Iminosugars are a class of small molecules defined by substitution of a sugar’s ring oxygen with nitrogen. Various chemical modifications of these basic structures (e.g. alkyl chain addition off of the ring nitrogen) have been developed during the last several decades. These molecules have been considered as therapeutics for a number of pathologies including viral infection, congenital disorders of glycosylation (of both glycoproteins and glycolipids), and diabetes. This thesis focuses on the application of a small subset of iminosugars, known as deoxynojirimycin derivatives, as therapeutics against dengue virus induced pathology. Dengue virus infection predominates in tropical climates, but autochthonous infection has recently emerged in areas of both southern Europe and the southern United States. With 390 million people infected annually, dengue is the most prevalent arthropod-borne viral infection worldwide, and the possibility of severe pathology including haemorrhage, shock, and/or death, necessitates development of effective antiviral therapies. Although the molecular mechanisms responsible for progression to severe dengue disease are not completely understood, there is considerable evidence for the role of both the innate and the adaptive immune responses in development of life-threatening complications. Excessive activation of the innate immune response, a phenomenon known as cytokine storm, has been hypothesised to explain development of symptoms related to vascular permeability, whereas the adaptive immune response has been implicated in severe disease through two hypotheses – the antibody dependent enhancement and original antigenic sin hypotheses. The evidence regarding each of these potential mechanisms of severe pathology is discussed throughout this thesis principally with respect to how iminosugar treatment could alter any detrimental effects of the immune response to dengue virus infection. The principal aim of this thesis is to consider the potential of deoxynojirimycin iminosugars as antiviral therapeutics in dengue infection with a focus on how these molecules exert their antiviral effects in primary human cells. I first consider the contributions of glycoprotein inhibition and glycolipid inhibition on production of infectious dengue virus. These experiments suggest that inhibition of glycoprotein folding is responsible for inhibition of infectious dengue virus production. I next consider the impact of treatment of a promising clinical candidate iminosugar, N9-methoxynonyl-deoxynojirimycin (MON-DNJ), on the primary human macrophage transcriptome. In uninfected macrophages as well as macrophages infected with dengue virus or treated with lipopolysaccharide to model bacterial sepsis, iminosugar treatment results in activation of the unfolded protein response and inhibition of several elements of the inflammatory response including signalling by the cytokines IFN-γ and TNF-α, and the inflammatory cascade mediated by NF-κB. Activation of the unfolded protein response as a result of treatment with MON-DNJ can be confirmed by analysis of phosphorylated (activated) NFE2L2, a transcription factor that functions principally to control oxidative stress in response to ER stress signals. Modulation of the inflammatory response of macrophages to dengue infection and bacterial sepsis is confirmed by analysis of secreted cytokines. As predicted by my transcriptomic experiments, levels of TNF-α and IFN-γ produced in response to dengue or lipopolysaccharide are reduced by treatment with MON-DNJ. Finally, I attempted to extend these observations to an animal model of dengue infection with a particular focus on TNF receptor and ligand superfamily members. Unfortunately, heterogeneity of cells types from tissue samples as well as limitations of the animal model complicate interpretation of these findings. Nevertheless, this thesis demonstrates that MON-DNJ is an effective dengue antiviral therapeutic and that this therapeutic activity may be related to both reduction of infectious virus as a consequence of inhibition of glycoprotein processing and as a result of changes to the host’s response to the pathogen. These results have been used in part to justify recently initiated clinical trials of MON-DNJ as a dengue antiviral therapy.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

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

