dans ces cellules. Dans un second temps, nous avons mis en évidence une corrélation inverse entre le niveau d'expression d'IP₃R3 et la morphologie arrondie de ces trois lignées cellulaires. En effet, plus la morphologie cellulaire est arrondie, plus l'expression d'IP₃R3 est importante. Par ailleurs, l'inhibition d'IP₃R3 induit un arrondissement des cellules migrantes et une diminution des protrusions membranaires accompagnés d’une diminution de leur adhésion. Ces résultats suggèrent fortement l'implication d'IP₃R3 dans la modulation des acteurs du cytosquelette. En effet, l'inhibition d'IP₃R3 induit une diminution de l'expression de l'ARHGAP18, de l'activité de RhoA et de l'expression de Cdc42. Ces Rho GTPases permettent à leur tour une diminution de la phosphorylation de FAK_⁸⁶¹ et la réorganisation du cytosquelette d'actine et de profiline. Enfin, dans un modèle migratoire, le profil oscillant s'établit dès réalisation d'une blessure et prédomine 3 h après dans les cellules du front de migration alors qu'il se met en place dans les cellules à l'arrière uniquement après 3 h. L'intervalle spatio-temporel de ce signal calcique oscillant reflète une dynamique calcique intracellulaire nécessaire aux processus migratoires et au remodelage du cytosquelette des cellules du cancer du sein. En conclusion, nos résultats révèlent un rôle clé de l'IP₃R3 dans les processus migratoires et dans le remodelage du cytosquelette de profilactine via la voie ARHGAP18/ RhoA/ FAK associé à une modulation du profil calcique intracellulaire
Breast cancer is the most common lethal cancer in women in worldwide. In spite of screening improvement in early stages of tumor development, it remains difficult to cure late stages when metastases are begun. Metastatic development depends on migratory capacities acquisition by epithelial cells, involving a cytoskeleton remodeling, highly depending of intracellular calcium concentration. While plasma membrane calcium channels have been highly studied in migration processes, the role of IP₃Rs remains misunderstood. Firstly, we highlighted a correlation between IP₃R3 expression level and migratory potential of three human breast cancer cell lines with different migratory potential. Indeed, the more the migratory profile increases, the more is the expression of IP₃R3. Moreover, IP₃R3 expression modulation regulates their migratory capacities. The migratory capacities decreased when silencing IP₃R3, whereas they increased by overexpressing IP₃R3 in the low migratory breast cancer cell line (MCF-7). Furthermore, IP₃R3 silencing reveals an oscillating calcium profile, while its overexpression induces a sustained calcium profile. Secondly, we demonstrated a reverse correlation between IP₃R3 expression level and rounded shape of these three cell lines. Indeed, the longer the cell has an elongated morphology, the greater the IP₃R3 expression is important. Moreover, IP₃R3 silencing induced a rounded shape of migrating cells and decreased of protrusions and adhesion. Our results suggest IP₃R3 implication in modulation of cytoskeleton actors. Indeed, knockdown of IP₃R3 induced a decrease of ARHGAP18 and Cdc42 expression, RhoA and FAK_⁸⁶¹ activity, and a reorganization of profilactin cytoskeleton. Furthermore, in a migratory model, oscillating profile is revealed at wound realization and predominates 3 h amer in cells of the front wound whereas it sets up in cells of back only amer 3 h. The spatio-temporal gap of this oscillating calcium signal reflects an intracellular calcium dynamic essential to migratory processes and cytoskeleton remodelling in breast cancer. In conclusion, our results reveal a key role of IP₃R3 in migratory processes by profilactin cytoskeleton remodeling through ARHGAP18/ RhoA/ FAK pathway thanks to intracellular calcium profile modulation

Breast cancer is the most prevalent type of cancer and second leading cause of death in women. Among others, the triple negative breast cancer (TNBC) is the most invasive as it has the highest recurrence and death rates with no targeted therapeutic available thus far. Epidermal Growth Factor Receptor (EGFR) is one of the important targets as more than fifty percent of the TNBC overexpress it but all the therapies designed against it have failed to show significant results. The juxtamembrane domain of EGFR has been explored comparatively recently and has been used to design a decoy peptide with the anticipation to affect the EGFR downstream functions. Previous research has shown it to cause cell death in cancer cells. This study is aimed towards deciphering the mechanism of action of the stapled form of this decoy peptide-SAH5. It presents evidence that the peptide leads to an immediate intracellular calcium release from the Inositol 1,4,5 triphosphate on the endoplasmic reticulum, an inhibition of which can rescue SAH5 induced cell death. The study also demonstrate that the peptide is able to increase the production of Reactive Oxygen Species (ROS) in mitochondria, part of which is triggered by the peptide-induced calcium release.

Grado de magister en fisiología
La plasticidad muscular es la capacidad que presentan las células musculares esqueléticas de adaptarse a diferentes estímulos externos, modificando su fenotipo. La frecuencia de estimulación eléctrica (EE) (in vitro como in vivo) induce la transición fenotípica de un músculo. De modo interesante, el EE de baja frecuencia induce marcadores de transición fenotípica rápido/lenta por una vía dependiente de la activación del IP3R. Además, se ha establecido al uniportador de Ca2+ mitocondrial como un regulador del trofismo muscular. Hipotetizamos que la regulación de los niveles de RNAm del uniportador de Ca2+ mitocondrial y de sus proteínas reguladoras en respuesta a EE de baja frecuencia es dependiente de la activación del IP3R. Fibras musculares adultas aisladas desde el músculo flexor digitorium brevis (fdb) de ratones C57BL/6J de 8 a 10 semanas de edad fueron expuestas a EE de baja frecuencia en presencia o ausencia de inhibidores de la vía propuesta. Se observó que el estímulo eléctrico de baja frecuencia resulta en una disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE, mientras que el EE de alta frecuencia no genera modificaciones. Las fibras musculares esqueléticas pre-incubadas con apirasa (enzima que degrada ATP) y xestospongina B (inhibidor de los receptores de IP3) previenen la disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE mediada por un EE de baja frecuencia. El ATP extracelular exógeno (agregado al medio de incubación) resulta en una disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE). Además, este efecto de EE no es prevenido por actinomicina D (inhibidor de la transcripción). Este trabajo contribuye a la comprensión de los mecanismos moleculares involucrados en la plasticidad muscular en respuesta a ejercicio físico y en particular, al rol de la mitocondria en estos mecanismos.
