Der Eintritt löslicher oder membranständiger Proteine in den Sekretionsweg beginnt mit dem Transport der Proteine durch die Membran des Endoplasmatischen Retikulums (ER). Die Translokationspore wird durch den heterotrimeren Sec61-Komplex gebildet. Homologe Membranproteine der alpha- und gamma-Untereinheit dieses Komplexes sind bislang in allen daraufhin untersuchten prokaryotischen und eukaryotischen Organismen gefunden worden. Es handelt sich somit um einen entwicklungsgeschichtlich hoch konservierten Mechanismus des Proteintransports durch Membranen. Zahlreiche Untersuchungen in den letzten Jahren haben uns mittlerweile ein komplexes Bild des Translokationsgeschehens vermittelt. Die vorliegende Arbeit beleuchtet einen völlig neuen Aspekt der Proteintranslokation: Es zeigt sich, daß Gene, die für Komponenten des Sec61-Komplexes kodieren, in sehr unterschiedlichen eukaryotischen Organismen unabhängig voneinander dupliziert wurden und sich im folgenden nebeneinander weiterentwickelten. Die Untersuchung dieser sog. paralogen Gene und ihrer Genprodukte zum einen in Säugerzellen und zum anderen in der Hefe Saccharomyces cerevisiae ist Gegenstand der Arbeit. In Säugerzellen finden sich zwei sehr ähnliche SEC61a-Gene (knapp 80% Identität auf Nukleinsäureebene, etwa 94% auf Aminosäureebene), deren Expression in verschiedenen Organen und Embryonalstadien der Maus analysiert wurde. In allen bislang getesteten Geweben wurden beide Gene exprimiert. Deutliche Unterschiede zeigten sich allerdings in der Stärke der Expression: Während die Expressionshöhe des bereits bekannten SEC61a-I-Gens relativ konstant war, variierte die des paralogen SEC61a-II-Gens zwischen den einzelnen Organen. Der selektive Vorteil zweier paraloger SEC61a-Gene für den Säugerorganismus liegt somit möglicherweise in der unterschiedlichen Regulierbarkeit ihrer Expression. In Saccharomyces cerevisiae finden sich paraloge Proteine und dafür kodierende Gene sowohl für die alpha-Untereinheit Sec61p als auch für die beta-Untereinheit Sbh1p des heterotrimeren Sec61p-Komplexes. Der Grad der Identität liegt mit 32% zwischen Sec61p und Ssh1p (Sec sixty-one homolog 1) allerdings erheblich niedriger als in Säugerzellen. Sbh1p und Sbh2p (Sec sixty-one beta homolog 2) sind zu etwa 50% identisch. Für die gamma-Untereinheit Sss1p wurde kein paraloges Protein gefunden. Es konnte gezeigt werden, daß die beiden, hier neu beschriebenen Proteine Ssh1p und Sbh2p zusammen mit Sss1p einen eigenständigen, heterotrimeren Komplex, den Ssh1p-Komplex, bilden, der ebenfalls in der ER-Membran lokalisiert ist und eine Funktion beim Proteintransport durch diese Membran hat. Im Gegensatz zu SEC61 ist SSH1 nicht essentiell, wenn auch das Ssh1p für normale Wachstumsraten erforderlich ist. Die Deletion des SBH2-Gens zeigt keinen Phänotyp, was allerdings für die des SBH1-Gens ebenfalls gilt. Erst das Fehlen beider Proteine führt zu einem temperatur-abhängigen Wachstumsphänotyp und zu Defekten in der Translokation. Der entscheidende Unterschied zwischen dem Sec61p- und dem Ssh1p-Komplex scheint darin zu bestehen, daß der Sec61p-Komplex modulartig einsetzbar ist: als trimerer Komplex bei der kotranslationalen Translokation und im heptameren Sec-Komplex beim posttranslationalen Proteintransport. Demgegenüber deuten die Untersuchungen des Ssh1p-Komplexes darauf hin, daß dieser nicht mit dem tetrameren Sec62p-Sec63p-Komplex zum heptameren Sec-Komplex assoziieren kann. Vielmehr läßt er sich in Assoziation mit membrangebundenen Ribosomen nachweisen, was auf eine ausschließliche Funktion des Ssh1p-Komplexes bei der kotranslationalen Translokation schließen läßt. / Soluble or membrane proteins entering the secretory pathway are first translocated across the endoplasmic reticulum (ER) membrane. The proteins move through a channel formed by the heterotrimeric Sec61-complex. Homologues of the alpha- and gamma-subunit of this complex have been found in a wide variety of procaryotic and eucaryotic organisms indicating an evolutionary highly conserved mechanism for translocating proteins across membranes. Several recent investigations contributed to a complex understanding of this process. The work presented here sheds light on a completely new aspect of protein translocation across the ER-membrane: Interestingly genes coding for components of the Sec61-complex are found to have been duplicated independently in diverse eucaryotic organisms giving rise to so called paralogous genes (= intraspecies homologues) co-evolving after duplication. These paralogous genes and their gene products in mammalian cells as well as in the yeast Saccharomyces cerevisiae have been analyzed in detail. Mammalian cells possess two very similar SEC61a-genes (about 80% identity on nucleic acid level, about 94% on protein level). Their expression has been analyzed for several murine organs and embryonic stages. All tissues tested so far showed expression of both genes though the level of expression differed significantly: whereas expression of the already known SEC61a-I-gene seemed to be more or less constant across different tested organs, expression levels