Visando contribuir para o conhecimento da dinâmica do estresse celular por agentes químicos e físicos e suas possíveis implicações sobre a mobilização de elementos transponíveis presentes em Drosophila, foram realizadas diferentes abordagens experimentais utilizando a espécie cosmopolita Drosophila melanogaster, e as espécies neotropicais do subgrupo willistoni, D. willistoni (três linhagens) D. equinoxialis, D. paulistorum, D. insularis e D. tropicalis. Foi verificado o efeito tóxico de disseleneto de difenila ((PhSe)2) durante diferentes estágios do ciclo de vida da Drosophila melanogaster e das espécies do subgrupo willistoni. Em D. melanogaster, as moscas adultas foram mais resistentes a intoxicação por (PhSe)2 do que larvas e pupas. De uma maneira geral as espécies do subgrupo willistoni são mais sensíveis à intoxicação por selênio do que a D. melanogaster, possivelmente explicado e de acordo com a recentemente demonstrada ausência de homologia de genes para selenoproteínas no genoma de D. willistoni com todas as onze outras espécies sequenciadas. O potencial do selênio para promover estresse celular em D. melanogaster foi evidenciado através da transcrição do gene hsp83 por Northern blot. Nas espécies do subgrupo willistoni a expressão deste gene e do hsp70 foi abordada através de Real Time PCR em resposta ao selênio e ao paraquat, que igualmente foi mais tóxico nos estágios iniciais de desenvolvimento. Os resultados da expressão dos genes de estresse, entretanto, não foram conclusivos, bem como a procura da transposase do elemento P em resposta aos tratamentos. Apesar disso foram estabelecidas às condições experimentais para a realização de experimentos futuros. / Aiming to contribute to the knowledge of the dynamics of cell stress caused by chemical and physical agents and the likely implications thereof in the mobilization of transposable elements present in Drosophila, different experimental approaches were adopted using the cosmopolitan species Drosophila melanogaster and the Neotropical species of the willistoni subgroup, D. willistoni (three strains), D. equinoxialis, D. willistoni, D. paulistorum, D. insularis and D. tropicalis. The toxic effect of diphenyl diselenide [(PhSe)2] was observed in the different life cycles of Drosophila melanogaster and of the species of the willistoni subgroup. D. melanogaster adult flies were more resistant to intoxication by (PhSe)2 as compared to larvae and pupae. Generally speaking the species belonging to the willistoni subgroup are more sensitive to intoxication by selenium as compared to D. melanogaster, which is possibly explained and in accordance to the recently demonstrated absence of gene homology of selenoproteins in the D. willistoni genome with all that of the other eleven species sequenced. The evidence of the potential exhibited by selenium to promote cell stress in D. melanogaster is shown by the hsp83 gene transcription analyzed by Northern blot. In the species of the willistoni subgroup, the expression of the hsp83 and of the hsp70 genes was analyzed by Real Time PCR in response to selenium and paraquat, which was equally more toxic in the early development stages. Nevertheless, the results of the stress gene expression were inconclusive, as well as the search for P element transposase in response to treatments. In spite of that, the experimental conditions required for future research have been established.

