O consumo mundial de cocaína vem crescendo e no Brasil já são estimados mais de 2 milhões de usuários, destes 370 mil usam regularmente o crack. A cocaína, em suas diversas formas, é um psicoestimulante com alto potencial de abuso e a forma fumada causa à seus usuários mais complicações de saúde do que as demais formas. Muitas dessas complicações estão relacionadas às funções cognitivas, como comprometimento da atenção e memória. O usuário de crack, no ato de fumar, está sujeito tanto à ação da cocaína volatilizada quanto a dos seus produtos de pirólise, principalmente da anidroecgnonina metil éster (AEME). Considerando que pouco se conhece a respeito da AEME, ou de sua associação com cocaína, que os distúrbios cognitivos podem estar relacionados à morte neuronal e que o hipocampo é uma das principais estruturas encefálicas relacionada com cognição e memória, este trabalho visou investigar as vias de ativação de morte celular decorrente das exposições à 1 mM de AEME, 2 mM de cocaína, bem como da associação de ambas (C + A), por 3, 6 e 12 h. Para tanto, utilizamos neurônios hipocampais de embriões de rato no 18º dia embrionário (E18) que foram mantidos em cultura por até 7 dias (DIV7), quando foram feitas as exposições. Nossos resultados mostraram que em 3 h a cocaína e a AEME promoveram aumento de atividade enzimática (pelo teste de MTT) que se reverteu ao longo de 12 h. Além disso, AEME aumentou na permeabilidade da membrana plasmática em 6 h que se manteve em 12 h. Embora essas alterações tenham ocorrido em 3 h e 6 h, caspase-8 se ativou apenas em 12 h, ativando também a sinalização apoptótica com a externalização de FS. A cocaína ativou o processo autofágico a partir de 3 h aumentando a quantificação de LC3 II, mas apresentou redução de células com vesículas ácidas em 6 h e 12 h, sugerindo que esta promova morte neuronal por causar falha no fluxo autofágico. A AEME apresentou somente aumento de células com vesículas ácidas em 3 h, revertendo-se já em 6 h, indicando que o processo autofágico só se fez presente no primeiro horário, dando vez à programação de apoptose celular, por ativação da via extrínseca. A associação dessas substâncias apresentou-se mais neurotóxica do que as substâncias isoladas, com redução de células íntegras a partir de 3 h de exposição, ativação de caspase-8 e externalização de FS em 6 h, sem envolver o sistema autofágico. Além disso, as características morfológicas observadas em 6 h, como o aumento do tamanho do núcleo e do corpo celular que se tornaram picnóticos em 12 h, podem sugerir que a neurotoxicidade induzida por C + A seja por necroptose, onde a ativação de caspases resulta em um processo tipo necrótico. Assim, concluímos que, embora a literatura mostre morte neuronal por apoptose a partir de 24 h de exposição para cocaína e para AEME, as respostas celulares que levam à este fim iniciam-se já em 12 h, por ativação da via extrínseca e a associação destas substâncias é ainda mais neurotóxica, iniciando a sinalização de morte já em 6 h e induzindo uma morfologia tipo necrótica. / Cocaine market is increasing all around the world. In Brazil it is estimated that almost 2 million people make usage of this substance which 370 thousand people use the crack form. Cocaine is a psychostimulant with large potential for abuse and the smokable form produces more health problems than the other routes of use, mainly in the cognitive field related to compromising attention, memory and decision take. The crack users are exposed to both volatized cocaine and their pyrolysis products, which the main product is the anhydroecgonine methyl ester (AEME). Considering that the cognitive disturbs could be related to neurons death, the memory functions are also related to the hippocampal functions, and little is known about the AEME neurotoxicity or even the combination of cocaine and AEME in cell fate, our study aims to characterize the time and pathways related to the hippocampal neurotoxicity induced by 2 mM of cocaine, 1 mM of AEME and the association (C + A) of both substances during 3 h, 6 h and 12 h of exposure. Our results showed that cocaine and AEME increased enzymatic activity (MTT test) in 3 h but it reversed during 12 h of exposure. Moreover, AEME increased cell permeability in 6 h keeping it until 12 h. Although theses early alterations, both substances activated caspase -8 after 12 h when early apoptosis was also observed by the FS externalization. Cocaine activated the autophagic process at 3 h increasing the LC3 II quantification, but decreased the number of cell with acid vesicle at 6 h and 12 h, suggesting neuronal death due to failure in the autophagic flux. AEME showed increased in cell number with acid vesicle only in 3 h which returned after 6 h suggesting that the autophagic process gave place to the apoptotic program starting from the extrinsic pathway. The association of cocaine and AEME was shown more neurotoxic than them alone, decreasing the number of integral cells after 3 h, activating caspase -8 and promoting FS externalization after 6 h without involving the autophagy. In addition, taking the C + A morphology in 6 h, where it was observed increasing of nucleus and soma size that became pyknotic at 12 h, we suggest that the neuronal death could occur by necroptosis because this composition activated caspase -8 and resulted in necrotic like morphology. Thus, we conclude that cocaine- and AEME-induced apoptosis neuronal death starts in 12 h of exposure by the extrinsic pathway and the association of both substances is more neurotoxic than they alone, starting earlier after 6 h and resulting in a necrotic-like morphology.