Muscular plasticity is the ability of the skeletal muscle cells of different external stimuli, modifying their phenotype. The frequency of electrical stimulation (ES) (in vitro as in vivo) induces the phenotypic transition of a muscle. Interestingly, low frequency ES induces rapid / slow phenotypic transition markers by a pathway dependent on the activation of IP3R. In addition, the mitochondrial Ca2 + uniporter has been established as a regulator of muscle trophism. We hypothesized that the regulation of mRNA levels of the mitochondrial Ca2 + uniporter and its regulatory proteins in response to low frequency EE is dependent on the activation of IP3R. Adult muscle fibers isolated from the flexor digitorium brevis muscle (fdb) of C57BL / 6J mice from 8 to 10 weeks of age were exposed to low frequency ES in the presence or absence of inhibitors of the proposed pathway. It was observed that the low frequency electrical stimulus results in a decrease in mRNA levels of MCU, MICU1, MICU2 and EMRE, while high frequency ES does not generate modifications. Skeletal muscle fibers pre-incubated with apirasa (enzyme that degrades ATP) and xestospongin B (inhibitor of IP3 receptors) prevent the decrease of mRNA levels of MCU, MICU1, MICU2 and EMRE mediated by a low frequency ES. Exogenous extracellular ATP (added to the incubation medium) results in a decrease in mRNA levels of MCU, MICU1, MICU2 and EMRE). In addition, this effect of ES is not prevented by actinomycin D (transcription inhibitor). This work contributes to the understanding of the molecular mechanisms involved in muscle plasticity in response to physical exercise and, in particular, to the role of mitochondria in these mechanisms.
30/08/2019

The principal purpose of this study is to provide a refined empirical investigation concerning country-specific determinants of horizontal and vertical intra-industry trade (IIT,) in relation to South Africa using the gravity model of trade in a panel data setting. Prior to investigating the case of South Africa's intra-industry trade a critical review of the relevant theoretical, methodologcal, and empirical literature is provided. The study operationalises the theoretical dstinction between horizontal and vertical IIT using the latest methodology of decomposing total IIT into horizontal intra-industry trade (HIIq and vertical intra-industry trade (WIT,). Thts study makes several advances on earlier empirical studies of intra-industry trade determinants. These include the introduction of new countq-specific determinants of intraindustry trade that previous studies have not examined. Furthermore, it is the first empirical study that traces the relationshp between intra-industry trade flows and intellectual property rights (IPRs). Moreover, to ensure the sensitivity and robustness of the results, several econometric approaches have been used in estimating the gravity model of South Africa's intra-industry trade: the consistent coefficient approach, the fixed effects approach, the random effects approach, and the between effects approach. The econometric results are generally satisfactory in terms of economic interpretation and statistical significance and thus offer new empirical validation to the theoretical explanatory variables. The key findngs suggest the following: the volume of South Africa's IIT has increased during the study period and its WIT exceeds its HIIT. The latter result reflects the nature of South Africa's trade as it imports high valued added products and exports primary and mineral products. South Africa's intra-industry trade and its two components are positively related to market size and standard of living, and negatively related to geographcal distance. Furthermore, separately, the IPRs and the imitation ability of South Africa's tradmg partners are not important factors in determining IIT flows; however, the interaction between them is an important factor. Thts study also reveals South Africa should pursue its intra-industry trade with rest of world concentrating on local industries that produce most competitive varieties, absorbing labor and other resources from the production of other varieties.

Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

La tesi ha ad oggetto le forme di tutela dei diritti di proprietà industriale sulle invenzioni aventi ad oggetto piante. La prima parte del lavoro è dedicata ad un’analisi delle linee evolutive degli strumenti specifici di protezione delle nuove varietà vegetali introdotti, dapprima, nell’ordinamento giuridico statunitense e, qualche tempo dopo, in quelli dei Paesi aderenti alla Convenzione UPOV. L’autore pone a confronto le suddette discipline ad hoc con la normativa brevettuale classica, evidenziandone le differenze sia sul piano dei rispettivi requisiti d’accesso che su quello del contenuto e della portata della tutela. Viene, dunque, valutato l’impatto prodotto dall’avvento delle moderne biotecnologie sui sistemi normativi vigenti in Europa e negli Stati Uniti in materia di protezione dell’innovazione conseguita in campo agricolo. Attraverso lo studio dei principali arresti giurisprudenziali e delle iniziative legislative succedutisi negli ultimi anni, vengono delineati i tratti caratteristici degli approcci adottati, rispettivamente, oltre-Atlantico e nel vecchio continente al fine di fornire risposta alle questioni determinate dalla rivoluzione biotecnologica, con particolare riguardo ai problemi di interfaccia fra tutela varietale e tutela brevettuale che quest’ultima ha determinato. L’autore ritiene che le vie seguite negli Stati Uniti ed in Europa, per quanto diverse tra loro sul piano formale, non siano poi così distanti nella sostanza e, de jure condendo, suggerisce di ridefinire in Europa le interrelazioni fra tutela varietale e tutela brevettuale, abrogando il divieto di brevettazione delle nuove varietà vegetali, vero elemento differenziale fra il contesto normativo europeo e quello statunitense.