Visando contribuir para o conhecimento da dinâmica do estresse celular por agentes químicos e físicos e suas possíveis implicações sobre a mobilização de elementos transponíveis presentes em Drosophila, foram realizadas diferentes abordagens experimentais utilizando a espécie cosmopolita Drosophila melanogaster, e as espécies neotropicais do subgrupo willistoni, D. willistoni (três linhagens) D. equinoxialis, D. paulistorum, D. insularis e D. tropicalis. Foi verificado o efeito tóxico de disseleneto de difenila ((PhSe)2) durante diferentes estágios do ciclo de vida da Drosophila melanogaster e das espécies do subgrupo willistoni. Em D. melanogaster, as moscas adultas foram mais resistentes a intoxicação por (PhSe)2 do que larvas e pupas. De uma maneira geral as espécies do subgrupo willistoni são mais sensíveis à intoxicação por selênio do que a D. melanogaster, possivelmente explicado e de acordo com a recentemente demonstrada ausência de homologia de genes para selenoproteínas no genoma de D. willistoni com todas as onze outras espécies sequenciadas. O potencial do selênio para promover estresse celular em D. melanogaster foi evidenciado através da transcrição do gene hsp83 por Northern blot. Nas espécies do subgrupo willistoni a expressão deste gene e do hsp70 foi abordada através de Real Time PCR em resposta ao selênio e ao paraquat, que igualmente foi mais tóxico nos estágios iniciais de desenvolvimento. Os resultados da expressão dos genes de estresse, entretanto, não foram conclusivos, bem como a procura da transposase do elemento P em resposta aos tratamentos. Apesar disso foram estabelecidas às condições experimentais para a realização de experimentos futuros. / Aiming to contribute to the knowledge of the dynamics of cell stress caused by chemical and physical agents and the likely implications thereof in the mobilization of transposable elements present in Drosophila, different experimental approaches were adopted using the cosmopolitan species Drosophila melanogaster and the Neotropical species of the willistoni subgroup, D. willistoni (three strains), D. equinoxialis, D. willistoni, D. paulistorum, D. insularis and D. tropicalis. The toxic effect of diphenyl diselenide [(PhSe)2] was observed in the different life cycles of Drosophila melanogaster and of the species of the willistoni subgroup. D. melanogaster adult flies were more resistant to intoxication by (PhSe)2 as compared to larvae and pupae. Generally speaking the species belonging to the willistoni subgroup are more sensitive to intoxication by selenium as compared to D. melanogaster, which is possibly explained and in accordance to the recently demonstrated absence of gene homology of selenoproteins in the D. willistoni genome with all that of the other eleven species sequenced. The evidence of the potential exhibited by selenium to promote cell stress in D. melanogaster is shown by the hsp83 gene transcription analyzed by Northern blot. In the species of the willistoni subgroup, the expression of the hsp83 and of the hsp70 genes was analyzed by Real Time PCR in response to selenium and paraquat, which was equally more toxic in the early development stages. Nevertheless, the results of the stress gene expression were inconclusive, as well as the search for P element transposase in response to treatments. In spite of that, the experimental conditions required for future research have been established.

Visando contribuir para o conhecimento da dinâmica do estresse celular por agentes químicos e físicos e suas possíveis implicações sobre a mobilização de elementos transponíveis presentes em Drosophila, foram realizadas diferentes abordagens experimentais utilizando a espécie cosmopolita Drosophila melanogaster, e as espécies neotropicais do subgrupo willistoni, D. willistoni (três linhagens) D. equinoxialis, D. paulistorum, D. insularis e D. tropicalis. Foi verificado o efeito tóxico de disseleneto de difenila ((PhSe)2) durante diferentes estágios do ciclo de vida da Drosophila melanogaster e das espécies do subgrupo willistoni. Em D. melanogaster, as moscas adultas foram mais resistentes a intoxicação por (PhSe)2 do que larvas e pupas. De uma maneira geral as espécies do subgrupo willistoni são mais sensíveis à intoxicação por selênio do que a D. melanogaster, possivelmente explicado e de acordo com a recentemente demonstrada ausência de homologia de genes para selenoproteínas no genoma de D. willistoni com todas as onze outras espécies sequenciadas. O potencial do selênio para promover estresse celular em D. melanogaster foi evidenciado através da transcrição do gene hsp83 por Northern blot. Nas espécies do subgrupo willistoni a expressão deste gene e do hsp70 foi abordada através de Real Time PCR em resposta ao selênio e ao paraquat, que igualmente foi mais tóxico nos estágios iniciais de desenvolvimento. Os resultados da expressão dos genes de estresse, entretanto, não foram conclusivos, bem como a procura da transposase do elemento P em resposta aos tratamentos. Apesar disso foram estabelecidas às condições experimentais para a realização de experimentos futuros. / Aiming to contribute to the knowledge of the dynamics of cell stress caused by chemical and physical agents and the likely implications thereof in the mobilization of transposable elements present in Drosophila, different experimental approaches were adopted using the cosmopolitan species Drosophila melanogaster and the Neotropical species of the willistoni subgroup, D. willistoni (three strains), D. equinoxialis, D. willistoni, D. paulistorum, D. insularis and D. tropicalis. The toxic effect of diphenyl diselenide [(PhSe)2] was observed in the different life cycles of Drosophila melanogaster and of the species of the willistoni subgroup. D. melanogaster adult flies were more resistant to intoxication by (PhSe)2 as compared to larvae and pupae. Generally speaking the species belonging to the willistoni subgroup are more sensitive to intoxication by selenium as compared to D. melanogaster, which is possibly explained and in accordance to the recently demonstrated absence of gene homology of selenoproteins in the D. willistoni genome with all that of the other eleven species sequenced. The evidence of the potential exhibited by selenium to promote cell stress in D. melanogaster is shown by the hsp83 gene transcription analyzed by Northern blot. In the species of the willistoni subgroup, the expression of the hsp83 and of the hsp70 genes was analyzed by Real Time PCR in response to selenium and paraquat, which was equally more toxic in the early development stages. Nevertheless, the results of the stress gene expression were inconclusive, as well as the search for P element transposase in response to treatments. In spite of that, the experimental conditions required for future research have been established.