of the paralogous SEC61a-II-gene were not. Consequently the ability to differentially regulate the expression of the two paralogous SEC61a-genes may be of selective advantage for the mammalian organism. In the yeast Saccharomyces cerevisiae paralogous proteins of the alpha-subunit Sec61p as well as for the beta-subunit Sbh1p of the heterotrimeric Sec61p-complex can be found. The paralogous alpha-subunits Sec61p and Ssh1p (Sec sixty-one homolog 1) are less well conserved (32% identity) than the mammalian paralogues. Sbh1p and Sbh2p (Sec sixty-one beta homolog 2) are 50% identical. No paralogous protein was found for the gamma-subunit Sss1p. Instead, it could be shown that the new proteins Ssh1p and Sbh2p together with Sss1p are found in a heterotrimeric complex, the Ssh1p-complex, which like the Sec61p-complex localizes to the ER-membrane and has a function in translocating proteins across this membrane. In contrast to Sec61p Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and show translocation defekts in vivo as well as in vitro. The most intriguing difference between the Sec61p-and the Ssh1p-complex might be that the Sec61p-complex has a role in both co- and post-translational translocation pathways, as a separate entity and as part of the heptameris Sec-complex, respectively. However, there was no evidence for the Ssh1p-complex being part of an heptameric complex together with the tetrameric Sec62p-Sec63p-complex, but association of the trimeric Ssh1p-complex with membrane-bound ribosomes could be shown, making it likely that the Ssh1p-complex functions exclusively in the co-translational pathway of protein transport.

Les ligands des récepteurs nucléaires PPAR[gamma] (15-deoxy-[delta]12,14-Prostaglandine J2 (15d-PGJ2) et les thiazolidinediones (TZDs) : troglitazone (TGZ), ciglitazone (CGZ)) exercent un effet antiprolifératif sur les lignées cancéreuses mammaires. Plusieurs études suggèrent que les effets anticancéreux sont liés à des effets PPAR[gamma]-indépendants. Notre travail s?inscrit dans la compréhension de tels mécanismes d?action. Notre étude a montré une induction du facteur de transcription EGR1 (Early Growth Response gene 1) par certains ligands de PPAR[gamma] (TGZ, CGZ, 15d-PGJ2 et [delta]2-TGZ (agoniste inactif de PPAR[gamma])) dans les cellules cancéreuses mammaires hormono-dépendantes MCF7. Cet effet est précoce et PPAR[gamma]-indépendant. Il est lié à une libération quasi immédiate de calcium intracellulaire suivie de l?activation des ERK1/2. L?induction d?EGR1 a aussi lieu dans les cellules hormono-indépendantes MDA-MB-231 exposées à la [delta]2-TGZ. Cependant, l?induction d?EGR1 ne joue qu?un rôle partiel dans l?effet antiprolifératif. Les données d?une analyse par puce à ADN ont suggéré l?induction d?un stress du réticulum endoplasmique (RE) dans les cellules MCF7 exposées à la [delta]2-TGZ. Des analyses complémentaires ont confirmé que la [delta]2-TGZ induit un tel stress dans les cellules MCF7 et MDA-MB-231. Cependant, le rôle du stress du RE dans l?effet antiprolifératif de la [delta]2-TGZ reste à déterminer. Nous avons enfin testé l?hypothèse d?un lien entre EGR1 et stress du RE. En effet, EGR1 est aussi induit précocement par d?autres inducteurs de stress du RE. Les diverses situations où l?induction d?EGR1 est inhibée suggèrent une régulation possible de l?expression du facteur de transcription ATF3 par EGR1 / The ligands of PPAR[gamma] nuclear receptors (15-deoxy-[delta]12,14-Prostaglandin J2 (15d-PGJ2) and thiazolidinediones (TZDs) : troglitazone (TGZ), ciglitazone (CGZ)) show antiproliferative effects on breast cancer cell lines. Several studies suggest that the anti-cancer effects are PPAR[gamma]-independent. Our work is focused on the comprehension of such mechanisms of action. Our study has shown the induction of the transcription factor EGR1 (Early Growth Response gene 1) by some PPAR[gamma] ligands (TGZ, CGZ, 15d-PGJ2, and [delta]2-TGZ (PPAR[gamma] inactive agonist)) in the hormone-dependent breast cancer cells MCF7. This early effect is PPAR[gamma]-independent. It is associated with the almost immediate release of intracellular calcium followed by the activation of ERK1/2. EGR1 induction also occurs in the hormone-independent breast cancer cells MDA-MB-231 treated with [delta]2-TGZ. However, EGR1 induction plays only a partial role in the antiproliferative effect. Data analysis of DNA array has suggested the induction of an endoplasmic reticulum stress (ER) in MCF7 cells treated with [delta]2-TGZ. Complementary data have confirmed this result in MCF7 cells and in MDA-MB-231 cells. However, the role of ER stress in the antiproliferative effect is still to be determined. Finally, we have tested the hypothesis of a link between EGR1 and ER stress. Indeed, EGR1 is also early induced by other ER stress inductors. Diverse conditions where EGR1 is inhibited suggest a possible regulation of ATF3 expression by EGR1