Intellectual property rights on plant-related inventions are the subject matter of this thesis. In the first part of this work, the author analyzes the evolution of the specific means of protection for new plant varieties first introduced in the United States of America and then in the member States of the UPOV Convention. The author makes a comparison between these ad hoc means of protection and the patent protection, by pointing out the differences in respect to the conditions for protection and the scope of protection. After that, an assessment of the impact of modern biotechnology on the existent European and American plant-related invention systems of protection is made. The author investigates the approaches which were chosen in Europe and in the United States of America in order to address the issues related to the biotechnological revolution, with particular focus on the interface problems between plant variety protection and patent protection, by means of a study of the principal case law and regulatory interventions adopted during the last years. The author believes that American and European approaches are not so different in substance and, de jure condendo, suggests redefining the relationship between plant variety protection and patent protection in Europe through the elimination of the ban on patent protection for plant varieties, which is the real differential factor between European and American normative contexts.

Ainda que limitadas enquanto expressão dos eventos ligados à saúde e apesar das deficiências em relação à cobertura e à qualidade dos dados, as estatísticas de mortalidade constituem um dos mais importantes subsídios para o planejamento e avaliação dos serviços de saúde. Tradicionalmente, a mortalidade de uma determinada população tem sido aferida por meio das taxas brutas e específicas de mortalidade. Porém, esses índices consideram apenas a magnitude das causas de óbito, sem qualificar o peso resultante dessas mortes para a sociedade. Neste sentido, tem-se enfatizado cada vez mais a importância da mortalidade prematura enquanto expressão social do valor da morte, pois esta, quando ocorre numa idade de altas criatividade e produtividade não só afeta o indivíduo e o grupo social que convive diretamente com ele, mas a sociedade como um todo, que é privada do seu potencial econômico e intelectual (REICHENHEIM; WERNECK, 1994). \"O indicador Anos Potenciais de Vida Perdidos (APVP), ao combinar a magnitude das mortes com a idade em que ocorreram os óbitos, qualifica essas mortes\" (KERR-PONTES; ROUQUAYROL, 1999 apud SAUER; WAGNER, 2003, p. 1520). Este estudo pretendeu avaliar a evolução dos APVPs nos municípios e regionais de saúde do Estado de São Paulo, no período de 2003 a 2013, para a população total. Pretendeu, também, analisar a evolução retrospectiva das 15 causas de óbito com as maiores taxas de APVP em 2013, para a população total do Estado de São Paulo. Para tanto, foi elaborada uma base de dados a partir dos óbitos de residentes do Estado de São Paulo ocorridos no período de 2003 a 2013 e processados pelo SIM (Sistema de Informação sobre Mortalidade), sendo as causas de morte classificadas de acordo com a Décima Revisão da Classificação Internacional de Doenças (CID 10). O cálculo dos APVPs foi realizado com base numa proposta feita por Romeder e McWhinnie (1988) e, após elaborados os dados, foram confeccionados cartogramas utilizando o programa Tabwin para visualização da evolução dos APVPs nas regionais de saúde do Estado. Foram construídos gráficos de linha para a observação da evolução das 15 causas de óbito com as maiores taxas de APVP de 2003 a 2013. Posteriormente, foram analisadas as Taxa de Mortalidade Geral (TMG), Taxa de Mortalidade Infantil (TMI) e Taxa de Mortalidade Materna (TMM) para os anos de 7 2003 a 2013 para o Estado de São Paulo. E, finalmente, foram avaliados: as dimensões escolaridade, longevidade e riqueza do Índice Paulista de Responsabilidade Social (IPRS) em cada um de seus grupos; frequência absoluta e relativa do IPRS em cada um de seus grupos; relação do IPRS segundo Redes Regionais de Atenção à Saúde (RRAS); relação IPRS segundo Grupos Populacionais; TAPVP por grupos de IPRS; TAPVP por Grupos Populacionais; TAPVP por RRAS; IPRS na sua dimensão Riqueza por TAPVP; IPRS na sua dimensão Longevidade por TAPVP; IPRS na sua dimensão Escolaridade por TAPVP; Correlação entre as dimensões do IPRS e TAPVP. Todas estas avaliações são válidas para o Estado de São Paulo para o ano de 2012 e foram obtidas utilizando-se o aplicativo Stata 9.0. A Taxa de Mortalidade Geral (TMG) para o Estado de São Paulo para o período de 2003 a 2013 em comparação com a do Brasil mostrou-se desfavorável, o mesmo acontecendo com a Taxa de Mortalidade Infantil (TMI), cujo predomínio, no Estado, foi do componente Pós-Neonatal. Já a Taxa de Mortalidade Materna (TMM) demonstrou boa assistência ao pré-natal, parto e puerpério no Estado no período citado. Observando-se a evolução das TAPVP nos cartogramas do Estado de São Paulo no período de 2003 a 2013 as RRAS onde as TAPVP foram maiores foram: 6, 7, 9, 10, 11, 12, 13 e 17. Das quinze maiores causas de óbito segundo TAPVP para o Estado no período, nove são passíveis de prevenção na atenção primária. Citou-se ainda o subregistro e a tripla carga de doenças. A maioria dos 645 municípios do Estado de São Paulo, no ano de 2012 apresentaram um IPRS de grupo 4. Houve 95% de probabilidade de que a maior TAPVP ocorreu para o IPRS 4 com um IC de 17.325,04 a 18.424,20. O Teste de Anova, com 4 gl mostrou diferença significativa (p<0,05) na TAPVP por grupos de IPRS. Com 5 graus de liberdade, o Teste de Kruskal-Wallis foi significativo (p<0,05) indicando que houve diferença entre os Grupos Populacionais quanto à TAPVP. Com 16 graus de liberdade, o Teste de Kruskal-Wallis foi significativo (p<0,05) indicando que existiu diferença entre as RRAS quanto às TAPVP. À medida que aumentou a riqueza do IPRS, diminuiu, ainda que discretamente, a TAPVP. Longevidade e TAPVP mantiveram-se estáveis. Conforme aumentou a escolaridade, aumentou a TAPVP. Por existir uma correlação positiva entre riqueza e longevidade e escolaridade, à medida que aumentou a riqueza, aumentaram a longevidade e escolaridade. Com relação a TAPVP e riqueza e longevidade, o coeficiente de correlação foi negativo, significando que à medida que aumentaram a riqueza e 8 longevidade, diminuiu a TAPVP. Porém, com relação à escolaridade, o coeficiente de correlação entre o mesmo e a TAPVP foi positivo, indicando que à medida que aumentou a escolaridade, aumentou a TAPVP. Por fim, essa dissertação poderia ser apresentada às autoridades de saúde do Estado como um projeto para redução da mortalidade prematura, com foco em melhoria da educação básica, instalação de mais serviços de saúde de qualidade e adequação dos serviços de segurança pública.