DCs são células especializadas na apresentação de antígenos (Ags) para linfócitos T virgens e indução de respostas imunes primárias. Deficiências no processo de eliminação de células apoptóticas relacionam-se com desenvolvimento de doenças auto-imunes. Para avaliar o efeito de células apoptóticas sobre os processos de maturação e atividade funcional de DCs derivadas in vitro de monócitos aderentes de doadores saudáveis, células não aderentes obtidas após uma 2ª etapa de aderência por 3 dias foram submetidas a aquecimento e/ou irradiação. 48h após, células somente irradiadas (37i) apresentaram maior porcentagem de apoptose que células aquecidas e irradiadas (43i), sugerindo que o calor protege da apoptose induzida. Na cocultura com iDCs ou mDCs (iDC+TNF), iDC+37i apresentaram aumento de CD1a, correlacionado com altos níveis de IL-10 e inibição de autoestimulação linfocitária. mDC+43i apresentaram níveis de CD1a semelhantes a iDC, baixos níveis de IL-10, altos níveis de IL-12p70 e altos índices de auto e aloestimulação linfocitária. Concluímos que o fenótipo e função de DCs é modulado diferencialmente na presença de 43i, que induzem ativação in vitro, enquanto 37i induzem DCs com características moduladoras dependente de lipídios. / DCs are specialized in presenting Ags to naïve lymphocytes, inducing primary immune responses or immunological peripheral tolerance, through tissue turnover by scavenging dying cells. Works with human cells are scarcer and controversial about the latter. Then, the aim of this work was to investigate the effect of apoptotic cells (Nadhs) on maturation and function of human monocyte-derived DCs in vitro. The results suggest that heating Nadhs before irradiation (43i) seems to protect them from apoptosis induced by irradiation (37i). DCs were cocultured with 37i or 43i, simultaneously to TNF-a addition in the 5th day of culture. In the 7th day, there was an association between high levels of CD1a, IL-10, low levels of IL-12p70 and decreased allostimulation induced by iDC+37i. mDC+43i had high levels of IL-12p70, CD86 and proliferation index, associated with low levels in CD1a expression. We conclude that DCs phenotype and function are differentially modulated in the presence of 43i, which induce DCs activation, or 37i, which induce regulatory phenotype and function in DCs. We suggest that these protocols for DCs activation with 43i or 37i could be used, respectively, as models of in vitro auto-reactivity or homeostatic immunomodulation of DCs in vitro.

