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.

A imunodeficiência comum variável (CVID) é caracterizada por hipogamaglobulinemia e infecções recidivantes. Neste estudo verificamos o papel da via Unfolded Protein Response (UPR) na patogênese da doença. Uma paciente com CVID apresentou expressão aumentada do RNAm XBP-1 unspliced e co-localização de IgM e BiP no retículo endoplasmático (RE). Verificamos a ausência de mutações nos produtos obtidos por RT-PCR de XBP-1 e nos domínios quinase/endonuclease da IRE-1a. Análises por Q-PCR dos RNAm XBP-1 spliced, IRE-1a e BiP após tratamento com LPS ou brefeldina A mostrou que, ao contrário dos controles saudáveis que respondem a estes estressores com ondas de transcrição destes três genes, esta paciente apresenta baixos níveis de transcrição, não atingindo o mesmo nível de resposta apresentado pelos indivíduos saudáveis. Nossos achados associam o splicing diminuído do RNAm XBP-1 ao acúmulo de IgM no RE e baixas taxas de transcrição de chaperonas, fornecendo um mecanismo para explicar a hipogamaglobulinemia observada em uma paciente com CVID. / Common Variable Immunodeficiency (CVID) is characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of Unfolded Protein Response (UPR) in the pathogenesis of the disease. Augmented unspliced XBP-1 mRNA concurrent with co-localization of IgM and BiP was found in one CVID patient. Sequencing of RT-PCR amplicons did not reveal any mutation on XBP-1 neither on the kinase/endonuclease domains of IRE-1a. Q-PCR analysis of spliced XBP-1, IRE-1a and BiP after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these ER stressors by presenting waves of transcription of these three genes, the cells presented lower rates of transcription, not reaching the same level of response of healthy subjects. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia.

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.

No description available.

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

A imunodeficiência comum variável (CVID) é caracterizada por hipogamaglobulinemia e infecções recidivantes. Neste estudo verificamos o papel da via Unfolded Protein Response (UPR) na patogênese da doença. Uma paciente com CVID apresentou expressão aumentada do RNAm XBP-1 unspliced e co-localização de IgM e BiP no retículo endoplasmático (RE). Verificamos a ausência de mutações nos produtos obtidos por RT-PCR de XBP-1 e nos domínios quinase/endonuclease da IRE-1a. Análises por Q-PCR dos RNAm XBP-1 spliced, IRE-1a e BiP após tratamento com LPS ou brefeldina A mostrou que, ao contrário dos controles saudáveis que respondem a estes estressores com ondas de transcrição destes três genes, esta paciente apresenta baixos níveis de transcrição, não atingindo o mesmo nível de resposta apresentado pelos indivíduos saudáveis. Nossos achados associam o splicing diminuído do RNAm XBP-1 ao acúmulo de IgM no RE e baixas taxas de transcrição de chaperonas, fornecendo um mecanismo para explicar a hipogamaglobulinemia observada em uma paciente com CVID. / Common Variable Immunodeficiency (CVID) is characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of Unfolded Protein Response (UPR) in the pathogenesis of the disease. Augmented unspliced XBP-1 mRNA concurrent with co-localization of IgM and BiP was found in one CVID patient. Sequencing of RT-PCR amplicons did not reveal any mutation on XBP-1 neither on the kinase/endonuclease domains of IRE-1a. Q-PCR analysis of spliced XBP-1, IRE-1a and BiP after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these ER stressors by presenting waves of transcription of these three genes, the cells presented lower rates of transcription, not reaching the same level of response of healthy subjects. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia.

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