Although limited as an expression of health-related events and despite problems concerning the coverage and quality of available data, mortality estimates are among the most important foundations for the planning and evaluation of health services. Traditionally, mortality has been estimated according to the gross and specific mortality rates in a given population. However, these indicators consider the impact of death causes alone, without qualifying the burden resulting from deaths to society. The importance of premature mortality as a social expression of the burden of death has therefore received increasing attention, as it occurs at an age range of high creativity and productivity and affects not only the individual and his direct social group, but society as a whole, whose economic and intellectual potential is affected (REICHENHEIM; WERNECK, 1994). The estimate of potential years of life lost (PYLL) provides a more detailed assessment of mortality by combining death rates and the age when death occurs (KERR-PONTES; ROUQUAYROL, 1999 apud SAUER; WAGNER, 2003, p. 1520). Our study was aimed at assessing the evolution of PYLL rates in the total population of cities and health districts in the State of São Paulo, Brazil, between 2003 and 2013. We also assessed the retrospective evolution of the 15 death causes with the greatest PYLL rates in 2013 for the total population of the State of São Paulo. In order to achieve this, we created a database with information on deaths occurred in the state between 2003 and 2013 which were processed by the Mortality Information System (MIS), with death causes classified according to the 10th revision of the International Classification of Diseases (ICD-10). PYLL rates were calculated according to the method proposed by Romeder and McWhinnie (1988) and data charts 9 were created in TabWin to display the evolution of PYLL rates in the health districts of the state. Line graphs were created to display the evolution of the 15 death causes with the highest PYLL rates between 2003 and 2013. We further assessed the general mortality rate (GMR), child mortality rates (CMR), and mother mortality rates (MMR). Finally, we assessed the education, longevity, and wealth dimensions of the São Paulo Index of Social Responsibility (SPISR) in each of its groups; absolute and relative frequency of the SPISR in each of its groups; relationship of the SPISR according to the Regional Health Care Networks (RHCN); SPISR relationship according to population groups; PYLL rates by SPISR group; PYLL rates by population groups; PYLL rates by RHCN; SPISR dimension \'wealth\' by PYLL rates; SPISR dimension \'longevity\' by PYLL rates; SPISR dimension \'education\' by PYLL rates; and correlations between SPISR dimensions and PYLL rates. All the analyses are valid for the State of São Paulo in the year of 2012 and were made using the Stata 9.0 software. The GMR in the State of São Paulo for the period of 2003-2013 was worse compared to Brazil, and so was the CMR, with a predominance of the post-neonatal component in the State. The MMR indicated the availability of adequate prenatal, delivery, and postpartum assistance in the State during the period. The data charts displaying the evolution of PYLL rates in the State of São Paulo show that the RHCNs with the highest PYLL rates were 6, 7, 9, 10, 11, 12, 13, and 17. From the main 15 death causes according to PYLL rates in the period, 9 can be prevented in primary care. Under-recording and the triple load of diseases were also detected. The SPISR of most of the 645 municipalities in the State of São Paulo in the year 2012 was 4. The probability that the highest PYLL rate was associated with a SPISR of 4 was 95%, with a confidence interval between 17325.04 and 18424.20. An ANOVA with 4 degrees of freedom showed significant differences (p<0.05) in PYLL rates by SPISR group. With 5 degrees of freedom, the test of Kruskal-Wallis provided significant results (p<0.05), indicating the existence of differences between population groups in respect to PYLL rates. With 16 degrees of freedom, the Kruskal-Wallis test indicated the existence of significant differences between the RHCNs in terms of PYLL rates. PYLL rates decreased, although subtly, with the increase of wealth in the SPISR. Longevity and PYLL rates remained stable. As education increased, PYLL rates also increased. Since there was a positive correlation between wealth, longevity, and education, increased wealth was associated 10 with increased longevity and education as well. Concerning the relationship between PYLL rates and wealth and longevity, we found a negative correlation coefficient, indicating that as wealth and longevity increased, PYLL rates decreased. In respect to education, however, the correlation with PYLL rates was positive, indicating that increases in education were associated with increases in PYLL rates. Finally, this dissertation could be presented to the health authorities of the State of São Paulo as a project to reduce early mortality, focused on improvements in basic education, expansion of high-quality health services, and improvements in public security

La contrefaçon est un phénomène de dimension internationale qui constitue aujourd’hui plus qu’hier un véritable fléau. Il apparaît que les principaux pays de provenance des contrefaçons saisies dans l’Union Européenne sont les pays d’Asie,notamment la Thaïlande. Cette recherche a pour but d'étudier les problèmes juridiques relatifs à la contrefaçon des droits de propriété intellectuelle. Ceci nous amène à poursuivre la recherche suivant deux axes. La première partie a pour but de mettre en évidence la comparaison de la définition de la contrefaçon en France et en Thaïlande. Elle se décompose en deux sous-parties, la première s'attachant à décrire la qualification de l’atteinte constitutive de contrefaçon selon les droits concernés (le terme « contrefaçon » en lui-même, tant en France qu’en Thaïlande, désignant différentes formes d’atteintes à un droit patrimonial de propriété intellectuelle). Pour identifier les atteintes constitutives de contrefaçon, notamment l’élément matériel, nous mettons l’accent sur 4 points: l’existence de la création, la diffusion de la création, l’usage de la création et la participation à l’action contrefaisante. S’agissant de l’élément intentionnel de contrefaçon, il semble présenter de multiples facettes. La seconde définit la preuve de la contrefaçon. On observe alors les moyens de preuve de la contrefaçon : la loi prévoit deux séries de mesures principales que nous pouvons regrouper en moyens de preuve en matière civile et pénale. Par ailleurs, il y a des moyens de