Les applications des cellules souches pluripotentes humaines (CSP) dans le domaine biomédical sont particulièrement prometteuses, aussi bien au niveau expérimental qu’au niveau clinique. Leur utilisation comme source inépuisable de cellules permettant de tester et développer de nouvelles molécules thérapeutiques (notamment par modélisation de pathologies in vitro, criblage haut-débit et tests de cytotoxicité) s’ajoute à l’important potentiel qu’elles présentent en médecine régénérative et en thérapie cellulaire. Utilisables comme matériel biologique permettant de restaurer partiellement ou totalement un organe ou un tissu défaillant, il reste essentiel de vérifier l’intégrité génétique des lignées cellulaires utilisées afin de garantir une utilisation sécurisée pour le patient. Parmi les facteurs responsables de l’apparition d’anomalies génétiques chez les CSP, les conditions cultures jouent un rôle essentiel. Des techniques de culture inadaptées peuvent facilement provoquer l’émergence d’une instabilité génomique. Toute altération doit être détectée et documentée afin de pouvoir définir des critères d’acceptation préalable à leur utilisation clinique.Les CSP sont des cellules particulièrement sensibles au stress qui peut résulter de techniques de repiquage inappropriées. La dérive génétique qui découle de ce stress peut être précoce et apparaître dès les premiers passages des lignées cultivées. Notre équipe a pu tester de nombreuses méthodes de repiquage sur différentes lignées cellulaires pluripotentes. Nous avons notamment observé que des anomalies génétiques majeures caryotypiques (trisomies) et infra-caryotypiques (SNPs) ainsi que des changements phénotypiques (survie augmentée, acquisition de mobilité) apparaissaient rapidement suite à l’utilisation de techniques de repiquage basées sur l’utilisation d’enzyme de dissociation (TryPLE). Ces altérations apparaissent dans des lignées qui s’adaptent progressivement à la dissociation en cellules uniques (dissociation « single-cell ») provoquées par ces enzymes.Notre équipe étudie les conséquences cellulaires liées à ce phénomène d’adaptation des CSP provoquée par la dissociation « single-cell ». Grâce à des techniques de séquençage dernière génération (RNA-Seq), nous avons comparé les profils transcriptomiques de CSP repiquées par des techniques standard (comme le passage mécanique) et par des techniques basées sur la dissociation « single-cell » (comme le passage enzymatique par TryPLE). Cette comparaison a montré au niveau transcriptionnel une surexpression spectaculaire d’ARNs non codants appartenant à une classe récemment décrite : les longs ARNs non codants (lncRNAs).L’objectif principal de ce travail de thèse a été d’évaluer le niveau d’implication de ces lncRNAs dans le processus d’adaptation des CSP en culture, et leur rôle fonctionnel potentiel. Nous avons ainsi dans un premier temps déterminé in silico quels lncRNAs étaient différentiellement exprimés dans les CSP adaptées, et après validation expérimentale par biologie moléculaire des candidats les plus prometteurs, nous avons utilisé des tests fonctionnels (notamment par RNA interférence (siRNA)) afin de déterminer le rôle de ces lncRNAs dans la machinerie cellulaire et la pluripotence des CSP. Autour de ce projet principal, nous avons essayé de comprendre les mécanismes régissant les changements phénotypiques et comportementaux provoquées par la dissociation « single-cell ». Nous avons notamment pu suggérer la mise en place d’un phénomène de transition épithélio-mésenchymateuse (EMT) chez des CSP dissociées. Enfin, l’attractivité que représente un sujet d’étude récent comme les lncRNAs et la disponibilité croissante de publications les concernant nous ont poussé à publier une revue approfondie ainsi qu’une méta-analyse sur l’implication des longs ARN non codants dans le développement précoce de l’embryon et dans les cellules souches pluripotentes. / The actual and future applications of human pluripotent stem cells (PSC) in the biomedical field are highly promising. Their use for the discovery of new therapeutic drugs through the development of high-throughput screening tests, cytotoxicity tests and in vitro disease modeling has been added to their tremendous interests in regenerative medicine and cellular therapy. As a source of biological material that can be used to restore partially or totally the lost functions of a damaged organ or tissue, or as a source of normal cells to study human development or test putative new drugs, their genomic integrity has to be thoroughly assessed. Therefore, an effective optimization of their culture conditions has to be considered, in