O melanoma é o tipo de câncer de pele mais letal, apesar de ser o menos incidente. Em virtude de sua alta letalidade e de sua crescente incidência, estudos sobre melanoma são de fundamental importância nos dias de hoje. Assim como células tumorais em geral, células de melanoma apresentam características metabólicas diferenciadas, como, por exemplo, altos níveis de espécies reativas de oxigênio e alta taxa de síntese proteica. Essas modificações no metabolismo dispararam vias de resposta a estresse, como a \"Unfolded Protein Response\" (UPR), contudo, essas células se adaptam a esse estresse constante, que não culmina com a morte das mesmas. Além disso, o padrão de glicosilação em células tumorais também é sabidamente alterado, entre outros motivos, pela expressão diferencial de enzimas da via de glicosilação, como a N-acetilglicosaminiltransferase 5 (MGAT5). Relacionando essas duas características de células de melanoma, propusemo-nos a avaliar se a alta expressão de MGAT5A ( e/ou MGAT5B) funcionaria como uma resposta adaptativa de células de melanoma ao estresse de retículo endoplasmático, e seria, portanto, responsável por manter o equilíbrio diferenciado nessas células. Durante o desenvolvimento desse estudo, foi possível comprovar que a indução de estresse de retículo por meio de tratamento com tunicamicina, um inibidor da N-glicosilação e indutor clássico de UPR, sensibilizou as células de melanoma ao posterior tratamento com cisplatina. Contudo, o tratamento com swainsonina, um inibidor do processamento dos N-glicanos que ocorre no complexo de Golgi, não foi capaz nem de disparar \"Unfolded Protein Response\" nem de induzir morte nessas células e, talvez por esse motivo, não apresentou efeito sensibilizador frente à cisplatina. Além disso, foi observado que as linhagens tumorigênicas apresentam maior expressão de MGAT5A em comparação à linhagem não-tumorigênica melan-a. As tentativas de realização de silenciamento de MGAT5A não foram exitosas. Informações relacionando estresse de retículo e N-glicosilação aberrante em células tumorais ainda serão foco de estudo em nosso grupo. Com os resultados apresentados, é possível concluir que o equilíbrio diferencial dos níveis de estresse de retículo em que se encontram as linhagens tumorigênicas do nosso modelo é importante para a sobrevivência das mesmas. Além disso, é de nosso interesse avaliar a dependência de células tumorais das vias ativadas pela superexpressão de MGAT5A, caso ela realmente exista / Melanoma is the most lethal skin cancer, despite being the least prevalent. Due to its lethality and resistance to a variety of known chemotherapeutic drugs, studies on melanoma are paramount. Tumor cells in general, and melanoma cells particularly, commonly present a disturbed metabolic rate, e.g., altered metabolism of reactive oxygen species and increased rates of protein synthesis. Altogether these perturbations trigger the Unfolded Protein Response (UPR); however, tumor cells are adapted to these conditions and are able to survive. Besides, glycosylation of tumor cells is commonly altered, due to differentiated expression rates of N-glycosylation enzymes, like N-acetylglucosaminyltransferase 5 (MGAT5). Considering these information together, we proposed that the sustained overexpression of MGAT5A (and/or MGAT5B) observed in Tm1 and Tm5 melanoma cells is part of an adaptive response to reticulum stress, maintaining an unstable equilibrium in tumor cells. In this work, we observed that the induction of endoplasmic reticulum stress caused by tunicamycin treatment, a N-glycosylation inhibitor and UPR inducer, sensitized melanoma cells to further cisplatin treatment. In contrast, swainsonine treatment, an inhibitor of Golgi N-glycan processing pathway, did not cause cell death nor UPR signaling, and this may be the reason why this treatment did not sensitize cells to cisplatin treatment. MGAT5A silencing was not successful yet. Altogether, the results above show that the unstable equilibrium under which Tm1 and Tm5 tumor cells are seems necessary for their survival. Therefore, it seems that upon malignant transformation, melanoma cells present dependence of MGAT5A expression. Its our interest exploit this melanoma model to understand the concept of oncogenic dependence for MGAT5A expression in the case of melanomas, if it exists

No description available.