preuve alternatifs entre les deux régimes. Ce sont des procédures douanières.La seconde partie a pour objet la prise en considération de la répression par les juridictions civiles et pénales. Elle se décompose donc en deux sous-parties : la première concerne les sanctions prononcées par les juridictions pénales. Nous avons déjà étudié la procédure pénale et les sanctions applicables à la contrefaçon. Il se trouve que la situation en Thaïlande est totalement différente de la situation française, notamment concernant la jurisprudence en matière pénale. Il semblerait que la majorité des décisions soient des sanctions prononcées par les juridictions pénales. La seconde sous-partie concerne quant à elle la réparation des atteintes à la propriété intellectuelle. Nous constatons que le préjudice subi, en France tout comme en Thaïlande constitue en un gain manqué . En outre l’évaluation du préjudice en France et en Thaïlande est difficile (préjudice subi en matière de marque, préjudice moral et fixation de l’indemnisation de peine privée)
Counterfeiting is an international problem. It appears that the main countries of origin of counterfeit goods seized in the European Union are the Asian countries, including Thailand. The research explains concisely the entire key factors to this whole problem. The research is divided into two parts; in the first place, I will outline pointly the definition of Intellectual Property Rights law (IPRs law) infringement between French and Thailand. Firstly, we focus on the structure of IPRs infringement. The term "counterfeit" in himself both in France and Thailand indicating different forms of an intellectual property rights liability conception. To identify violations constitute infringements, including the material element, we focus on four points, the existence of the creation, dissemination of the creation, use of creation, participation in the infringing action . With regard to the intentional element of infringement, iconcerned the intention of counterfeiter by the civil and criminal aspects as well as the objectives of my research would analysis on two components. First, the application of substantive issues embodies in the civil action. The second is the criminal action. The intention of counterfeiter are also intersect into two parts of action. The secondly,, we research to the proof of infringement. There provides two measures of proof in civil matters and evidence incriminal matters. In addition, there are a customs procedures as an alternative measure of proof . In the second place, we mainly concerned the IPRs law enforcement: Firstly we concerns the penalties imposed by criminal courts. We have already studied the criminal proceedings. In addition, we studied the penaltiesfor counterfeiting. We find that the criminal proceedings in Thailand is totally different from the criminal proceedings in France. In addition, we studied the penalization of IPRs law. We find that the situation in Thailand is totally different from a France, especially in criminal jurisprudence. It seems that the majority of decisions are the penalties imposed by criminal courts. Secondly were search about categories of damages and criteria for proof of damages. We find that the damages, in France as well as in Thailand, is the recovery of profit. Also the difficulty of assessing the damage, in France as well as in Thailand, are the damage of Trademark law, moral right damage and punitive damage

碩士
國立臺灣大學
資訊工程學研究所
94
The things we normally do in daily living including any daily activity we perform for self-care (such as feeding ourselves, bathing, dressing, grooming), work, homemaking, and leisure. The ability or inability to perform activities of daily living (ADLs) can be used as a very practical measure of ability/disability in many disorders. Modeling human ADLs via contextual information is gaining increasing interest in the artificial intelligent and ubiquitous communities. There are several studies on tracking ADLs, such as using video cameras, or microphones. Many people are uncomfortable living with cameras and microphones. Furthermore, video cameras, especially in non-public place spaces, provoke strong privacy concerns. Those approaches sometimes cannot process sensor data with minimal computational resources (e.g., a personal digital assistant (PDA)). Our developed system: Intelligent Portable Activity Recognition System (IPARS) performs activity recognition online with minimal computational resources. Sensors should be low-maintenance, easy to replace and maintain. Tagging objects with a remotely readable identification tag is adopted in our system. In addition, we develop a wearable wrist, based on Radio Frequency Identification (RFID) reader to detect everyday objects. In addition, we use WiFi positioning system to capture a person’s current position. IPARS is equipped with an RFID reader, which connects to a PDA. The way to obtain contexts to infer the current activity of a person is by detecting person-object interactions, and movement. Our approach uses a general framework for activity recognition by building upon and extending multiway tree structure (trie) to model ADLs via contextual information. There are two steps for IPARS to achieve activity recognition. First, by using the interface provided by IPARS, the person can train his activities easily. Therefore, while the person performs activities, IPARS models sequential sensor readings involved in these activities. Second, after modeling human activities, IPARS makes inferences for activity recognition by collecting current contexts and extracting features to map trained activity models. There are two phases in our experiments. In the first phase, we conducted our experiments in the Computer Science and Information Engineering at National Taiwan University. The first goal is using IPARS to test object recognition and location tracking. In the second phase, the experiment is run in a real home. The second goal is to evaluate the proposed solution of the activity recognition problem. We found that a discriminative relational approach for activity recognition based on the framework of multi-tries models to be well-suited to model sequence of contexts for activity recognition. IPARS detected 80 percent correctly for activity recognition. The results are promising. In the future, we focus on how to detect activities about healthcare. We plan to extend our model in a number of ways. First, by collecting data from more subjects, we can learn a set of generic models by clustering the subjects based on their similarities; then we can use a mixture of these models to better recognize activities of a new person.

