京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21415号 / 医博第4405号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 坂井 義治, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O linfoma difuso de grandes cÃlulas B (LDGCB) corresponde a 50 % dos casos de linfoma nÃo-Hodgkin (LNH). Seu tratamento de escolha à a quimioterapia de associaÃÃo, em especial o esquema CHOP (ciclofosfamida, doxorrubicina, vincristina e prednisona), considerado o tratamento inicial padrÃo dos LDGCB. VariaÃÃes deste esquema, como o protocolo CHOP-Bleo (ciclofosfamida, doxorrubicina, vincristina, prednisona e bleomicina) tem sido utilizadas com a intenÃÃo de se obter maiores taxas de remissÃo completa pelos pacientes. No Brasil, pouco se conhece a respeito da incidÃncia, do comportamento clÃnico, da resposta Ãs terapÃuticas utilizadas e da sobrevida de pacientes com LDGCB. Este estudo teve como objetivos traÃar o perfil epidemiolÃgico dos pacientes portadores de linfoma difuso de grandes cÃlulas B, atendidos no Hospital UniversitÃrio Walter CantÃdio (HUWC), com data de primeiro atendimento de janeiro de 1989 a dezembro de 2003, e que fizeram uso dos esquemas quimioterÃpicos CHOP e/ou CHOP-Bleo; avaliar a eficÃcia e seguranÃa terapÃutica dos esquemas propostos atravÃs da anÃlise do tipo de resposta terapÃutica, achados clÃnicos e laboratoriais destes pacientes. A coleta dos dados foi realizada a partir dos prontuÃrios mÃdicos dos 31 pacientes analisados. Destes, 21 (67,74%) eram do sexo masculino e 10 (32,26%) do feminino, com idade mÃdia de 45,81  16,3 anos. A ocupaÃÃo trabalhador agrÃcola representou 25,82% (8/31). O estÃdio III da classificaÃÃo de Ann Arbor foi o mais freqÃente (32,26%), mas apenas 45% dos pacientes apresentaram sintomas B. A lactato desidrogenase (LDH) sÃrica de 49% dos pacientes encontrava-se elevada à Ãpoca do diagnÃstico, sendo que outros 16% dos pacientes nÃo apresentavam resultado desta enzima em seus prontuÃrios. Quanto ao IPI, 71% foram classificados como de risco baixo e intermediÃrio, 13% de alto risco intermediÃrio, nenhum dos pacientes do estudo apresentou IPI compatÃvel com de alto risco e em 16% dos pacientes nÃo foi possÃvel estabelecer a classificaÃÃo devido à ausÃncia de dados nos prontuÃrios. Quanto à utilizaÃÃo dos protocolos quimioterÃpicos, 58% (18/31) dos pacientes fizeram uso do esquema CHOP, 36% (11/31) utilizaram CHOP-Bleo e 6% (2/31) utilizaram os dois esquemas quimioterÃpicos. Entre os pacientes que utilizaram o esquema CHOP, 78% atingiram a remissÃo completa (RC), 17% apresentaram recidiva da doenÃa e apenas 5 % foram a Ãbito. No grupo que utilizou o esquema CHOP-Bleo, 63% atingiram a RC, 18% apresentaram recidiva da doenÃa e 19% foram a Ãbito. Os 2 pacientes que utilizaram os dois esquemas como tratamento apresentaram recidiva da doenÃa. Os valores de LDH dos pacientes apÃs a quimioterapia apresentam-se reduzidos tanto em pacientes que atingiram a remissÃo completa como naqueles que tiveram recidiva. Verificamos que a sobrevida global (SG) e a sobrevida livre de doenÃa (SLD) nÃo foram influenciadas pelo estÃdio clÃnico e LDH inicial dos pacientes. A regressÃo logÃstica nÃo mostrou significÃncia estatÃstica quando analisou a remissÃo completa dos pacientes a partir dos resultados das variÃveis em estudo pÃs QT, com exceÃÃo da proporÃÃo de reduÃÃo da LDH e a resposta ao tratamento. Os resultados mostraram a eficÃcia e seguranÃa dos esquemas terapÃuticos CHOP e CHOP-Bleo em nossa populaÃÃo de estudo. Os resultados demonstram ainda que se faz necessÃrio o estudo epidemiolÃgico de diferentes