order to control the absence of genomic instability and prevent their potential emergence. Any genetic or epigenetic alteration resulting from cell culturing must be detected in order to define and characterize acceptance criteria for scientific and medical purposes.PSC are particularly sensitive to stress resulting from unappropriated passaging techniques, which cause rapid genetic drift. Indeed, our team observed that many genomic abnormalities arise from aggressive single cell, enzymatic based, passaging methods, and that substantial phenotypical changes such as increased survival after cell dissociation and variation in cell shape can then occur.In order to understand the mechanisms governing the emergence of those adverse alterations, the team focused on the consequences resulting from the adaptation of PSC to single-cell dissociation. By using new generation sequencing techniques as RNA-Seq, we compared transcriptomics of PSC passaged by standard techniques (such as mechanical passaging) versus single-cell enzymatic dissociation (such as TRyPLE-based single-cell passaging). This comparison showed that the most striking difference in the gene expression pattern between adapted and non adapted cells concerned the dramatic overexpression of RNAs from a recently discovered class: long non-coding RNAs (lncRNAs).The aim of this thesis work was to determine to which extent some of these lncRNAs were functionally linked to adaptation of PSC. In order to address this matter, we first investigated in silico which lncRNAs were upregulated by single-cell dissociation, and after experimental validation of lncRNA candidates by molecular biology, we performed functional in vitro analysis (notably by siRNA-mediated loss of function) and sought their cellular localization in order to decipher their role in the cellular machinery and their level of implication. Beside this main project, other auxiliary projects were grafted. The observation of major changes in cell phenotype and behavior led to the investigation of the global mechanisms governing these modifications, underlining the potential role of epithelial-to-mesenchymal transition provoked by single-cell dissociation. Finally, the global attractiveness of lncRNAs and the emergence of exponential documentation concerning non-coding RNAs prompted the writing of an extensive review and meta-analysis concerning the implications of lncRNAs during embryo development and in pluripotent stem cells.

Os mecanismos pelos quais a produção in vitro de embriões (PIVE) bovinos gera embriões com excessivo acúmulo lipídico, com elevado estresse celular e com reduzida criotolerância ainda são desconhecidos. Também permanece desconhecido quando essas alterações acontecem, se acontecem desde a maturação oocitária e o papel que as células do cumulus possuem neste mecanismo. O objetivo do presente trabalho foi estudar e comparar o metabolismo lipídico e homeostase celular, além dos perfis de miRNAs, durante a maturação oocitária e o desenvolvimento embrionário inicial in vivo e in vitro em bovinos. Para isso, o trabalho foi dividido em 4 estudos. No Estudo 1, intitulado \"A maturação in vitro gera complexos cumulus-oócitos metabolicamente desregulados e estressados em bovinos\" foram analisadas as células do cumulus e oócitos de complexos cumulus-oócitos (COCs) imaturos e maturados in vivo e in vitro. Foram realizadas análises de quantificação de lipídeos, espécies reativas de oxigênio (EROs), glutationa reduzida (GSH), razão ATP/ADP assim como expressão de mRNAs e miRNAs relacionados com as vias de metabolismo e homeostase celular. A partir dos dados obtidos neste estudo, concluímos que a maturação in vitro (MIV) provoca aumento de lipídeos no COC, diminuição de GSH e da atividade mitocondrial nos oócitos, acompanhado por uma desregulação massiva das vias relacionadas a metabolismo e homeostase nas células do cumulus da MIV. No Estudo 2, intitulado \"A proteína ligadora de ácidos graxos 3 (Fatty Acid Binding Protein 3 - FABP3) e as projeções transzonais estão envolvidas no acúmulo lipídico durante a maturação in vitro em oócitos bovinos\" foi constatado aumento do conteúdo lipídico e da expressão da FABP3 nas células do cumulus da MIV, quando comparado ao sistema in vivo. Ainda, imunolocalizamos a FABP3 dentro das projeções transzonais (TZPs) e verificamos um aumento concomitante da FABP3 e dos lipídeos oocitários nas primeiras 9 horas da MIV. Mediante a remoção das TZPs às 9 horas da MIV e consequente diminuição lipídica observada no oócito, concluímos que existe um possível transporte de ácidos graxos a partir das células do cumulus até o oócito mediante FABP3 e