Une carence en donneurs de méthyle (CDM) est fréquente dans les maladies inflammatoires chroniques intestinales (MICI) et induit un stress du réticulum endoplasmique (RE). La désacétylase SIRT1 (Sirtuine 1) régule la réponse cellulaire suite à un stress nutritionnel par le biais d’un stress du RE. Nous avons étudié pour la première fois l’impact d’une CDM sur le stress du RE et les effets de l'activation de SIRT1 dans un modèle expérimental de colite chez le rat. Pour restaurer l’homéostasie du réticulum endoplasmique, la cellule met en place la réponse UPR « Unfolded Protein Response ».Les quantités de formes actives des acteurs de l’UPR (effecteurs et régulateurs), ainsi que du facteur de transcription (HSF1) et SIRT1 ont été étudiés par western blot, immunoprécipitation et RT-qPCR sur des tissus coliques (rats et humains) et sur des lysats cellulaires. Les effets de l'activation de SIRT1 ont été étudiés in vitro et in vivo. Une CDM aggrave la colite induite par le DSS cliniquement, endoscopiquement et histologiquement. Une CDM induit un stress du RE en augmentant le niveau d’expression des formes active des acteurs de l’UPR : p-PERK, p-eIF-2a, p-IRE-1a, ATF6, XBP1-S et l’ARNm d’ATF4. Ceci est accompagné d’une diminution dramatique du niveau d’expression des protéines chaperonnes, d’une réduction du niveau d'expression de SIRT1 et d’une hyperacétylation de HSF1. L’ajout de la vitamine B12, de la S-Adénosine Methionine (SAM), ou un activateur de SIRT1 (SRT1720) réduit l'UPR in vitro. L’immunohistochimie a montré une diminution de l’expression de SIRT1 dans l'épithélium du côlon de patients atteints de MICI. Chez le rat, l’activation de SIRT1 prévient la colite en réduisant le niveau acétylé de HSF1 et en augmentant le niveau d’expression des molécules chaperonnes. L'activation de SIRT1 prévient la colite en réduisant le stress du RE causé par la CDM. Ces résultats suggèrent que SIRT1 pourrait être une cible thérapeutique dans les MICI où les CDM sont fréquentes / Methyl donor deficiency (MDD) aggravates experimental colitis in rats and decreases SIRT1 activity in cell models. Endoplasmic reticulum (ER) stress plays a key role in inflammatory bowel disease (IBD) pathogenesis. We investigated whether the influence of MDD on colitis resulted from an ER stress response triggered by decreased SIRT1 expression. The Unfolded protein response (UPR), chaperone proteins, heat-shock factor protein 1 (HSF1) and SIRT1 were examined in rats with MDD and dextran sulfate sodium (DSS)- induced colitis, in a Caco-2 cell model with stable expression of transcobalamin-oleosin chimera (TO), which impairs cellular availability of vitamin B12, and in human IBD. The effects of SIRT1 activation were studied both in vitro and in vivo. MDD aggravated DSS-induced colitis clinically, endoscopically and histologically. MDD activated ER stress pathways, with increased p-PERK, p-eiF-2a, p-IRE-1a, ATF6, XBP1-S protein and ATF4 mRNA expression levels in rats. This was accompanied by reduced SIRT1 expression level and greater acetylation of HSF1, in relation with a dramatic decrease of chaperons (BIP, HSP27 and HSP90). Adding either vitamin B12, Sadenosylmethionine, or a SIRT1 activator (SRT1720) reduced the UPR in vitro. In rats, SIRT1 activation by SRT1720 prevented colitis by reducing acetylation of HSF1 and increasing the expression of BIP, HSP27 and HSP90. Immunohistochemistry showed impaired expression of SIRT1 in the colonic epithelium of IBD patients. SIRT1 is a master regulator of ER stress and severity of experimental colitis in case of MDD. It could deserve further interest as a therapeutic target of IBD

Les mitochondries et le réticulum endoplasmique (RE) interagissent au niveau de points de contacts appelés « Mitochondria-Associated ER Membranes » (MAM), afin d'échanger du Ca2+ via le complexe TP3Rl/Grp75/VDACl et maintenir l'homéostasie énergétique. Bien que des dysfonctions mitochondriales, un stress du RE et des altérations de l'homéostasie du Ca2+ participent au développement de l'insulino-résistance, on ne sait pas si ce sont des facteurs indépendants ou s'ils sont inter-reliés par une altération des MAM. Mes travaux de thèse ont permis de mettre en évidence un nouveau rôle des MAM dans l'insulino-résistance hépatique. J'ai mis au point et validé la technique d'in situ PLA pour visualiser et quantifier les interactions mitochondrie-RE dans les cellules. J'ai montré que l'intégrité des MAM était nécessaire pour la signalisation de l'insuline dans le foie, et qu'un défaut d'intégrité des MAM était impliqué dans l'insulino-résistance hépatique. Des données préliminaires suggèrent qu'une altération des MAM est également associée à l'insulino-résistance musculaire. J'ai ensuite mis en évidence la présence de la protéine kinase B, une protéine clé de la signalisation de l'insuline, dans les MAM, et démontré que sa phosphorylation par l'insuline est altérée dans cette fraction dans le foie de souris diabétique. Enfin, j'ai participé à la mise en évidence l) de la présence de la cyclophilin D à l'interface des MAM régulant les échanges calciques entre les deux organites dans les cardiomyocytes et les hépatocytes, et 2) d'une régulation des MAM par le glucose dans le foie qui permet un contrôle de la dynamique et de la fonction mitochondriale au