populaÃÃes com LDGCB para que haja seguranÃa na escolha de esquemas quimioterÃpicos, bem como a uniformidade em descrever e classificar os linfomas e os seus fatores prognÃstico por parte dos patologistas e oncologistas. / Diffuse Large B-Cell Lymphomas (DLBCL) corresponds to 50% of non-Hodgkinâs lymphomas (LNH). Their treatment of choice is the association chemotherapy, in special the CHOP therapy (cyclophosphamide, doxorubicin, vincristine and prednisone) considered the standard treatment initial of the DLBCL. Variations of this therapy, with the CHOP-Bleo protocol (cyclophosphamide, doxorubicin, vincristine, prednisone and bleomycin) have been used with the intention of obtaining complete response rates for the patients. In Brazil, little is known about the incidence, clinical behavior, response to therapy and survival of the patients with DLBCL. This study aimed to set out the epidemiological profile of patients with diffuse large B-Cell lymphomas, who received medical care at Hospital UniversitÃrio Walter CantÃdio (HUWC), outline in Cearà state, with the first attendment from January 1989 to December 2003, who the used the CHOP and/or CHOP-Bleo therapy; Evaluating the security and efficiency of the protocols proposed by analysis of the kind of therapeutical response, clinical and laboratorial outcomes of these patients. The data collection was performed from medical recording of the 31 patients analyzed. These, 21 (67,74%) were the men and 10 (32,26%) women. The average age was 45,81  16,3 anos. Agriculturists represented 25,82% (8/31) of all patients. The stage III the Ann Arbor classification were the most frequent (32,26%), but only 45% of the patients had B symptoms. The values of lactate dehydrogenises (LDH) enzyme were elevated in 49% of the patients at diagnosis, but in 16% of the patients these values at diagnosis were unknown. As much as the IPI, 71% were classified as an IPI low and intermediate risk, 13% as an IPI intermediate-high risk, none of the study patients showed as an IPI high risk and 16% there is not possible the classification to establish due to the data is unknown. As much as the chemotherapy protocols used, 58% (18/31) of the patients was received CHOP chemotherapy, 36% (11/31) CHOP-Bleo chemotherapy and 6% (2/31) received CHOP associated with CHOP-Bleo chemotherapy. Among the patients who used CHOP chemotherapy, 78% was achieving complete response (CR), 17% was achieving relapse of the disease and only 5% were the death. In the group who used CHOP-Bleo chemotherapy, 63% was achieving RC, 18% was achieving relapse of the disease and 19% died. The 2 patients who used CHOP and CHOP-Bleo chemotherapy were achieving relapse of the disease. The values of the LDH after chemotherapy showed decreased in patients with RC as much as the relapsed patients. We verified that the overall survival (OS) and disease-free survival (DFS) were not influenced by the clinic stage and initial values of the LDH patients. The logistic regression did not show statistical differences when the complete response was analyzed comparing to outcomes the studied variables after QT, except for the proportion of reduction of LDH levels and response to the treatment. The results stress the security and efficiency of the protocols CHOP e CHOP-Bleo in our study population. The data obtained also the need epidemiological studies in different DLBCL populations for the security in the choice chemotherapy, well as standardized the classification and description of the DLBCL and prognoses factures by pathologists and oncologists.