Considerando que várias mutações gênicas estão envolvidas no estabelecimento dos tumores, surge a idéia de que o alcance da melhor eficiência no tratamento do câncer é dado pela entrega de múltiplos genes. Este trabalho apresenta a construção, produção e caracterização funcional in vitro e in vivo do vetor adenoviral bicistrônico Adp16IRESp53 e dos monocistrônicos Adp16 e Adp53 em modelo de câncer de pulmão. Nossos resultados indicam uma forte indução de morte celular nas células H358 transduzidas com Adp16IRESp53 em comparação com vetores monocistrônicos Adp16, Adp53 ou o reporter AdeGFP e/ou AdLacZ. Nos ensaios in vivo, utilizando modelo xenografico onde as células H358 foram implantadas no subcutâneo de camundongos atímicos Balb/C nude, pudemos confirmar também in vivo a significativa inibição do crescimentos dos tumores tratados com Adp16IRESp53. Em conclusão, a remediação simultânea de p16INK4a e p53, mediada pelo arranjo bicistrônico, pode ser considerada como uma estratégia promissora para terapia gênica do câncer de pulmão. / This work presents the construction, production and functional evaluation in vitro and in vivo of the bicistronic adenoviral vector Ap16IRESp53 as well as the monocistronic vectors Adp16 and Adp53 in a lung cancer model. Considering that several mutation events are involved in tumorigenesis, comes the idea that a greater efficiency in cancer treatment would be reached with delivery of multiples genes. Our data demonstrate a strong cell death effect in H358 cells transduced with Adp16IRESp53 when compared with Adp16, Adp53 or the reporter AdeGFP and/or AdLacZ. For the in vivo studies, we have used H358 cells implanted subcutaneously in athymic Balb/c nude mice. Our data show significant suppression of tumors treated with the therapeutic adenoviral vector, Adp16IRESp53. In conclusion, the simultaneous replacement of p16INK4a and p53, mediated by the bicistronic vector, may prove to be a promising strategy for gene therapy of lung cancer.

Cellular senescence is a distinct cell state, characteristic by cessation of cell proliferation and it is accompanied by specific morphological and biochemical alterations. Increasing and persisting incidence of senescence cells has been shown to have detrimental effect on an organism largely contributing to its ageing. Senescent cells also positively support tumour growth and can even stimulate carcinogenic transformation of surrounding cells. Moreover, senescence can be induced even in tumour cells spontaneously or by chemotherapy. Regardless of an initial stimuli and type of cells, there are two main senescence inducing pathways p16/pRb and p53/p21. Both senescent cells as well as senescent cancer cells seems to have modified apoptotic signalling at the level of mitochondria and Bcl-2 family proteins. In this study, we aimed to analyse effect of senescent state as well as pre-senescent (growth arrested state) induced by p16/pRb and p53/p21 signalling pathways on the response of H28 mesothelioma cancer cells-derived clonal cultures to various cell death-inducing stimuli. By inducible expression of p16 and p21 proteins in doxycycline-dependent manner, we forced cells to acquire senescent-like phenotype, which we detailly characterised. Our results showed that senescent-like phenotype, manifests...

Abstract available through The FASEB Journal.