Contexte: La physiopathologie de la fibrillation auriculaire (FA) a été caractérisée par des changements de concentration cellulaire de Ca2+ et des processus connexes menant à l'apparition et au maintien de la maladie. Les récepteurs de trisphosphate d'inositol (IP3R) sont des canaux calciques ligand-dépendants pour lesquels la surexpression dans la FA a été liée à un remodelage cardiaque. Les microARN (miR, miARN), petits ARN non codants, sont d'une longueur d'environ 22 nucléotides et régulent l'expression des gènes par déstabilisation de l'ARN ou inhibition de sa traduction. De plus en plus de preuves ont été apportées sur le rôle des miARN dans la physiopathologie des troubles cardiaques, y compris le remodelage défavorable induit par la FA. Objectif: Notre laboratoire a montré que le niveau nucléaire IP3R1 est régulé à la hausse dans le modèle canin de FA, ce qui produit une augmentation de la charge nucléaire en calcium. Cette étude vise donc à étudier le rôle des miARN dans la régulation d'IP3R1 qui initie et/ou perpétue la FA dans les cardiomyocytes auriculaires du modèle de FA chez le chien. Méthodes: Nous avons utilisé un modèle canin de AF établi par méta-cardiographie auriculaire pendant 600 bpm × une semaine; des cœurs perfusés par Langendorff pour isoler les cardiomyocytes auriculaires pour des expériences moléculaires; le criblage des miRs qui ciblent le gène ITPR1, codant IP3R1, en utilisant des bases de données en ligne; RT-qPCR pour mesurer l'expression de l'ARNm de ITPR1 et confirmer le niveau d'expression des miARN criblés; l'analyse Western Blot pour évaluer le niveau de protéine d’IP3R1; le test de la double luciférase reporter, la surexpression et l'abattement des miARN en culture primaire de cardiomyocytes isolées ou de lignées cellulaires appropriées; et l'imagerie par fluorescence calcique Fluo-4 AM pour évaluer le rôle potentiel des miARN sur la manipulation du Ca2+. Pour les expériences de manipulation des miARN, les cellules ont été transfectées avec 1) un miARN non codant (miR-NC, groupe témoin), 2) un miARN mimétique et 3) un inhibiteur du miARN (AMO). La signification statistique est calculée avec le test t de Student ou l'analyse unidirectionnelle de variance (ANOVA) suivie par le test de Tukey à comparaisons multiples en utilisant le logiciel GraphPad Prism version 6.00. Résultats: Nos données indiquent une augmentation du niveau de la protéine IP3R1 sans changement apparent de l'expression du gène ITPR1 dans les cardiomyocytes de l'oreillette gauche par rapport à notre modèle canin de FA. Sur la base de l'analyse informatique, il a été prédit que miR-26a ciblerait l'ARNm de l'ITPR1. La FA a considérablement réduit la régulation du miR-26a dans les cardiomyocytes de l'oreillette gauche. Le dosage de la double luciférase reporté dans les cellules H9C2 a montré que le miR-26a agissait directement sur la région non traduite 3′ (3′UTR) de l'ARNm ITPR1. De plus, la surexpression de miR-26a a réduit le niveau de la protéine IP3R1 et a diminué le taux diastolique [Ca2+] dans le noyau et le cytosol des cardiomyocytes de chien, des transistors de Ca2+ stimulés électriquement; tandis que le knockdown de miR-26a a inversé ces effets. L'expression de l'ARNm de l'ITPR1 est restée inchangée dans les cardiomyocytes de chien isolées après la transfection avec l'imitateur et l'inhibiteur de l'ARNm. Conclusion: La régulation à la hausse d'IP3R1 dans la FA est due à l'inhibition de la traduction par le miR-26a, qui est régulé à la baisse dans les cardiomyocytes auriculaires du modèle canin de FA. Ce changement est associé à une altération de la manipulation du Ca2+, qui se traduit par une augmentation des taux de Ca2+ diastolique nucléaire. Nos résultats suggèrent que la régulation à la baisse de miR-26a augmente l'expression de l’IP3R1, contribuant au remodelage pro-arythmique dans la FA.