Scs2p ist ein integrales Membranprotein des Endoplasmatischen Retikulums (ER), dessen Deletion zu einer geringeren Expression der Inositol-1-P Synthase INO1 in Inositol-freiem Medium führt und dessen Überexpression die Inositol-Auxotrophie eines Stammes mit einer defekten "unfolded protein response" supprimiert. scs2 Mutanten weisen zudem eine erhöhte Sensitivität gegen Tunicamycin auf, eine Substanz, welche die N-Glykosylierung von Proteinen im ER inhibiert. Für die mutmaßlichen Orthologen von Scs2p in höheren Eukaryoten wurde eine Funktion dieser Proteine im vesikulären Transport postuliert. In dieser Arbeit wurde Scs2p als mit Cue1p, einer Komponente der ER-assoziierten Proteindegradation, quervernetzbares Protein identifiziert, und daraufhin begonnen, die Funktion von Scs2p näher zu charakterisieren. Eine Deletion von SCS2 hatte keinen Einfluß auf die bekannten Cue1p-abhängigen Degradationswege. Auch die Induktion der "unfolded protein response" (UPR) infolge einer Akkumulation von falsch gefalteten Proteinen wurde durch eine Deletion von SCS2 nicht beeinträchtigt. Eine Beeinträchtigung des Transportes durch den sekretorischen Weg konnte in scs2 Hefen ebenfalls nicht nachgewiesen werden. scs2 Mutanten zeigten eine generell verminderte Expression von Genen der Glycerolipid Biosynthese, deren Transkription über Ino2p/Ino4p reguliert wird. Dabei beeinträchtigte eine Deletion von SCS2 nicht die Induktion der UPR in Inositol-armem Medium und die darüber vermittelte Induktion der INO1 Expression. Ein analoger Phänotyp wurde auch in Mutanten beobachtet, in denen die Gene von Ubc7p, einer Komponente der ER-Degradation oder Lcb3p, einer Sphingosin-P Phosphatase, deletiert waren. Dabei bestand in bezug auf die Ino2p/Ino4p-abhängige Expressionsregulation keine epistatische Beziehung zwischen den drei Gendeletionen. Untersuchungen des Einflusses verschiedener Mutanten und Inhibitoren des Sphingolipid Stoffwechsels zeigten, daß Änderungen der Synthese von komplexen Sphingolipiden die Regulation der Inositol Synthese nur indirekt beeinflußten und gaben Hinweise auf eine Regulation des Glycerolipid Stoffwechsels durch Sphingosin. scs2 Mutanten erwiesen sich als sensitiv gegen Inhibitoren der Sphingolipid Biosynthese. Untersuchungen von Stämmen, in denen verschiedene Gene der Sphingolipid Synthese deletiert wurden, sowie Analysen der Lipidzusammensetzung zeigten, daß in scs2 Zellen eine Regulation beeinträchtigt ist, welche die IPC Synthase Aktivität bei einem verringerten Sphingolipid Gehalt der Zelle erhöht. Die geringere Expression von Genen der Glycerolipid Synthese und die niedrigere Kapazität zur Synthese von komplexen Sphingolipiden erwiesen sich als voneinander unabhängige Auswirkungen eines komplexen Phänotyps von scs2 Deletionsmutanten, wobei die primäre Funktion von Scs2p vermutlich nicht im Lipidstoffwechsel liegt. / Scs2p is an integral membrane protein of the endoplasmic reticulum (ER), whose deletion leads to a reduced expression of INO1 encoding inositol-1-phosphate synthase and whose over-expression suppresses the inositol auxotrophy of a strain with a defect unfolded protein response (UPR). scs2 mutants display an enhanced sensitivity to Tunicamycin, an inhibitor of protein N-glycosylation in the ER. It has been proposed that the putative orthologs of Scs2p in higher eukaryotes function in vesicular transport. In this work Scs2p, which was identified as a protein that can be crosslinked to Cue1p, a protein involved in ER-associated protein degradation, was further characterised. A deletion of SCS2 did not affect the known Cue1p-dependend degradation pathways. Cells lacking Scs2p normally induced the UPR upon an accumulation of misfolded proteins. An impairment of protein transport through the secretory pathway could also not be detected in scs2 cells. scs2 mutants displayed a generally reduced expression of genes involved in glycerolipid synthesis, whose transcription is regulated by Ino2p/Ino4p. The induction of the UPR in inositol-free medium and the subsequent upregulation of INO1 expression were not impaired in these cells. A similar effect was observed in strains lacking Ubc7p, a component of the ER -associated protein degradation system and in cells lacking the sphingoid base-1-phosphate phosphatase Lcb3p. Regarding the Ino2p/Ino4p-dependend gene expression no epistatic relationship was observed between Scs2p, Ubc7p and Lcb3p. An examination of the influence of various mutants and inhibitors of the sphingolipid pathway indicated that alterations of the synthesis of complex sphingolipids have only an indirect influence on the regulation of inositol synthesis and pointed towards a control of glycerolipid synthesis by sphingoid bases. scs2 cells were found to be sensitive to inhibitors of sphingolipid biosynthesis. The examination of strains lacking various enzymes involved in sphingolipid synthesis and direct analysis of lipid composition showed that a deletion of SCS2 impairs an upregulation of IPC synthase activity under conditions where the sphingolipid content of the cell is diminished. The reduced expression of genes involved in glycerolipid synthesis and the lower capacity for the synthesis of complex sphingolipids turned out to be mutually independent consequences of the complex phenotype of cells lacking Scs2p, whose primary function may not be in the lipid metabolism.