Indiana University-Purdue University Indianapolis (IUPUI) / The protein kinase General control non-derepressible-2 (GCN2) is a key regulator of the Integrated stress response that responds to various stress signals, including nutritional deprivation. As a result of high levels of uncharged tRNAs during amino acid depletion, GCN2 phosphorylates serine-51 of the α subunit of eukaryotic initiation factor-2 (eIF2), a translation factor that delivers initiator tRNA to ribosomes. Phosphorylation of eIF2α inhibits general translation, which conserves energy and nutrients and facilitates reprogramming of gene expression for remediation of stress damage. Phosphorylation of eIF2α also directs preferential translation of specific transcription factors, such as ATF4. ATF4 reprograms gene expression to alleviate stress damage; however, under chronic stress, ATF4 directs the transcriptional expression of CHOP, which can trigger apoptosis. Because multiple stresses can induce eIF2α phosphorylation and translational control in mammals, this pathway is referred to as the Integrated stress response. GCN2 and CHOP are subject to alternative splicing that results in multiple transcripts that differ in the 5'-end of the gene transcripts. However, the effect of the different GCN2 and CHOP isoforms on their function and regulation have not been investigated. Our data suggests that GCN2 is alternatively spliced into five different transcripts and the beta isoform of GCN2 is most abundant. Also alternative splicing of CHOP creates two CHOP transcripts with different 5'-leaders encoding inhibitory upstream open reading frames that are critical for translational control of CHOP during stress. This study suggests that alternative splicing can play an integral role in the implementation and regulation of key factors in the Integrated stress response.

Chez les mammifères, les carences nutritionnelles telles que les carences en acides aminés constituent un stress nutritionnel important. Pour faire face à ces situations, l'organisme dispose de processus adaptatifs tels que l'autophagie, régulés par de multiples voies de signalisation. Au niveau cellulaire, plusieurs voies de signalisation sont impliquées dans la régulation de ces processus adaptatifs qui permettent la survie cellulaire lors de différentes conditions de stress environnementaux y compris la carence en acides aminés. En particulie,r la voie eIF2α/ATF4 joue un rôle crucial dans l'adaptation des cellules à ces différents stress notamment en régulant la transcription de nombreux gènes cibles spécifiques. L'objectif de ce travail était donc de déterminer le rôle de la voie eIF2α/ATF4 dans la régulation de la transcription des gènes impliqués dans l'autophagie en réponse à carence en acides aminés. En utilisant p62 comme un modèle de travail, nous avons montré que la kinase GCN2 qui phosphoryle eIF2α et les facteurs de transcription ATF4 et CHOP jouent un rôle clé dans la régulation de la transcription d'un grand nombre de gènes impliqués dans le processus autophagique en réponse à une carence en acides aminés. Nous avons en particulier identifié 3 classes de gènes de l'autophagie selon leur dépendance à ATF4 et CHOP et la liaison de ces facteurs sur les éléments spécifiques de leurs promoteurs en fonction de l'intensité du stress. PLus généralement, nous avons démontré que ce mécanisme pouvait également être activé par la kinase PERK lors d'un stress du réticulum endoplasmique. Enfin, nous avons pu montrer que durant les 6 premières heures de la carence en acides aminés, la voie eIF2α/ATF4/CHOP n'est pas impliquée dans la diminition de la viabilité cellulaire. Cependant, lorsque la carence en acides aminés est prolongée (16-48h), CHOP joue un rôle clé dans la régulation de l'apoptose et dans la répression du processus autophagique en contrôlant la transcription des gènes cibles spécifiques. Ainsi, ce travail a permis de mettre en évidence qu'en cas de carence en acides aminés, la voie eIF2α/ATF4/CHOP joue un rôle clé dans le devenir de la cellule. En fonction de la durée et de l'intensité du stress, la régulation très coordonnée de ces mécanismes moléculaires va permettre successivement la survie de la cellule et ensuite l'apoptose. / In mammals nutritional deficiencies such as amino acid limitation are an important nutritional stress. To deal with these situations, the body has adaptive processes such as autophagy regulated by multiple signaling pathways. At the cellular