TZPs. No Estudo 3, intitulado \"Alterações no metabolismo lipídico e homeostase celular entre embriões bovinos produzidos in vivo e in vitro\", verificamos que, mesmo utilizando um cultivo in vitro com baixa tensão de oxigênio e sem soro, os blastocistos da PIVE possuem maiores níveis de lipídeos e EROs que aqueles produzidos in vivo. Além disso, constatamos que essas alterações nos lipídeos durante a PIVE não estão acompanhadas de alterações de expressão, porém, existe um aumento na expressão de genes relacionados com estresse. No último estudo, \"O sistema in vitro altera o perfil de miRNAs durante a maturação oocitária e desenvolvimento embrionário inicial em bovino\", constatamos as diferenças no perfil de miRNAs e nas vias reguladas por estes nas células do cumulus e oócitos maturados in vivo e in vitro e em blastocistos produzidos in vivo e in vitro. Verificamos uma maior regulação por miRNAs durante a maturação nas células do cumulus comparado ao oócito. Além do mais, tanto no COC quanto nos blastocistos, os miRNAs mostraram regular vias importantes do metabolismo, comunicação celular e vias de sinalização importantes para a maturação e desenvolvimento embrionário inicial. Conjuntamente, este trabalho permitiu elucidar as diferenças que a PIVE provoca no metabolismo e homeostase celular e na expressão de miRNAs. / The mechanism through which in vitro embryo production (IVEP) generates embryos with higher lipid accumulation, overstressed and with reduced cryotolerance is unknown. It is also unknown when these alterations take place, if occurs since the oocyte maturation and the role of cumulus cells in this mechanism are also unknown. The aim of the present work was to study and compare the lipid metabolism and cellular homeostasis, as well as the miRNAs profile during oocyte maturation and early in vivo and in vitro embryo development in bovines. For that, the present work was divided in 4 studies. In Study 1, entitled \"in vitro maturation generates metabolically disrupted and overstressed cumulus-oocyte complexes in bovines\" cumulus cells and oocytes from immature and in vivo and in vitro matured cumulus-cells complexes (COC) were analyzed. Analysis of lipid stores, oxygen reactive species (ROS), reduced glutathione and ATP/ADP ratio quantification, as well as mRNAs and miRNAs involved with metabolism and cellular homeostasis pathways were performed. From the obtained data in this study, we concluded that in vitro maturation (IVM) leads to an increase of lipids in the COC, a decrease of oocyte GSH and mitochondrial activity, as well as a massive deregulation in metabolism and homeostasis related pathways in MIV cumuls cells. In Study 2, \"Fatty Acid Binding Protein 3 and transzonal projections are involved on lipid accumulation during in vitro maturation in bovine oocytes\" we observed an increase of lipid accumulation and FABP3 expression in MIV cumulus cells when compared to the in vivo system. Furthermore, we immunolocalized the FABP3 inside the transzonal projections (TZPs) and verified a concomitant increase of FABP3 and oocyte lipids on the first 9 hours of MIV. By removing the TZPs at 9 hours of MIV and by the consequent lipid decrease observed in the oocyte, we concluded that there is a possible traffic of fatty acids from the cumulus cells to the oocyte mediated by FABP3 and TZPs. In Study 3, entitled \"Alterations in lipid metabolism and cellular homeostasis between in vivo and in vitro produced bovine embryos\", we observed that, even though under serum free and low oxygen tension used during in vitro culture was used, IVP blastocysts had higher lipid and ROS levels than the counterparts produced in vivo. Moreover, we found that these lipid alterations during IVP are not followed by corresponding genes expression alterations, however, there is an increase of the expression of stress related genes. In the last study, \"in vitro system alters miRNAs profile during oocyte maturation and early embryo development in bovines\", we observed differences in miRNAs profiles and in pathways modulated by them in in vivo and in vitro matured cumulus cells and oocytes and in in vivo and in vitro produced blastocysts. We observed an intense miRNAs regulation during maturation in cumulus cells when compared to the oocyte. Furthermore, in both COC and blastocysts, miRNAs regulated important metabolism, cellular communication pathways, as well as important signaling pathways for maturation and early embryo development. Taken together, the findings of the present work allowed us to elucidate the differences caused by IVEP on cell metabolism and homeostasis as well as on miRNAs expression.