cours des transitions nutritionnelles. Par conséquent, mes travaux ont permis d'améliorer les connaissances actuelles sur les partenaires, la fonction et la régulation des MAM et de dévoiler les MAM comme une nouvelle cible pour moduler la signalisation de l'insuline et le métabolisme hépatique / Mitochondria-associated endoplasmic reticulum membranes (MAM) are functional domains between both organelles involved in Ca2+ exchange, through the voltage-dependent anion channel (VDAC)-1/glucose regulated protein 75 (Grp75)/inositol 1,4,5-triphosphate receptor (TP3R)-1 complex, and regulating energy metabolism. Whereas mitochondrial dysfunction, ER stress, and altered Ca2+ homeostasis are associated with altered insulin signalling, the implication of MAM dysfunctions in insulin resistance is unknown. During my PhD, my work has underlined a new role of MAM in hepatic insulin- resistance. T have developed a quantitative method called in situ Proximity Ligation Assay to visualise and quantify the interactions between ER and mitochondria. T have shown that MAM integrity is required for insulin signalling and that disruption of MAM is implicated in hepatic insulin resistance. Preliminary data also suggest that MAM alterations are also associated with muscle insulin resistance. T have also identified the presence of the protein kinase B (PKB), a key protein involved in metabolic effects of insulin, at the MAM interface, and demonstrated that its phosphorylation by insulin is altered in this fraction in liver of diabetic mice. Lastly, T have also participated to the identification of: 1) the presence of cyclophilin D (CypD) at MAM interface which regulates calcium transfer from ER to mitochondria in both cardiomyocytes and hepatocytes, and 2) a regulation of MAM by glucose in liver, which is involved in the regulation of mitochondria dynamics and function during nutritional transitions. Consequently, my work improved the knowledge on the composition, function and regulation of MAM, and highlighted MAM as a potential new target for the modulation of hepatic insulin action and metabolism

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.

Como a clorexidina (CHX) tem sido recomendada como solução irrigadora de canais radiculares e como curativo de demora, o objetivo do presente estudo foi caracterizar in vivo a lesão induzida pela injeção de CHX a 0,125, 0,25, 0,5 e 1,0% na pata de camundongos em intervalos de tempo selecionados (24 e 48 horas e 7 e 14 dias) e in vitro o modo e a causa morte celular (necrose e/ou apoptose) e o estresse causado pela exposição de fibroblastos L929 em cultura a concentrações crescentes da droga (0,000125, 0,00025, 0,0005, 0,001, 0,002, 0,004, 0,008 e 0,016%) por 24 horas. A proliferação celular foi avaliada por meio da incorporação de metil-3H-timidina ao DNA das células e imunomarcação para PCNA. A ultraestrutura foi analisada em microscópio eletrônico de transmissão e de varredura e o citoesqueleto das células por meio de marcação para actina e α-tubulina. Citometria de fluxo (Anexina-V FITC/iodeto de propídeo) foi empregada para diferenciar células necróticas de apoptóticas. Também, foi efetuada marcação para retículo endoplasmático, Bcl-2 (B-célula CLL/linfoma 2), Hsp70 (proteína de choque térmico 70) e Grp78 (glucose-regulated protein 78). Quando injetada no espaço subplantar da pata traseira de camundongos, a CHX induziu alterações necróticas na epiderme, derme e tecido subcutâneo em associação com uma resposta inflamatória reacional, particularmente nas concentrações mais elevadas. Em cultura de fibroblastos, a CHX induziu a diminuição da proliferação celular, causou morte celular por apoptose e necrose, desestruturação do citoesqueleto, alteração da morfologia celular, aumento da área das células, dilatação do retículo endoplasmático rugoso e acúmulo de proteínas nas cisternas. Além disso, a CHX causou aumento da expressão de Hsp70, de Grp78 (indicadores de estresse celular) e de Bcl-2 (proteína anti-apoptótica). Em conclusão, a CHX injetada no espaço subplantar da pata traseira de camundongos induz efeitos tóxicos severos. Além disso, a CHX adicionada em cultura de fibroblastos causou estresse do retículo endoplasmático como consequência do acúmulo de proteínas nas cisternas e induziu a morte celular por necrose e apoptose via estresse do retículo endoplasmático, além de causar estresse celular. Os resultados sugerem que a CHX poderia ter um efeito desfavorável na resolução de lesões periapicais em decorrência de sua ação tóxica sobre as células do tecido em torno do ápice dentário. / Because chlorhexidine (CHX) has been recommended as either endodontic irrigant or root canal dressing, this study aimed to characterize in vivo the lesion induced by injection of CHX at concentrations of 0.125, 0.25, 0.5 and 1.0% in the paw of mice at selected time intervals (24 and 48 hours and 7 and 14 days) and in vitro the mode and the cause of cell death (necrosis and /or apoptosis), and the cellular stress caused by exposition of cultured L929 fibroblasts to ascending concentrations of the drug ( 0.000125, 0.00025, 0.0005, 