level, several signaling pathways are involved in the regulation of these adaptive processes that allow cell survival in different conditions of environmental stress, including amino acid deficiency. In particular, the eIF2α/ATF4 pathways plays a crucial role in the adaptation of these cells to various stresses such as the transcriptional regulation of many specific target genes. The aim of this work was to identify the role of the eIF2α/ATF4 pathway in the stress-regulated transcription of mammalian autophagy genes. Using p62 as a working model, we have shown that the GCN2 eIF2α-kinase and ATF4 and CHOP transcription factors are required to increase transcription of a set of autophagy genes implicated in the formation, elongation and function of the autophagosome. We also identify 3 classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Furthermore, we have demonstrated that the same mechanism can also be activated by ER stress through PERK eIF2α-kinase activation. We also show that during the first 6h of starvation, CHOP up-regulates a number of autophagy genes while cell viability is not affected. By contrast, when the amino acid starvation is prolonged (16-48h), we demonstrated that CHOP has a dual role in both limiting autophagy and inducing apoptosis through the transcriptional activation of specific target genes. Thus, this work establishes that following amino acid starvation, the eIF2α/ATF4 pathway plays a key role in the cell fate. Depnding on the duration and intensity of the stress, the highly coordinated regulation of these molecular mechanisms sequentially will allow the survival of the cell and subsequently apoptosis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18147号 / 医博第3867号 / 新制||医||1002(附属図書館) / 31005 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 千葉 勉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This triggers a series of signaling pathways known as the unfolded protein response (UPR). The response helps to both enhance protein folding capacity and initiate mis-folded protein degradation, reducing E.R. stress. Alternatively, misfolded proteins are degraded and nutrients are recycled through autophagy. Thus, E.R. homeostasis depends on both UPR and autophagy. However, if E.R. stress is not resolved, UPR and autophagy can also cause apoptosis by mechanisms that are not fully understood. In chicken embryo fibroblasts, gamma-aminobutyric acid receptor-associated protein or GABARAP (a protein involved in autophagy) can promote apoptosis in conditions of prolonged starvation (Maynard et al. 2015). In these conditions, the down-regulation of GABARAP by shRNA/RNA interference reduces the expression of the pro-apoptotic CHOP (CAAT-enhancer-binding protein homologous protein) transcription factor (a marker of E.R. stress) and enhances cell survival. This suggests that elevated levels of autophagy compromises E.R. homeostasis and promotes the expression of CHOP in UPR lethal pathways. While GABARAP induction and processing/activation has been linked to the expression of CHOP upon prolonged starvation (Maynard et al. 2015), nothing is known about the pathway mediating CHOP expression and the relationship with other pathways of the UPR in cells with GABARAP mis-expression. Understanding these pathways will allow us to determine if GABARAP is a general determinant of E.R. stress or acts specifically on the expression of CHOP to control cell survival. Elucidating mechanisms which are involved in E.R. stress and the cellular transition between pro-survival to pro-apoptotic roles can allow understanding of processes associated with several pathological conditions like cancer and neuro-degenerative diseases. Additionally, establishing a role for GABARAP tumor suppressor in the control of the UPR and cell fate is also important. / Thesis / Bachelor of Science (BSc) / In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This activates both the Unfolded Protein Response (UPR) and Autophagy as both processes help to reduce E.R. stress. GABARAP, a protein involved in autophagy, has been shown to be involved in the promotion of apoptosis in conditions of prolonged starvation as its downregulation reduces apoptosis and CHOP expression (Maynard et al. 2015). However, how GABARAP regulates apoptosis remains unknown. Here, we investigate if GABARAP mis-expression affects multiple pathways in the UPR relieving global E.R. stress or if its specifically involved in blocking CHOP expression.