Phakopsora pachyrhizi Syd.& P.Syd. est le plus important fléau du soja (Glycine max (L.) Merrill). Introduit au Brésil dans les années 2000, ce champignon s’est rapidement répandu sur les deux continents Américains. Seule l’utilisation de fongicides associée à des pratiques culturales strictes permet de maintenir le niveau de production. L’utilisation répétitive de ces fongicides ainsi que la plasticité génétique de ce champignon ont rapidement entraîné une diminution d’efficacité de certaines molécules. Par ailleurs, la plupart des résistances verticales identifiées dans les ressources naturelles du soja restent inefficaces contre certains isolats du champignon. La compréhension des mécanismes de l’immunité des plantes permet de proposer des solutions biotechnologiques pour le contrôle des maladies. L’utilisation antérieure du système barnase/barstar induisant une mort cellulaire artificielle, a permis de générer des pommes de terre résistantes à Phytophtora infestans. Cette technologie est basée sur l’expression de la barnase une ribonucléase toxique pour les cellules, et la barstar un inhibiteur de la barnase. Il a été proposé d’évaluer ce système pour le contrôle de P. pachyrhizi. Le point critique de cette approche est de trouver le bon rapport de l’expression des gènes barnase/barstar. Pour ce faire la barnase sera placée sous le contrôle d’un promoteur induit par le pathogène, permettant une régulation spatiotemporelle. La recherche de tels promoteurs a été effectuée en utilisant des données transcriptomiques et bibliographiques. Des sojas stables exprimant les différentes fusions promoteur:GFP ont été créées afin d’étudier l’ expression spatiotemporelle de ces promoteurs en présence du champignon. Les promoteurs pGmCHIT1 (de G. max) et pgst1 (de Solanum tuberosum) contrôlant respectivement l’expression d’une chitinase et d’une glutathione-S-transférase ont été identifiés comme induits par le pathogène. L’impact de différents stress sur ces deux promoteurs a été évalué. Les constructions génétiques « barnase/barstar » comprenant les différentes combinaisons des promoteurs ont été générées. Nicotiana benthamiana a été utilisé pour exprimer transitoirement les construits et évaluer leur phytotoxicité en absence du pathogène. Un seul construit contenant le promoteur gst1 s’est avéré non phytotoxique. Il a été transféré avec succès dans le soja. Ces sojas n’ont pas montré de gain de tolérance à la rouille. Une proposition d’amélioration du système barnase/barstar est discutée afin de mieux cerner les possibilités et les limites de ce système pour le contrôle de la rouille du soja / Phakopsora pachyrhizi Syd.& P.Syd, the fungus responsible for Asian soybean rust, is the most devastating soybean (Glycine max (L.) Merrill) pathogen. First observed in the 2000s in Latin America, the pathogen has spread throughout the Americas. The control of this pathogen depends on the use of fungicides and strict agricultural practices. The repetitive use of the 3 classes of fungicides and the genome plasticity of the pathogen have led to a decreased efficacy of certain molecules. Although vertical resistance genes have been mapped in the soybean germplasm, most of them are not effective against all Asian soybean rust isolates. A deeper understanding of plant immunity facilitates the development of biotechnological approaches for plant disease control. Artificial cell death was previously developed to control Phytophthora infestans development in potato. The technology was based on a barnase ribonuclease that is highly toxic to the plant cell and that consequently needed to be expressed only in the presence of the pathogen. The lethal expression of barnase was counterbalanced by barstar, a highly specific inhibitor of barnase. We propose to evaluate this technology in soybean to control P. pachyrhizi. The key objective is the modulation of the ratio of barnase/barstar based on the identification of an adequate inducible promoter to control the expression of barnase. The previous literature and transcriptomic data were used to identify candidate promoters for barnase expression. Stable transgenic soybean expressing the different promoter:GFP fusions were generated to test the spatiotemporal activity of the promoters in the presence of the pathogen. pGmCHIT1 (from G. max) and pgst1 (from Solanum tuberosum) promoters controlling a chitinase and a glutathione-Stransferase, respectively, were identified as induced by soybean rust. The impacts of different stresses on these promoters were evaluated. Molecular constructs with different promoters driving the barnase and barstar gene combination were generated. Nicotiana benthamiana was used to evaluate construct toxicity in the absence of the pathogen. One single construct containing the promoter pgst1 was shown to be non-phytotoxic. This construct was successfully introduced in soybean plants. The generated soybeans were challenged with rust, but no protection was observed. Based on these results, we discuss how to improve the barnase/barstar system to control soybean rust

<p>Apoptosis is a genetically regulated cell death program, which shows distinct morphological characteristics. It takes place during neuronal development and in some neurodegenerative diseases. During apoptosis, the intracellular proteins are degraded by various caspases, cysteine aspartases, which are regulated by pro- and anti-apoptotic signals. This thesis elucidates the role of anti-apoptotic proteins in nerve cell survival and neurodegeneration. Studies have focused on Bcl-2 family members and Inhibitor of Apoptosis Proteins (IAP).</p><p>XIAP and RIAP-2 are IAP proteins, which are expressed by neurons in the central nervous system. Kainic acid, a glutamate receptor agonist that induces seizures, increased XIAP immunoreactivity in rat hippocampus, whereas RIAP-2 expression in the same time decreased in degenerating neurons. Both XIAP and RIAP-2 were absent in dying neurons indicating that these proteins have a protective role in kainic acid induced neurodegeneration.</p><p>NAIP, another IAP family member, was shown to interact with the calcium binding protein Hippocalcin using the yeast two-hybrid system and immunoprecipitation experiments. Hippocalcin-NAIP interaction increased motoneuron survival in caspase-3 independent and dependent manners.</p><p>The anti-apoptotic Bcl-2 proteins, Bcl-2 and Bcl-x, were studied using cultured neurons and human neuronal progenitor cells. In the progenitor cells, Bcl-2 overexpression enhanced cell survival and induced downregulation of Caspase-2 (ICH-1) and caspase-3 (YAMA/CPP32). These results suggest a novel mechanism for the action of Bcl-2.</p><p>Estrogen was shown to inhibit death of cultured dorsal root ganglion neurons (DRG) after nerve growth factor withdrawal. The hormone increased the levels of Bcl-x, which may explain the known neuroprotective function of estrogen.</p>