Background: The pathophysiology of atrial fibrillation (AF) has been characterized by changes in the cellular concentration of Ca2+ and related processes leading to the initiation and maintenance of the condition. Inositol trisphosphate-receptors (IP3Rs) are ligand-gated calcium channels for which overexpression in AF has been linked to cardiac remodeling. microRNA (miR, miRNA)s, small non-coding RNAs, are around 22 nucleotides in length and regulate gene expression by mRNA destabilization or inhibition of its translation. A growing body of evidence has emerged about miRNA's role in the pathophysiology of cardiac disorders, including AF-induced adverse remodeling. Objective: Our laboratory has shown that nuclear IP3R1 level is upregulated in the dog AF model, producing increased nuclear calcium loading. Hence, this study aims to investigate the role of miRNAs in the regulation of IP3R1 initiating and/or perpetuating AF in atrial cardiomyocytes of the dog AF model. Methods: We used AF dog model established by atrial-tachypacing for 600 bpm × one week; Langendorff-perfused hearts to isolate atrial cardiomyocytes for molecular experiments; screening miRs that target ITPR1 gene, encoding IP3R1, using online databases; RT-qPCR to measure ITPR1 mRNA expression and confirm the expression level of the screened miRNAs; western blot analysis to evaluate the protein level of IP3R1; dual-luciferase reporter assay, overexpression and knockdown of miRNAs in primary culture of isolated cardiomyocytes or appropriate cell lines; and Fluo-4 AM calcium fluorescence imaging to assess the potential role of the miRNA on Ca2+ handling. For miRNA manipulation experiments, cells were transfected with 1) non-coding miRNA (miR-NC, control group), 2) miRNA mimic, and 3) inhibitor of the miRNA (AMO). Statistical significance is calculated with Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test using GraphPad Prism software version 6.00. Results: Our data indicated a rise in IP3R1 protein level with no apparent change in ITPR1 gene expression in left atrial cardiomyocytes from our dog AF model. Based on the computational analysis, miR-26a was predicted to target the ITPR1 mRNA. AF significantly downregulated miR-26a in left atrial cardiomyocytes. The dual-luciferase reporter assay in H9C2 cells showed that miR-26a directly acted on the 3′ untranslated region (3′UTR) of ITPR1 mRNA. In addition, miR-26a overexpression reduced the IP3R1 protein level and decreased the diastolic [Ca2+] in both nucleus and cytosol of the electrically-stimulated Ca2+ -transients, dog cardiomyocytes, while miR-26a knockdown reversed these effects. ITPR1 mRNA expression remained unaltered in isolated dog cardiomyocytes after transfection with the miRNA mimic and inhibitor. Conclusion: IP3R1 upregulation in AF is due to translation inhibition by miR-26a, which is downregulated in the atrial cardiomyocytes of the dog AF model. This change is associated with altered Ca2+ handling, reflected as enhanced nuclear diastolic Ca2+ levels. Our results suggest that miR-26a downregulation enhances the IP3R1 expression, contributing to pro-arrhythmic remodeling in AF.

碩士
國立臺灣大學
商學研究所
101
This thesis examined the relationships between corporation patent assets and four patent strategies - patent infringement lawsuit, patent unilateral licensing, patent cross license and patent purchase - in the North America region. Utilizing 4-digit IPC code of the patent is to extract the core technology areas of the smartphone firm. The comparison of the patent counts with average patent citations in the both smartphone firms’ core technology fields can determine which smartphone firm has competitive advantage in these core technology areas. The results showed that smartphone firms with abundant patent asset in quantity and quality usually take patent infringement lawsuit strategy to block rivals outside the market and to obtain more market share. On the contrary, smartphone firms with scarce patent asset usually need to acquire external patents for the purpose of protecting their products. Patent unilateral licensing strategy, in the case of smartphone firms with huge R&D investments, plays a significant role in activating intangible intellectual asset, since smartphone firms can earn loyalty via unilateral patent license activity. On the other hand, in cases where one of any two smartphone firms possesses predominant patents, whatever in quantity or quality, in the other’s core technology fields, the thesis reveals empirical outcome that it would take patent cross license strategy so as to obtain a win-win situation.

It is widely accepted that activating mutations of genes encoding the Notch family of transmembrane receptors, specifically Notch1, are associated with oncogenic transformation. Previous data from our lab has shown that an active form of Notch1 (NICD) provides protection against apoptosis in D011.10 T cells; and that this effect may be attributed to NICD binding the pro-apoptotic protein Nur77. Nur77 is an immediate early gene that is upregulated during both negative selection of thymocytes and activation-induced apoptosis in D011.10 T cells. Nur77 upregulation is tightly regulated and requires MEF2D, NFAT, and the transcriptional co-activator, p300, to effectively respond to apoptotic stimuli. Here, we show that NICD has the ability to interfere with the transcription of Nur77, and that this interference is directly related to the inability of p300 to bind the Nur77 promoter in the presence of NICD. We also show that blocking Notch activation, through inhibition of gamma secretase or shRNA directed against Notch1, in T cell acute lymphoblastic leukemia (T-ALL) cell lines restores Nur77 expression in response to apoptotic stimuli. These observations support a mechanism by which NICD over-expression can suppress the activation of a known pro-apoptotic molecule, and further suggests this mechanism may operate in T-ALL.

碩士
東吳大學
法律學系
106
According to the survey, for the time being these enterprises obtain needed capital mainly from government or guaranteed by the Small and Medium Enterprise Credit Guarantee Fund of Taiwan. They can hardly make it through project finance or syndication loan. As a result, these knowledge-intensive enterprises are unable to take advantage of their Intellectual Property Right to acquire the loan to reinvest and thrive and go to international market. Based on the analysis of the theory of intangible assets value and related evaluation theory, this paper combines the development status of foreign intellectual property rights financing business, and illustrates the research framework of the scope of intellectual property rights and the value evaluation of intellectual property rights. Firstly, this paper analyzes the background and significance of the research on the value of intellectual property financing, and introduces the status quo of foreign related research.Secondly, it expounds the relevant theories of intellectual property financing research; then, analyzes the special value of the value evaluation of intellectual property rights financing; Finally, in order to realize the purpose of intellectual property financing, this paper proposes and expounds the foreign value realization system for intellectual property rights financing, and hopes to provide positive reference for China's intellectual property rights financing business.