Indiana University-Purdue University Indianapolis (IUPUI) / Disturbances in membrane composition and protein folding in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR). Three UPR sensory proteins, PERK (PEK/EIF2AK3), IRE1, and ATF6 are each activated by ER stress. PERK phosphorylation of the alpha subunit of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with the preferential translation of ATF4 (CREB2). Results from cultured cells demonstrate that ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterized two ATF4 knockout mouse models and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but rather ATF6 is a primary inducer. RNA-sequence analysis indicated that ATF4 was responsible for a small portion of the PERK-dependent genes in the UPR. This smaller than expected subset of gene expression lends itself to the relevance of UPR crosstalk, with ATF6, XBP1, and CHOP being capable of upregulating UPR genes in the absence of ATF4. RNA-sequence analysis also revealed a requirement for expression of ATF4 for expression of a comparable number of genes basally, including those involved in oxidative stress response and cholesterol metabolism. Consistent with this pattern of gene expression, loss of ATF4 in our mouse model resulted in enhanced oxidative damage and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera. Taken together, this study highlights both an expansion of the role of ATF4 in transcriptional regulation of genes involved in metabolism in the basal state and a more specialized role during ER stress. These findings are important for understanding the variances of the UPR signaling between cell culture and in vivo and for a greater understanding of all the roles ATF4 plays within the cell.

Le mélanome est caractérisé par un remodelage métabolique important, participant à la métastase et à la résistance aux traitements. Les coactivateurs 1 du récepteur activé par le proliférateur du peroxisome (PPARγ), ou PGC-1s, constituent une famille de coactivateurs qui potentialisent la transcription de gènes du métabolisme et de la biogenèse mitochondriale, participant ainsi à la reprogrammation métabolique des cellules cancéreuses. Les différents cancers de la peau expriment des niveaux variables de PGC-1s et les tumeurs qui expriment fortement les PGC-1s présentent un profil pro-inflammatoire et immunosuppresseur favorisant l’évasion immunitaire. Ceci suggère un rôle des PGC-1s dans la réponse à l’immunothérapie. Nous montrons d’abord que la déplétion en PGC-1β diminue la prolifération de lignées cellulaires de mélanome en plus d’induire l’expression de cytokines pro-inflammatoires et de transcrits immunosuppresseurs. À ce jour, les mécanismes permettant de réguler l’expression et l’activité des PGC-1s demeurent inconnus. Nous montrons que différents stress cellulaires affectant des procédés métaboliques, épigénétiques ou la traduction diminuent l’expression des PGC-1s et la viabilité de lignées cellulaires de mélanome. Nous montrons également un mécanisme de régulation de PGC-1β par une protéine de liaison à l’ARN soutenant le rôle de PGC-1β dans la réponse à différents stress et la survie des cellules de mélanome. Cette régulation serait importante pour l’immunosuppression du mélanome et dicterait la réponse à l’immunothérapie. En conclusion, ces travaux illustrent bien le rôle de PGC-1β dans la reprogrammation métabolique et la réponse au stress du mélanome, ces processus pouvant influencer l’inflammation et l’immunosuppression du microenvironnement afin d’assurer la progression tumorale. / Melanoma is characterized by a significant metabolic remodeling, which contributes to metastasis and treatment resistance. Peroxisome proliferator-activated receptor gamma (PPARγ) coactivators 1, or PGC-1s, are a family of coactivators that potentiate the transcription of genes involved metabolism and mitochondrial biogenesis, thus participating in the metabolic reprogramming of cancer cells. Different skin cancers express varying levels of PGC-1s and tumors that strongly express PGC-1s exhibit a proinflammatory and immunosuppressive profile that favors immune evasion. This suggests that PGC-1s play a role in melanoma response to immunotherapy. We first show that PGC-1β knockdown decreases the proliferation of melanoma cell lines. Moreover, it induces the expression of proinflammatory cytokines and immunosuppressive transcripts. To date, the mechanisms that regulate the expression and activity of PGC-1s remain unknown. We show that different cellular stresses affecting metabolic and epigenetic processes or translation decrease PGC-1s expression and the viability of melanoma cell lines. We also show a mechanism of regulation of PGC-1β by an RNA-binding protein enhencing the role of PGC-1β in the response to different stresses and the survival of melanoma cells. This regulation could contribute to melanoma immunosuppression and determine the tumor’s response to immunotherapy. In conclusion, this work illustrates well the role of PGC-1β in melanoma metabolic reprogramming and stress response, processes that regulate inflammation and create an immunosuppressive tumor microenvironment in order to ensure tumor progression.