0.001, 0.002, 0.004, 0.008 and 0.016%) for 24 hours. The cell proliferation assay was performed by measuring incorporation of metil-3H-thymidine to cell DNA and immunocytochemical staining analysis of PCNA. The cell ultrastructure was analyzed in transmission and scanning electron microscopes and the cell cytoskeleton by florescence analysis of actin and α- tubulin. Apoptosis and necrosis were discriminated by flow cytometry with annexin V-FITC and PI. In addition endoplasmic reticulum, Bcl-2 (B-cell CLL/lymphoma 2), Hsp70 (heat shock protein 70) and Grp78 (glucoseregulated protein 78) were detected by fluorescence. CHX injected in the sub plantar space of the hind paw of mice induced necrotic changes in the epidermis, dermis and subcutaneous tissue in association with reactive inflammatory response, particularly at higher concentrations. In cultured fibroblasts, CHX decreased cellular proliferation, caused cell death by apoptosis and necrosis, disruption of the cytoskeleton, morphological cellular alterations, increased cells size (area), rough endoplasmic reticulum dilatation with accumulation of cysternal protein. In addition, CHX induced increased expression of Hsp 70, Grp78 (indicators of cellular stress) and Bcl-2 (anti-apoptotic protein). It was concluded that CHX injected in the sub plantar space of the hind paw of mice could induce severe toxic effect. In addition CHX caused endoplasmic reticulum stress as a consequence of accumulation of proteins in the endoplasmic reticulum cysterns and induced cell death by apoptosis and necrosis via endoplasmic reticulum stress and caused cellular stress. Taken together, these findings suggest that CHX may have an unfavorable effect on the resolution of apical periodontitis, due a toxic effect on the periapical tissue cells.

The cytochrome P450 enzyme system is a multicomponent electron-transfer chain composed of a haem-containing monooxygenase cytochrome P450 (CYP) and one or more redox partners. Eukaryotic CYPs and their redox partner NADPH-dependent cytochrome P450 oxidoreductase (CPR) are involved in many biological processes. Each protein has one N- terminal membrane anchor domain for location within the endoplasmic reticulum (ER). In mammals, CYPs and CPR are especially abundant in liver cells, where they play important roles in the metabolism of steroids, fatty acids, and xenobiotic compounds including numerous drugs of pharmaceutical importance. Incorporation into lipid membranes is an important aspect of CYP and CPR function, influencing their kinetic properties and interactions. In this thesis, soluble nanometer-scale phospholipid bilayer membrane discs, "nanodiscs", were used as a reconstitution system to study the influence of lipid membrane composition on the activities of the abundant human CYP3A4 and human CPR. Both enzymes were expressed and purified from bacteria, and assembled into functionally active membrane-bound complexes in nanodiscs. Nanodisc assembly was assessed by a combination of native and denaturing gel electrophoresis, and a fluorimetric assay was developed to study CYP3A4 reaction kinetics using 7-benzyloxyquinoline as substrate. Kinetic properties were investigated with respect to different lipid membrane compositions: phosphatidyl choline; a synthetic lipid mixture resembling the ER; and natural lipids extracted from liver microsomes. Full activity of the CYP3A4 system, with electron transfer from NADPH via CPR, could only be reconstituted when both CYP3A4 and CPR were membrane-bound within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated seperately into naodiscs then mixed together, or when soluble forms of CPR were mixed with pre-assembled CYP3A4-nanodiscs. Thus, assembly of the two proteins within the same membrane was shown to be essential for the function of the CPR-CYP3A4 electron transfer system. Comparison of the reaction kinetics in different membrane compositions revealed liver microsomal lipid to have an enhancing effect both on the activity of the assembled CPR-CYP3A4 nanodisc complex, and on the activity of CPR alone incorporated in nanodiscs, when compared either to the synthetic lipid mixture or to phosphatidyl choline alone. Thus, natural lipids appear to possess properties or include components important for the catalytic function of the CYP system, which are absent from synthetic lipid. Input of electrons, measured by NADPH consumption, exceeded product formation rate by the CPR-CYP3A4 complex in nanodiscs, indicating "leakage" in the electron flow, possibly due to uncoupling of the two enzymes. Uncoupling was shown to occur by developing a novel fluorimetric method using the dye MitSOX to detect superoxide production. The significance of this, and to what extent control of coupling could be a natural means of regulation of the CPR-CYP system, remains to be determined. Thus, phospholipid bilayer nanodiscs prove a powerful tool to enable detailed analysis of the reaction kinetics of membrane-reconstituted CPR-CYP systems, and to allow pertinent questions to be addressed concerning the integral significance of the membrane environment.