The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

O melanoma é o tipo de câncer de pele mais letal, apesar de ser o menos incidente. Em virtude de sua alta letalidade e de sua crescente incidência, estudos sobre melanoma são de fundamental importância nos dias de hoje. Assim como células tumorais em geral, células de melanoma apresentam características metabólicas diferenciadas, como, por exemplo, altos níveis de espécies reativas de oxigênio e alta taxa de síntese proteica. Essas modificações no metabolismo dispararam vias de resposta a estresse, como a \"Unfolded Protein Response\" (UPR), contudo, essas células se adaptam a esse estresse constante, que não culmina com a morte das mesmas. Além disso, o padrão de glicosilação em células tumorais também é sabidamente alterado, entre outros motivos, pela expressão diferencial de enzimas da via de glicosilação, como a N-acetilglicosaminiltransferase 5 (MGAT5). Relacionando essas duas características de células de melanoma, propusemo-nos a avaliar se a alta expressão de MGAT5A ( e/ou MGAT5B) funcionaria como uma resposta adaptativa de células de melanoma ao estresse de retículo endoplasmático, e seria, portanto, responsável por manter o equilíbrio diferenciado nessas células. Durante o desenvolvimento desse estudo, foi possível comprovar que a indução de estresse de retículo por meio de tratamento com tunicamicina, um inibidor da N-glicosilação e indutor clássico de UPR, sensibilizou as células de melanoma ao posterior tratamento com cisplatina. Contudo, o tratamento com swainsonina, um inibidor do processamento dos N-glicanos que ocorre no complexo de Golgi, não foi capaz nem de disparar \"Unfolded Protein Response\" nem de induzir morte nessas células e, talvez por esse motivo, não apresentou efeito sensibilizador frente à cisplatina. Além disso, foi observado que as linhagens tumorigênicas apresentam maior expressão de MGAT5A em comparação à linhagem não-tumorigênica melan-a. As tentativas de realização de silenciamento de MGAT5A não foram exitosas. Informações relacionando estresse de retículo e N-glicosilação aberrante em células tumorais ainda serão foco de estudo em nosso grupo. Com os resultados apresentados, é possível concluir que o equilíbrio diferencial dos níveis de estresse de retículo em que se encontram as linhagens tumorigênicas do nosso modelo é importante para a sobrevivência das mesmas. Além disso, é de nosso interesse avaliar a dependência de células tumorais das vias ativadas pela superexpressão de MGAT5A, caso ela realmente exista / Melanoma is the most lethal skin cancer, despite being the least prevalent. Due to its lethality and resistance to a variety of known chemotherapeutic drugs, studies on melanoma are paramount. Tumor cells in general, and melanoma cells particularly, commonly present a disturbed metabolic rate, e.g., altered metabolism of reactive oxygen species and increased rates of protein synthesis. Altogether these perturbations trigger the Unfolded Protein Response (UPR); however, tumor cells are adapted to these conditions and are able to survive. Besides, glycosylation of tumor cells is commonly altered, due to differentiated expression rates of N-glycosylation enzymes, like N-acetylglucosaminyltransferase 5 (MGAT5). Considering these information together, we proposed that the sustained overexpression of MGAT5A (and/or MGAT5B) observed in Tm1 and Tm5 melanoma cells is part of an adaptive response to reticulum stress, maintaining an unstable equilibrium in tumor cells. In this work, we observed that the induction of endoplasmic reticulum stress caused by tunicamycin treatment, a N-glycosylation inhibitor and UPR inducer, sensitized melanoma cells to further cisplatin treatment. In contrast, swainsonine treatment, an inhibitor of Golgi N-glycan processing pathway, did not cause cell death nor UPR signaling, and this may be the reason why this treatment did not sensitize cells to cisplatin treatment. MGAT5A silencing was not successful yet. Altogether, the results above show that the unstable equilibrium under which Tm1 and Tm5 tumor cells are seems necessary for their survival. Therefore, it seems that upon malignant transformation, melanoma cells present dependence of MGAT5A expression. Its our interest exploit this melanoma model to understand the concept of oncogenic dependence for MGAT5A expression in the case of melanomas, if it exists