de tumorigênese em melanomas, ainda não há tratamento eficaz para melanomas metastáticos. Esta ineficácia terapêutica pode estar relacionada com a adaptação e seleção de células de melanoma à indução de estresse de RE. Ultrapassar os níveis sustentados de estresse de RE, interferindo nas vias de adaptação a este estresse, foi o alvo deste estudo na tentativa de propor uma nova estratégia terapêutica para sensibilizar células de melanoma a morte induzida por cisplatina. Mostramos que GADD153, um dos componentes da via de UPR (Unfolded Protein Response) responsável por induzir apoptose em reposta ao estresse de RE, está excluída do núcleo em melanomas primários, metástases ganglionares e viscerais. Este dado sugere que a localização citoplasmática do fator de transcrição GADD153 possa estar envolvida na resposta adaptativa de melanomas ao estresse de RE, uma vez que se sabe que GADD153 se acumula no núcleo em resposta a este estresse. Investigamos se a indução de estresse de RE seria capaz de induzir a translocação de GADD153 para o núcleo e resultar na sensibilização de células de melanoma a morte induzida por cisplatina (CDDP). Realizamos o tratamento de células de melanoma (SbCl2, Mel85, SK-MEL- 29, SK-MEL-28 e SK-MEL-147) com tunicamicina (Tuni), indutor clássico de estresse de RE, previamente ao tratamento com CDDP. Demonstramos que em todas as linhagens exceto em SK-MEL-29, houve um aumento na porcentagem de células hipodiploides (>50%) no tratamento combinado (Tuni>CDDP) comparado ao tratamento com CDDP. As células SK-MEL-147 se mostraram mais sensíveis à indução de estresse de RE e as células SK-MEL-29 mais resistentes. Algumas diferenças entre estas linhagens como a expressão de GRP78 de superfície e presença de oligossacarídeos ?1-6 ligados de superfície podem estar relacionadas com esta resposta diferencial ao estresse de RE. Em todas as linhagens verificamos a acúmulo dos marcadores de UPR, GRP78 e GADD153, após o tratamento com tunicamicina. Além disso, GADD153 foi direcionada para o núcleo em reposta ao tratamento com tunicamicina. O acúmulo de vacúolos acídicos, da proteína autofágica LC3-II e de ROS após o tratamento com Tuni>CDDP, sugerem que tanto a autofagia quanto o estresse oxidativo parecem estar envolvidos na resposta de sensibilização. A inibição de autofagia com cloroquina aumentou a morte induzida por Tuni>CDDP, sugerindo que autofagia desempenha função protetora neste esquema terapêutico. Testamos um segundo agente genotóxico, temozolomida (TMZ), uma droga equivalente à dacarbazina, e a mesma capacidade de sensibilização foi observada pelo prévio tratamento com tunicamicina. A validação deste conceito in vivo foi dificultada pela acentuada toxicidade apresentada por tunicamicina. Avaliamos alguns candidatos a agentes estressores do RE que poderiam apresentar menor toxicidade celular, como swainsonina, atorvastatina, metformina e o composto de cobre [Cu2(apyhist)2(dpam)](ClO4)4. No entanto, não obtivemos resultados promissores com nenhum destes candidatos. Estes resultados mostram que as células tumorais podem ser pré-condicionadas à morte celular se expostas a um prévio estressor de RE, como Tuni, o que leva ao comprometimento da resposta adaptativa a indutores de morte celular como CDDP e TMZ. No entanto, ainda é necessário o estudo de agentes indutores de estresse de RE pouco tóxicos para que esta estratégia terapêutica possa ser utilizada em pacientes com melanoma / Melanoma is among the most aggressive malignancies with increasing worldwide incidence and there is no effective treatment for the metastatic disease. The absence of an effective therapy may be due to adaptation and selection of melanoma cells to endoplasmic reticulum (ER) stress. We showed that GADD153, one of the components of the ER stress-mediated apoptosis pathway, was mostly excluded from the nucleus of primary and metastatic melanoma cells compared to nevus cells. These data suggest that the unexpected GADD153 cellular localization could be involved in melanoma cell adaption to ER stress, since GADD153 accumulates in the nucleus during ER stress. Unfolded protein response (UPR) signaling induced in response to ER stress, is a dual process that induces a protective response to restore ER homeostasis or cell death if ER stress is severe or persistent. We investigated if induction of ER stress was a potential strategy to chemosensitize melanoma cells to a second insult by surpassing the adaptive levels to ER stress. We first treated human melanoma cells (SbCl2, SK-MEL-28, Mel85, SK-MEL-29 and SK-MEL-147) with tunicamycin (Tuni), an ER