As doenças cardiovasculares são a principal causa de morte no mundo, sendo a cardiomiopatia isquêmica a mais prevalente. Independente da sua etiologia, a via final comum da maioria das doenças cardiovasculares é a insuficiência cardíaca. Nos últimos anos, tem sido reportado que o acúmulo de proteínas mal enoveladas no retículo endoplasmático (estresse do RE) pode contribuir para redução da função cardíaca e instalação da insuficiência cardíaca. Apesar do mecanismo responsável pela disfunção contrátil induzida pelo estresse do RE ainda não ser conhecido, evidências sugerem que a inibição da via de sinalização PI3K/AKT pela proteína JNK pode estar envolvida nessa resposta. Na primeira parte desta tese, verificamos que a indução do estresse do RE em cardiomiócitos isolados ativou a JNK, mas não inibiu a via de sinalização PI3K/AKT. A inativação de JNK reverteu a disfunção contrátil e a redução da amplitude do transiente de Ca+2 de cardiomiócitos causados pelo estresse do RE. Pelo fato da via sinalização PI3K/AKT não estar envolvida na disfunção contrátil causada pelo estresse do RE, analisamos outro alvo de JNK, a proteína BNIP3, proteína pró-apoptótica e envolvida no controle de qualidade mitocondrial promovendo mitofagia quando ativada. O estresse do RE aumentou a expressão de BNIP3, a qual foi atenuada pela inibição de JNK. A depleção de BNIP3 impediu a disfunção contrátil dos cardiomiócitos e a redução da amplitude do transiente de Ca+2 induzidos pelo estresse do RE. Na segunda parte da tese, o objetivo foi avaliar se os efeitos observados em cardiomiócitos submetidos a estresse do RE poderiam ser observados em modelo experimental de doença cardiovascular. Nesse sentido, observamos que a disfunção cardíaca provocada pelo infarto do miocárdio em ratos foi acompanhada pelo quadro de estresse do RE e pela ativação da via de sinalização JNK/BNIP3. Entretanto, o treinamento físico aeróbico (TFA), uma das principais terapias não farmacológicas mais eficazes das doenças cardiovasculares, foi capaz de atenuar o estresse RE, a ativação da via de sinalização JNK/BNIP3 e a disfunção cardíaca de ratos infartados. Na terceira parte da tese, verificamos que o TFA aumentou a expressão proteica de DERLIN-1, uma proteína que atua retro-translocando proteínas mal enoveladas para o citosol, no miocárdio de ratos saudáveis. O aumento dos níveis proteicos de DERLIN-1 observado em ratos infartados foi atenuado pelo TFA. Apesar do aumento da proteína DERLIN-1, observamos que nos animais infartados as proteínas mal enoveladas acumulavam na forma de oligômeros e que o TFA atenuou essa resposta. Em conjunto, os resultados da presente tese sugerem que a ativação da via de sinalização JNK/BNIP3 pelo estresse do RE causa disfunção contrátil de cardiomiócitos e que o TFA é capaz de atenuar essa resposta no coração de ratos infartados, melhorando o controle de qualidade de proteína no músculo cardíaco / Cardiovascular diseases are currently the main cause of death worldwide, with the ischemic cardiomyopathy as the most prevalent ethiology. This is of particular interest, since ischemic cardiomyopathy advances to heart failure, a common endpoint of the most cardiovascular disease. In the last years, it has been showed that accumulation of unfolded protein in the endoplasmic reticulum (ER stress) may cause cardiac dysfunction and heart failure development. Despite the mechanisms behind this cardiac deterioration is still unknown, evidences suggest that ER stress-induced cardiomyocytes contractile dysfunction results from PI3K/AKT signaling pathway inhibition, which would be caused by JNK activation. In the first part of this thesis, we found that the ER stress activated JNK, but different from our hypothesis it was not accompanied by an inactivation of PI3K/AKT signaling pathway. The inhibition of JNK mitigated the reduction in cardiomyocytes shortening and amplitude of Ca+2 transient caused by ER stress. Once the PI3K/AKT signaling pathway was not involved in the ER stress-induced cardiomyocytes contractile dysfunction, we have analyzed protein expression of BNIP3, another JNK target involved in apoptosis and mitochondria quality control. We observed that the elevation in BNIP3 proteins levels after ER stress induction was prevented by inhibition of JNK. BNIP3 depletion attenuated the reduction in cardiomyocytes contractility and amplitude of Ca+2 transient induced by ER stress. In the second part of the thesis, we found that myocardial infarction-induced cardiac dysfunction in rats was accompanied by ER stress and activation of JNK/BNIP3 signaling pathway. However, the AET mitigated ER stress, activation of JNK/BNIP3 signaling pathway and cardiac dysfunction in infarcted rats. In third part of the thesis, we have identified that AET increased the protein expression of DERLIN-1 an ER membrane protein that retro-translocates unfolded proteins to cytosol in the myocardial of healthy rats. We observed that the increased DERLIN-1 protein levels in infarcted rats were mitigated by AET. Despite increased DERLIN-1 protein expression, we found high levels of oligomers in the myocardium of infarcted rat, which was reduced by AET. It suggests that unfolded protein degradation was reduced in infarcted hearts. Taken together, these results suggest that ER stress causes cardiomyocytes contractile dysfunction through JNK/BNIP3 signaling pathway activation and that AET mitigates the myocardial infarction-induced ER stress and activation of JNK/BNIP3 signaling pathway by restoring of ER-associated protein quality control in the cardiac muscle