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones that play a vital role in maintaining protein homeostasis in cells. They are the first line of defence against the detrimental effects of cellular stress conditions like fluctuations in temperature, pH, oxidative and osmotic potentials, heavy metal toxicity, drought and anoxia. Many sHSPs are also constitutively expressed during developmental stages of different plant tissues. Members of this family are ATP-independent chaperones, with monomeric masses varying from 12-40 kDa. A characteristic feature of sHSPs is their ability to assemble into large oligomers, ranging from dimers to 48-mers. Under stress conditions, these oligomers dissociate and/or undergo drastic conformational changes to facilitate their binding to misfolded substrate proteins in the cell. This interaction prevents the substrate from aggregating during stress. When physiological conditions are restored, the substrates are transferred to other ATP-dependent heat shock proteins for refolding. Thus sHSPs do not refold their substrates, but instead prevent them from aggregating and maintain them in a „folding-competent‟ state. The clientele of sHSPs includes proteins with a wide range of molecular masses, secondary structures and pIs. This promiscuity has led to sHSPs occupying key positions in the protein quality control network. As molecular chaperones that protect proteins, sHSPs prevent disease. Concomitantly, mutations in sHSPs have also been linked to various human diseases. Till date, high resolution crystal structures are available only for 3 sHSP oligomers. This insufficiency of structural information has hindered our understanding of the mechanism of chaperone function, the link between the oligomeric status and chaperone activity, identification of substrate binding sites and the role of the flexible terminal segments in mediating both the oligomerization and chaperone function. We undertook structural and functional characterization of plant and bacterial sHSPs in order to address some of these questions. Chapter 1 of this thesis gives an overview of the sHSP family, with special emphasis on the oligomeric assemblies of sHSPs of known structures. We highlight what we know about this family through mutational studies, what is as yet unknown, and why it is important to study this family. Chapter 2 describes our efforts at structural and functional characterization of 5 sHSPS in rice, each targeted to a different organelle. We probed the role played by the N-terminal region in mediating oligomer assembly and in the chaperone activity of the protein. Rice sHSPs displayed a wide range of hydrodynamic radii, from 4 nm to 14 nm, suggesting that their oligomeric assemblies are likely to be diverse. In chapter 3, we discuss our attempts at the structural characterization of a bacterial sHSP, Aggregation suppressing protein A, or AgsA from Salmonella enterica. We obtained a high resolution crystal structure of the dimer of the core sHSP domain. We compared this dimer with other known sHSP dimers, reported the deviations that we observed and analysed the structure to account for these differences. We used this dimer structure to successfully obtain solutions for low resolution X-ray diffraction data for oligomers of different truncated constructs of AgsA. We observed that a C-terminal truncated construct formed an octahedral 24¬mer (4.5 Å resolution), whereas a construct truncated at both termini formed a triangular bipyramidal 18-mer (7.7 Å resolution), an assembly hitherto unobserved for any sHSP. A similar 18-mer was obtained when the C-terminal truncated construct was incubated with a dipeptide prior to crystallisation (6.7 Å resolution). The cryo-EM map of the wild type protein (12 Å resolution) could be fitted with a different 18-mer. The low resolution of the data pre-empted an atomic-level description of the interfaces of the assemblies. However, our work highlights the structural plasticity of this protein and probes the sensitivity of the oligomeric assembly to minor differences in construct length.