stress inducer, before cisplatin (CDDP) treatment. CDDP is a low cost chemotherapeutic drug currently used in Brazil as a second line for melanoma treatment, especially in youngsters. All cell lines, except SK-MEL-29, demonstrated an >50% increase in the percentage of hypodiploid cells with Tuni>CDDP treatment when compared to CDDP only. The same results were obtained with temozolomide (TMZ), equivalent drug to the active form of dacarbazine, the first line of cytotoxic treatment of melanomas. UPR markers, GRP78 and nuclear translocation of GADD153 were induced by Tuni. Differences between SK-MEL-29 and SK-MEL-147 as cell surface GRP78 and ?1-6 oligossacharides can be related with the differential ER stress sensitization observed in these cells. One of the cellular mechanisms that are regulated by ER stress is autophagy. Accordingly, we observed an increase in the acidic vesicular organelles and accumulation of LC3II in response to Tuni>CDDP treatment. Autophagy inhibition with chloroquine increased Tuni>CDDP induced-cell death, suggesting that autophagy plays a protective role in this response. Oxidative stress can be involved in this scenario since we demonstrated an accumulation of reactive oxygen species in response to Tuni>CDDP. Tunicamycin was cytotoxic in vivo and we investigated alternatives to this antibiotic as swainsonine, atorvastatin, metformin and [Cu2(apyhist)2(dpam)](ClO4)4 but we did not observed ER stress induction. These results indicate that tumor cells could be preconditioned to cell death if exposed to a first ER stressor, such as Tuni, which would compromise an effective adaptive response to a cell death inducer, as CDDP and TMZ. This combined approach may be a promising strategy for melanoma therapy but further studies are necessary to find noncytotoxic alternatives to tunicamycin

de tumorigênese em melanomas, ainda não há tratamento eficaz para melanomas metastáticos. Esta ineficácia terapêutica pode estar relacionada com a adaptação e seleção de células de melanoma à indução de estresse de RE. Ultrapassar os níveis sustentados de estresse de RE, interferindo nas vias de adaptação a este estresse, foi o alvo deste estudo na tentativa de propor uma nova estratégia terapêutica para sensibilizar células de melanoma a morte induzida por cisplatina. Mostramos que GADD153, um dos componentes da via de UPR (Unfolded Protein Response) responsável por induzir apoptose em reposta ao estresse de RE, está excluída do núcleo em melanomas primários, metástases ganglionares e viscerais. Este dado sugere que a localização citoplasmática do fator de transcrição GADD153 possa estar envolvida na resposta adaptativa de melanomas ao estresse de RE, uma vez que se sabe que GADD153 se acumula no núcleo em resposta a este estresse. Investigamos se a indução de estresse de RE seria capaz de induzir a translocação de GADD153 para o núcleo e resultar na sensibilização de células de melanoma a morte induzida por cisplatina (CDDP). Realizamos o tratamento de células de melanoma (SbCl2, Mel85, SK-MEL- 29, SK-MEL-28 e SK-MEL-147) com tunicamicina (Tuni), indutor clássico de estresse de RE, previamente ao tratamento com CDDP. Demonstramos que em todas as linhagens exceto em SK-MEL-29, houve um aumento na porcentagem de células hipodiploides (>50%) no tratamento combinado (Tuni>CDDP) comparado ao tratamento com CDDP. As células SK-MEL-147 se mostraram mais sensíveis à indução de estresse de RE e as células SK-MEL-29 mais resistentes. Algumas diferenças entre estas linhagens como a expressão de GRP78 de superfície e presença de oligossacarídeos ?1-6 ligados de superfície podem estar relacionadas com esta resposta diferencial ao estresse de RE. Em todas as linhagens verificamos a acúmulo dos marcadores de UPR, GRP78 e GADD153, após o tratamento com tunicamicina. Além disso, GADD153 foi direcionada para o núcleo em reposta ao tratamento com tunicamicina. O acúmulo de vacúolos acídicos, da proteína autofágica LC3-II e de ROS após o tratamento com Tuni>CDDP, sugerem que tanto a autofagia quanto o estresse oxidativo parecem estar envolvidos na resposta de sensibilização. A inibição de autofagia com cloroquina aumentou a morte induzida por Tuni>CDDP, sugerindo que autofagia desempenha função protetora neste esquema terapêutico. Testamos um segundo agente genotóxico, temozolomida (TMZ), uma droga equivalente à dacarbazina, e a mesma capacidade de sensibilização foi observada pelo prévio tratamento com tunicamicina. A validação deste conceito in vivo foi dificultada pela acentuada toxicidade apresentada por tunicamicina. Avaliamos alguns candidatos