Como a clorexidina (CHX) tem sido recomendada como solução irrigadora de canais radiculares e como curativo de demora, o objetivo do presente estudo foi caracterizar in vivo a lesão induzida pela injeção de CHX a 0,125, 0,25, 0,5 e 1,0% na pata de camundongos em intervalos de tempo selecionados (24 e 48 horas e 7 e 14 dias) e in vitro o modo e a causa morte celular (necrose e/ou apoptose) e o estresse causado pela exposição de fibroblastos L929 em cultura a concentrações crescentes da droga (0,000125, 0,00025, 0,0005, 0,001, 0,002, 0,004, 0,008 e 0,016%) por 24 horas. A proliferação celular foi avaliada por meio da incorporação de metil-3H-timidina ao DNA das células e imunomarcação para PCNA. A ultraestrutura foi analisada em microscópio eletrônico de transmissão e de varredura e o citoesqueleto das células por meio de marcação para actina e α-tubulina. Citometria de fluxo (Anexina-V FITC/iodeto de propídeo) foi empregada para diferenciar células necróticas de apoptóticas. Também, foi efetuada marcação para retículo endoplasmático, Bcl-2 (B-célula CLL/linfoma 2), Hsp70 (proteína de choque térmico 70) e Grp78 (glucose-regulated protein 78). Quando injetada no espaço subplantar da pata traseira de camundongos, a CHX induziu alterações necróticas na epiderme, derme e tecido subcutâneo em associação com uma resposta inflamatória reacional, particularmente nas concentrações mais elevadas. Em cultura de fibroblastos, a CHX induziu a diminuição da proliferação celular, causou morte celular por apoptose e necrose, desestruturação do citoesqueleto, alteração da morfologia celular, aumento da área das células, dilatação do retículo endoplasmático rugoso e acúmulo de proteínas nas cisternas. Além disso, a CHX causou aumento da expressão de Hsp70, de Grp78 (indicadores de estresse celular) e de Bcl-2 (proteína anti-apoptótica). Em conclusão, a CHX injetada no espaço subplantar da pata traseira de camundongos induz efeitos tóxicos severos. Além disso, a CHX adicionada em cultura de fibroblastos causou estresse do retículo endoplasmático como consequência do acúmulo de proteínas nas cisternas e induziu a morte celular por necrose e apoptose via estresse do retículo endoplasmático, além de causar estresse celular. Os resultados sugerem que a CHX poderia ter um efeito desfavorável na resolução de lesões periapicais em decorrência de sua ação tóxica sobre as células do tecido em torno do ápice dentário. / Because chlorhexidine (CHX) has been recommended as either endodontic irrigant or root canal dressing, this study aimed to characterize in vivo the lesion induced by injection of CHX at concentrations of 0.125, 0.25, 0.5 and 1.0% in the paw of mice at selected time intervals (24 and 48 hours and 7 and 14 days) and in vitro the mode and the cause of cell death (necrosis and /or apoptosis), and the cellular stress caused by exposition of cultured L929 fibroblasts to ascending concentrations of the drug ( 0.000125, 0.00025, 0.0005, 0.001, 0.002, 0.004, 0.008 and 0.016%) for 24 hours. The cell proliferation assay was performed by measuring incorporation of metil-3H-thymidine to cell DNA and immunocytochemical staining analysis of PCNA. The cell ultrastructure was analyzed in transmission and scanning electron microscopes and the cell cytoskeleton by florescence analysis of actin and α- tubulin. Apoptosis and necrosis were discriminated by flow cytometry with annexin V-FITC and PI. In addition endoplasmic reticulum, Bcl-2 (B-cell CLL/lymphoma 2), Hsp70 (heat shock protein 70) and Grp78 (glucoseregulated protein 78) were detected by fluorescence. CHX injected in the sub plantar space of the hind paw of mice induced necrotic changes in the epidermis, dermis and subcutaneous tissue in association with reactive inflammatory response, particularly at higher concentrations. In cultured fibroblasts, CHX decreased cellular proliferation, caused cell death by apoptosis and necrosis, disruption of the cytoskeleton, morphological cellular alterations, increased cells size (area), rough endoplasmic reticulum dilatation with accumulation of cysternal protein. In addition, CHX induced increased expression of Hsp 70, Grp78 (indicators of cellular stress) and Bcl-2 (anti-apoptotic protein). It was concluded that CHX injected in the sub plantar space of the hind paw of mice could induce severe toxic effect. In addition CHX caused endoplasmic reticulum stress as a consequence of accumulation of proteins in the endoplasmic reticulum cysterns and induced cell death by apoptosis and necrosis via endoplasmic reticulum stress and caused cellular stress. Taken together, these findings suggest that CHX may have an unfavorable effect on the resolution of apical periodontitis, due a toxic effect on the periapical tissue cells.