Tese de doutoramento em Ciências da Saúde
Os astrócitos desempenham múltiplas funções desde a homeostasia cerebral ao controlo e processamento da atividade sináptica. Eles integram sinais neuronais por elevações complexas de cálcio (Ca2+) com impacto na comunicação neurónio-astrócito. As elevações de Ca2+ nos astrócitos podem ser divididas em dois tipos: globais (presentes no soma e principais processos) e/ou focais (presentes nos microdomínios). Apesar de estar descrito que as elevações globais de Ca2+ nos astrócitos podem modular a comunicação sináptica, continuam por esclarecer quais os mecanismos moleculares envolvidos. Desta forma, urge uma caracterização comportamental, estrutural e molecular detalhada para compreender esses mecanismos. Nesta tese, utilizámos o modelo de murganho que apresenta a deleção constitutiva do receptor 2 do inositol 1,4,5-trifosfato (IP3R2 KO), no qual as elevações globais de Ca2+ nos astrócitos estão ausentes. Na primeira parte deste trabalho (Capítulo 2) demonstramos que os murganhos IP3R2 KO têm um desenvolvimento somático e neurológico normal. Em seguida, a caracterização comportamental deste modelo transgénico (Capítulo 3) revelou que os murganhos IP3R2 KO apresentam uma melhoria do desempenho cognitivo em tarefas dependentes do hipocampo. Identificámos o factor de transcrição Foxo1 como modulador da expressão de genes específicos de astrócitos, responsáveis pela regulação do citoesqueleto e de espinhas dendríticas. A sobre-expressão do FOXO1 em astrócitos do hipocampo de murganho C57BL/6J foi suficiente para mimetizar a melhoria cognitiva verificada no modelo IP3R2 KO. Este resultado levou-nos a avaliar o papel da sinalização global de Ca2+ no contexto da depressão, uma doença que afeta comportamento dependente das regiões cortico-límbicas (Capítulo 4). Os murganhos IP3R2 KO apresentam uma surpreendente resiliência ao efeito ansiogénico do stress crónico. Por fim, explorámos o papel da sinalização de Ca2+ nos astrócitos no envelhecimento cognitivo (Capítulo 5). Os nossos resultados demonstram uma preservação do desempenho cognitivo em murganhos IP3R2 KO envelhecidos, caracterizado por alteração do rácio neurónio/astrócito e por refinamento dendrítico dos neurónios da camada V do córtex pré-frontal. Em suma, este trabalho contribuiu para uma melhor compreensão do papel da sinalização global de Ca2+ nos astrócitos desde o desenvolvimento até ao envelhecimento, num contexto de saúde e doença. Os resultados revelaram um alvo terapêutico específico em astrócitos com potencial aplicação em contextos de depressão e envelhecimento cognitivo.
Astrocytes are responsible for distinct functions ranging from brain homeostasis to the modulation of synaptic functioning. They integrate neuronal signals by complex calcium (Ca2+) elevations that control intracellular mechanisms that in turn drive the neuron-astrocyte dialogue, modulating the activity of cells and networks. It is now recognized that Ca2+ elevations in astrocytes appear spatially distributed in global (soma and main processes) and/or focal regions (microdomains). Although it is observed that global astrocytic Ca2+ signaling contributes to synaptic communication, its role in circuit computation and behavioral performance is still poorly understood. A detailed behavioral, structural and molecular characterization should provide us with putative mechanisms underlying the roles of astrocytic Ca2+. In this thesis, we took advantage of the inositol 1,4,5-trisphosphate receptor type 2 knockout (IP3R2 KO) mouse model, which lacks global Ca2+ signaling in astrocytes. In the first part of this work (Chapter 2), we demonstrate that IP3R2 KO mice retain a normal developmental maturation, as compared with WT littermates. Next, a detailed behavioral characterization of this mouse model (Chapter 3) showed that IP3R2 KO mice display enhanced cognitive performance in hippocampal-dependent tasks. We found Foxo1 as the most active transcription factor controlling the increased expression of astrocyte-specific genes related with fine cytoskeleton modulation and spinogenesis, which could underlie the cognitive enhancement observed. Moreover, specific overexpression of FOXO1 in hippocampal astrocytes of C57BL/6J mice was enough to recapitulate the enhanced fear memory observed in IP3R2 KO mice. This striking observation prompted us to test the role of global Ca2+ signaling in the context of depression, which affects cortico-limbic regions (Chapter 4). IP3R2 KO mice present an unexpected resilience to the installation of stress effects, namely translated into an increased self-care and a reduced anxious-like phenotype. Finally, we explored the role of astrocytic Ca2+ signaling in cortico-limbic performance in aged mice that display cognitive decline (Chapter 5). We observed a preserved cognitive performance in aged IP3R2 KO mice, an altered neuron/astrocyte ratio and a dendritic refinement of mPFC neurons. Overall, this work contributed to a better understanding on the role of global astrocytic Ca2+ signaling from development to aging, both in a health and disease context. We found a putative astrocyte-specific therapeutic target that could be used to prevent depression- and aging-related deficits.
The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), at the School of Medicine, University of Minho. Financial support was provided by a PhD grant (SFRH/BD/101298/2014 to SGG), FCT Investigator grants (IF/00328/2015 to JO, IF/01079/2014 to LP) and PTDC/MED-NEU/31417/2017 from the FCT – Foundation for Science and Technology, by BIAL Foundation grants (207/14 to JO and 427/14 to LP), by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013); FEDER Funds, through the Competitiveness Factors Operational Programme (COMPETE), and The National Fund, through the FCT (POCI-01-0145-FEDER-007038).

La réaction acrosomique de Mammifère nécessite l'ouverture successive de trois canaux calciques : un canal calcique activé par de faibles dépolarisations (LVA), le récepteur à l'IP3 et un canal activé par la vidange des stocks (SOC) TRPC2. Nos données montrent une intéressante interaction fonctionnelle entre le canal LVA et les protéines dont l'activité est liée au niveau de remplissage du réticulum (vraisemblablement les canaux TRPCs et l'IP3R). Toute la signalisation calcique de la RA est sous le contrôle du canal LVA qui est le premier à s'activer. En utilisant les souris déficientes pour Cav3.1, Cav3.2, nous avons montré que la sous-unité α1H est la sous-unité majoritaire dans les cellules spermatogéniques sauvages. Nos données fournissent de nouvelles hypothèses concernant l'activation de TRPC2 : celle-ci pourrait être due à des modifications de son interaction avec la junctate et l'IP3R, induite par la vidange des stocks.