a agentes estressores do RE que poderiam apresentar menor toxicidade celular, como swainsonina, atorvastatina, metformina e o composto de cobre [Cu2(apyhist)2(dpam)](ClO4)4. No entanto, não obtivemos resultados promissores com nenhum destes candidatos. Estes resultados mostram que as células tumorais podem ser pré-condicionadas à morte celular se expostas a um prévio estressor de RE, como Tuni, o que leva ao comprometimento da resposta adaptativa a indutores de morte celular como CDDP e TMZ. No entanto, ainda é necessário o estudo de agentes indutores de estresse de RE pouco tóxicos para que esta estratégia terapêutica possa ser utilizada em pacientes com melanoma / Melanoma is among the most aggressive malignancies with increasing worldwide incidence and there is no effective treatment for the metastatic disease. The absence of an effective therapy may be due to adaptation and selection of melanoma cells to endoplasmic reticulum (ER) stress. We showed that GADD153, one of the components of the ER stress-mediated apoptosis pathway, was mostly excluded from the nucleus of primary and metastatic melanoma cells compared to nevus cells. These data suggest that the unexpected GADD153 cellular localization could be involved in melanoma cell adaption to ER stress, since GADD153 accumulates in the nucleus during ER stress. Unfolded protein response (UPR) signaling induced in response to ER stress, is a dual process that induces a protective response to restore ER homeostasis or cell death if ER stress is severe or persistent. We investigated if induction of ER stress was a potential strategy to chemosensitize melanoma cells to a second insult by surpassing the adaptive levels to ER stress. We first treated human melanoma cells (SbCl2, SK-MEL-28, Mel85, SK-MEL-29 and SK-MEL-147) with tunicamycin (Tuni), an ER stress inducer, before cisplatin (CDDP) treatment. CDDP is a low cost chemotherapeutic drug currently used in Brazil as a second line for melanoma treatment, especially in youngsters. All cell lines, except SK-MEL-29, demonstrated an >50% increase in the percentage of hypodiploid cells with Tuni>CDDP treatment when compared to CDDP only. The same results were obtained with temozolomide (TMZ), equivalent drug to the active form of dacarbazine, the first line of cytotoxic treatment of melanomas. UPR markers, GRP78 and nuclear translocation of GADD153 were induced by Tuni. Differences between SK-MEL-29 and SK-MEL-147 as cell surface GRP78 and ?1-6 oligossacharides can be related with the differential ER stress sensitization observed in these cells. One of the cellular mechanisms that are regulated by ER stress is autophagy. Accordingly, we observed an increase in the acidic vesicular organelles and accumulation of LC3II in response to Tuni>CDDP treatment. Autophagy inhibition with chloroquine increased Tuni>CDDP induced-cell death, suggesting that autophagy plays a protective role in this response. Oxidative stress can be involved in this scenario since we demonstrated an accumulation of reactive oxygen species in response to Tuni>CDDP. Tunicamycin was cytotoxic in vivo and we investigated alternatives to this antibiotic as swainsonine, atorvastatin, metformin and [Cu2(apyhist)2(dpam)](ClO4)4 but we did not observed ER stress induction. These results indicate that tumor cells could be preconditioned to cell death if exposed to a first ER stressor, such as Tuni, which would compromise an effective adaptive response to a cell death inducer, as CDDP and TMZ. This combined approach may be a promising strategy for melanoma therapy but further studies are necessary to find noncytotoxic alternatives to tunicamycin

Nowadays clusters are one of the most used platforms in High Performance Computing and most programmers use the Message Passing Interface (MPI) library to program their applications in these distributed platforms getting their maximum performance, although it is a complex task. On the other side, OpenMP has been established as the de facto standard to program applications on shared memory platforms because it is easy to use and obtains good performance without too much effort. So, could it be possible to join both worlds? Could programmers use the easiness of OpenMP in distributed platforms? A lot of researchers think so. And one of the developed ideas is the distributed shared memory (DSM), a software layer on top of a distributed platform giving an abstract shared memory view to the applications. Even though it seems a good solution it also has some inconveniences. The memory coherence between the nodes in the platform is difficult