<p>Apoptosis or programmed cell death is crucial for the resolution of inflammation, and phagocytosis of apoptotic cells initiates the release of actively anti-inflammatory responses from the phagocytes. Eosinophils are one of the most potent inflammatory cells in the body and is involved in a number of diseases, most commonly associated with parasitic infections and allergic diseases. Apoptosis in eosinophils is therefore one of the most important systems to avoid inflammation. This aim of the present investigation was to examine the mechanisms behind, and the consequences of this process in eosinophils. Apoptotic eosinophils have a unique surface receptor expression that indicates abilities to communicate with T-, B- and antigen presenting cells. They have a novel expression of CD49f, indicating an importance for binding to laminin or unknown functions of the VLA-6 receptor, possibly in the concept of phagocytosis of the apoptotic cell. </p><p>In apoptotic eosinophils the granules are translocated to the periphery of the cell, probably through a disruption of the cytoskeleton. This translocation makes the granules easily accessible and the apoptotic eosinophil can release considerable amounts of granule proteins in response to specific stimuli. The spontaneous release however, is decreased as compared with living cells. </p><p>Furthermore, the survival of eosinophils in response to an allergen challenge is increased in healthy subjects, but not in allergic patients. Mechanistically, this needs further investigation, but one theory is that it is due to the presence of specific IgE in patients in combination with differences in the response from the epithelial cells.</p>

Apoptosis is a genetically regulated cell death program, which shows distinct morphological characteristics. It takes place during neuronal development and in some neurodegenerative diseases. During apoptosis, the intracellular proteins are degraded by various caspases, cysteine aspartases, which are regulated by pro- and anti-apoptotic signals. This thesis elucidates the role of anti-apoptotic proteins in nerve cell survival and neurodegeneration. Studies have focused on Bcl-2 family members and Inhibitor of Apoptosis Proteins (IAP). XIAP and RIAP-2 are IAP proteins, which are expressed by neurons in the central nervous system. Kainic acid, a glutamate receptor agonist that induces seizures, increased XIAP immunoreactivity in rat hippocampus, whereas RIAP-2 expression in the same time decreased in degenerating neurons. Both XIAP and RIAP-2 were absent in dying neurons indicating that these proteins have a protective role in kainic acid induced neurodegeneration. NAIP, another IAP family member, was shown to interact with the calcium binding protein Hippocalcin using the yeast two-hybrid system and immunoprecipitation experiments. Hippocalcin-NAIP interaction increased motoneuron survival in caspase-3 independent and dependent manners. The anti-apoptotic Bcl-2 proteins, Bcl-2 and Bcl-x, were studied using cultured neurons and human neuronal progenitor cells. In the progenitor cells, Bcl-2 overexpression enhanced cell survival and induced downregulation of Caspase-2 (ICH-1) and caspase-3 (YAMA/CPP32). These results suggest a novel mechanism for the action of Bcl-2. Estrogen was shown to inhibit death of cultured dorsal root ganglion neurons (DRG) after nerve growth factor withdrawal. The hormone increased the levels of Bcl-x, which may explain the known neuroprotective function of estrogen.

Apoptosis or programmed cell death is crucial for the resolution of inflammation, and phagocytosis of apoptotic cells initiates the release of actively anti-inflammatory responses from the phagocytes. Eosinophils are one of the most potent inflammatory cells in the body and is involved in a number of diseases, most commonly associated with parasitic infections and allergic diseases. Apoptosis in eosinophils is therefore one of the most important systems to avoid inflammation. This aim of the present investigation was to examine the mechanisms behind, and the consequences of this process in eosinophils. Apoptotic eosinophils have a unique surface receptor expression that indicates abilities to communicate with T-, B- and antigen presenting cells. They have a novel expression of CD49f, indicating an importance for binding to laminin or unknown functions of the VLA-6 receptor, possibly in the concept of phagocytosis of the apoptotic cell. In apoptotic eosinophils the granules are translocated to the periphery of the cell, probably through a disruption of the cytoskeleton. This translocation makes the granules easily accessible and the apoptotic eosinophil can release considerable amounts of granule proteins in response to specific stimuli. The spontaneous release however, is decreased as compared with living cells. Furthermore, the survival of eosinophils in response to an allergen challenge is increased in healthy subjects, but not in allergic patients. Mechanistically, this needs further investigation, but one theory is that it is due to the presence of specific IgE in patients in combination with differences in the response from the epithelial cells.

The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.