Atualmente, a obesidade é considerada uma epidemia mundial. Está associada a um estado de inflamação crônica e ativação do estresse do retículo endoplasmático (ERE), relacionados à patogênese de diversas doenças como diabetes mellitus tipo 2, doenças cardiovasculares, câncer, hipertensão, dentre outras. Nesse contexto, são necessários estudos para encontrar alternativas que melhorem o processo inflamatório. Vários estudos em humanos e animais já demostraram as propriedades anti-inflamatórias do ácido graxo ômega-3. O objetivo do nosso trabalho foi avaliar os efeitos da suplementação do ácido alfa-linolênico (ALA) no metabolismo e no estresse do retículo endoplasmático em obesos grau III. Foi conduzido um estudo prospectivo, randomizado, placebo-controlado, duplo-cego. No total, 52 pacientes foram randomizados para a suplementação com 3g/dia de ALA ou placebo, sendo 27 indivíduos do grupo ômega-3/ALA e 25 do grupo controle. Foi avaliado perfil lipídico, glicídico e inflamatório antes e após suplementação. O tecido adiposo visceral (TAV) foi coletado durante cirurgia bariátrica após suplementação. O perfil de ácidos graxos incorporados no TAV foi avaliado por cromatografia gasosa. Os genes foram avaliados através de PCR em tempo real. Não houve alteração nos níveis séricos de IL-6 (p=0,2201), TNF-? (0.7703) e PCR (p=0,57) após suplementação com ômega-3/ALA, porém observamos diminuição nos níveis séricos de Leptina (p=0.0154) e IP-10 (p=0.0410), citocinas inflamatórias, e aumento na IL-4 (p=0,0211), citocina anti-inflamatória. Foi observado incorporação significativa do ALA (p=0,0002), EPA (p<0,0001) e DHA (p=0,0005) no TAV. Avaliação molecular evidenciou um aumento da expressão gênica do XBP1 (p=0,0013), sXBP1 (p<0,0001), EIF2-? (p=0,0063) e da chaperona CCT4 (p=0,0001) e diminuição na expressão gênica da leptina (p=0,0410). Podemos concluir que o ALA pode modular o ERE através da via da IRE1/XBP, levando ao aumento das chaperonas (CCT4), o que pode demonstrar um potencial terapêutico do ALA em pacientes obesos. / Currently, obesity is considered a worldwide epidemic. It is associated with chronic inflammation and stress activation of the endoplasmic reticulum (ERE), related to the pathogenesis of various diseases such as type 2 diabetes mellitus, cardiovascular diseases, cancer, hypertension, among others. In this context, studies are needed to find alternatives that improve the inflammatory process. Several studies in humans and animals have already demonstrated the anti-inflammatory properties of omega-3 fatty acid. The objective of our study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation on the metabolism and stress of the endoplasmic reticulum in obese patients. A prospective, randomized, placebo-controlled, double-blind study was conducted. In total, 52 patients were randomized to supplementation with 3 g / day of ALA or placebo, 27 individuals from the omega-3 / ALA group and 25 from the control group. Lipid, glycidic and inflammatory profile were evaluated before and after supplementation. Visceral adipose tissue (TAV) was collected during bariatric surgery after supplementation. The fatty acid profile incorporated in the TAV was evaluated by gas chromatography. Genes were evaluated by real-time PCR. There was no change in serum levels of IL-6 (p = 0.2201), TNF-? (0.7703) and CRP (p = 0.57) after supplementation with ALA, but we observed a decrease in serum Leptin levels (P = 0.0154) and IP-10 (p = 0.0410), inflammatory cytokines, and increase in IL-4 (p = 0.0211), anti-inflammatory cytokine. Significant incorporation of ALA (p = 0.0002), EPA (p <0.0001) and DHA (p = 0.0005) into the TAV was observed. Molecular evaluation showed an increase in the gene expression of XBP1 (p = 0.0013), sXBP1 (p <0.0001), EIF2-? (p = 0.0063), GADD34 (p=0,0117) and CCT4 chaperone (p = 0.0001), decrease in the gene expression of leptin (p = 0.0410) and ATF-6 (p=0,0305) and a tendency to decrease the gene expression of CHOP. We can conclude that ALA can modulate ERE through the IRE1 / XBP, PERK and ATF-6 pathways, leading to increased chaperones (CCT4), which may demonstrate a therapeutic potential of ALA in obese patients.