to maintain (complex management, scalability issues, high overhead and others) and the latency of the remote-memory accesses which can be orders of magnitude greater than on a shared bus due to the interconnection network. Therefore this research improves the performance of OpenMP applications being executed on distributed memory platforms using a DSM with sequential consistency evaluating thoroughly the results from the NAS parallel benchmarks. The vast majority of designed DSMs use a relaxed consistency model because it avoids some major problems in the area. In contrast, we use a sequential consistency model because we think that showing these potential problems that otherwise are hidden may allow the finding of some solutions and, therefore, apply them to both models. The main idea behind this work is that both runtimes, the OpenMP and the DSM layer, should cooperate to achieve good performance, otherwise they interfere one each other trashing the final performance of applications. We develop three different contributions to improve the performance of these applications: (a) a technique to avoid false sharing at runtime, (b) a technique to mimic the MPI behaviour, where produced data is forwarded to their consumers and, finally, (c) a mechanism to avoid the network congestion due to the DSM coherence messages. The NAS Parallel Benchmarks are used to test the contributions. The results of this work shows that the false-sharing problem is a relative problem depending on each application. Another result is the importance to move the data flow outside of the critical path and to use techniques that forwards data as early as possible, similar to MPI, benefits the final application performance. Additionally, this data movement is usually concentrated at single points and affects the application performance due to the limited bandwidth of the network. Therefore it is necessary to provide mechanisms that allows the distribution of this data through the computation time using an otherwise idle network. Finally, results shows that the proposed contributions improve the performance of OpenMP applications on this kind of environments.

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in chronic and acute neurodegenerative diseases. Indeed, markers of ER stress are common features of neurons destined to die in these conditions. In the present study we demonstrate that PUMA, a BH3-only member of the Bcl-2 family is essential for ER stress-induced cell death. PUMA is known to be a key transcriptional target of p53, however we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism involving instead the ER stress inducible transcription factor ATF4. Specifically, we demonstrate that ectopic expression of ATF4 sensitizes neurons to ER stress induced apoptosis, and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP, and that CHOP in turn directly activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER stress induced neuronal death involving ATF4-CHOP mediated transactivation of the pro-apoptotic Bcl-2 family member PUMA. Protein aggregates and markers of ER stress response have also been observed in dying neurons in several animal models of cerebral ischemia. Therefore, to decipher the significance of the ER stress apoptotic response, we investigate the role of ATF4-CHOP signaling pathway in ischemic neuronal injury. Ischemic stroke results from a transient or permanent reduction in cerebral blood flow in the brain. In spite of much research in trying to develop therapeutic strategies, most clinical trials have failed. These failures demonstrate that effective treatments require a more complete understanding of molecular signals that lead to neuronal death. However, stroke is a complex scenario since distinct mechanisms may involve in rapid and/or delayed neuronal death. The signaling pathways regulating these mechanisms however are not fully defined. Previous studies had suggested that ER stress playing a pivotal role in post-ischemic neuronal death. Yet, the relevance of ER stress signals was not fully known in ischemic neuronal injury. Accordingly, this thesis research attempts to explore the functional role of ER stress -inducible pathway, ATF4-CHOP axis, in different models of neuronal death (delayed and excitotoxic cell death) evoked by ischemia. The data indicates that ATF4 is essential in delayed type of death in vitro. In focal ischemia model (tMCAO) ATF4 also plays a role as a mediator of death signal in vivo. However, CHOP function looks more complex, and our data did not support the role of CHOP in ischemic neuronal death.