The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown. Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis. In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%). The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop. We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate. The third objective of this work was to obtain a broad picture of the pancreatic beta-cell molecular responses during and after (recovery period) a severe ER stress. For this purpose, we utilized an “in home” spotted microarray, the APOCHIP, containing nearly 600 probes selected for the study of beta-cell apoptosis. Time-dependent gene expression profiles were measured in INS-1E cells exposed to CPA. CPA-induced ER-stress modified expression of 183 genes in at least one of the time points studied. Most of theses genes returned to control levels 3h after CPA removal from the culture medium. We observed full beta-cell recovery and survival, indicating that these cells trigger efficient defenses against ER stress. Beta-cell recovery is associated with a sustained increase in the expression of ER chaperones and a rapid decrease of pro-apoptotic mRNAs following CPA removal. Two groups of genes were particularly affected by CPA, namely those related to the cellular responses to ER stress, which were mostly up-regulated, and those related to differentiated beta-cell functions, which were down-regulated. Among this last group, we observed a 40-90% decrease of the mRNAs for insulin-1 and -2. These findings were confirmed in INS-1E cells exposed to cytokines or thapsigargin (another SERCA blocker), and in primary beta-cells exposed to the same treatments. This decrease in insulin mRNA expression is due to transcript degradation, most probably caused by IRE-1 activation and triggering of its endoribonuclease activity, as recently described in Drosophila cells. In conclusion, our work enabled a better understanding of the pancreatic beta-cell responses to ER stress: 1.)We identified a sensitizing effect of IFN-gamma to ER stress in beta-cells via downregulation of key ER chaperones. 2.)We observed a differential regulation of Chop transcription by different treatments suggesting distinct responses of pancreatic beta-cells to diverse ER stress inducers. 3.)We provided the first global analysis of gene expression modifications in pancreatic beta-cells following ER stress. 4.)We demonstrated a high capacity of beta-cells to cope and recover from a severe ER stress. 5.)We identified a new protective mechanism against ER stress, namely the degradation of insulin mRNA which limits the load posed on the ER by insulin synthesis. This, coupled to a marked increase in ER chaperones and a fast degradation of pro-apoptotic mRNAs, enables beta cells to recover from ER stress after the causes of this stress are removed.

The heat shock or stress response is a transient response to stressful stimuli that protects vital cellular proteins from damage and irreversible aggregation. Heat shock proteins (Hsps) are molecular chaperones that bind to unfolded protein and inhibit their aggregation, thereby maintaining their solubility until they can be refolded to their native conformation. Hsp47 is an endoplasmic reticulum (ER)-resident protein that serves as a molecular chaperone during collagen production. Collagen is the major class of insoluble fibrous protein found in the extracellular matrix and in connective tissues. It is the single most abundant protein of the animal kingdom; at least 14 different forms exist, each with distinct structures and binding properties. The various types of collagen all possess protein regions with the distinct triple helical conformation. This complex physical structure requires very organized assembly and HSP47 has been established as an integral component of this process for collagen types I-V. Most of the previous studies examining the expression and function of hsp47 have been conducted with mammalian cultured cells. The present study represented the first investigation of the expression of hsp47 in the poikilothermic vertebrate, <i>Xenopus laevis</i>. Full-length <i>Xenopus</i> hsp47 nucleotide and amino acid sequences were obtained from Genbank and compared with hsp47 from chicken, mouse, rat, human and zebrafish. <i>Xenopus</i> HSP47 protein had an identity of approximately 77% with chicken, 73% with mouse, 72% with rat and human, and 70% with zebrafish. Most of the sequence identity between HSP47 from all investigated organisms occurred centrally in the amino acid sequence and in several carboxyl terminal regions. Three key features were conserved between HSP47 proteins from most species investigated: a hydrophobic leader sequence, two potential glycosylation sites and the ER-retention signal, RDEL. A partial cDNA clone encoding <i>Xenopus</i> hsp47 was obtained from the American Type Culture Collection (ATCC) and used to generate hsp47 antisense riboprobe for the purpose of investigating hsp47 mRNA accumulation in <i>Xenopus</i> A6 kidney epithelial cells and embryos. Northern blot analysis detected hsp47 mRNA constitutively in A6 cells. The expression pattern for hsp47 mRNA was compared with two other <i>Xenopus</i> heat shock proteins that have been previously characterized in our laboratory: hsp70, a cystolic/nuclear hsp and BiP, an ER-resident hsp. The results of hsp47 mRNA accumulation in A6 cells suggested that the expression pattern for <i>Xenopus</i> hsp47 was unique but, with respect to some stressors, resembled that of a cytosolic hsp rather than an ER-resident hsp. HSP47 protein levels were also examined in A6 cells. Heat shock, sodium arsenite and b-aminopropionitrile fumerate treatments enhanced hsp47 accumulation. In some experiments, western blot analysis revealed the presence of two closely sized protein bands. It is possible that minor differences in HSP47 protein size may be due to post-translational modification, namely phosphorylation or glycosylation. The present study also examined the accumulation and spatial pattern of hsp47 mRNA accumulation during <i>Xenopus laevis</i> early development. Hsp47 was constitutively expressed throughout <i>Xenopus</i> early development. Constitutive levels of hsp47 mRNA in unfertilized eggs, fertilized eggs and cleavage stage embryos indicated that these transcripts were maternally inherited. Constitutive hsp47 mRNA accumulation was enhanced in neurula and tailbud embryos compared to earlier stages. This finding may be explained by the shift towards organogenesis during these stages. Whole mount <i>in situ</i> hybridization revealed hsp47 message along the dorsal region of the embryo, in the notochord and somites, as well as in the head region including the eye vesicle. Hsp47 mRNA induction in <i>Xenopus</i> embryos was also examined in response to heat shock. Hsp47 mRNA accumulated in response to heat shock immediately following the midblastula transition (MBT). In tailbud stages, hsp47 mRNA accumulated in the notochord, somites and head region. Northern blot analysis and whole mount <i>in situ</i> hybridization results revealed an expression pattern that coincided well with the development of collagen-rich tissues thereby substantiating the proposed role of HSP47 as a procollagen molecular chaperone.

No description available.

Obligate intracellular pathogens must acquire host cell-derived nutrients to facilitate their survival. One such bacterial pathogen, Anaplasma phagocytophilum, replicates within neutrophils and non-phagocytic cells in a bacterial-modified, host cell-derived vacuole. The bacterium exploits host cell vesicular trafficking pathways to route nutrients to its vacuole and utilizes Rab GTPases, guanine nucleotide-dependent, vesicular trafficking regulators, to do so. We previously discovered that the A. phagocytophilum vacuolar membrane is decorated with a specific subset of Rab GTPases - Rab1, Rab4A, Rab10, Rab11, Rab14, Rab22A and Rab35. Rab1 is exclusively found on the endoplasmic reticulum (ER) and thus its localization suggests that the bacterium intercepts the ER. Rab10, which is found on the ER, trans-Golgi and recycling endosomes, localizes to the vacuolar membrane in a guanine nucleotide-independent and bacterial protein synthesis-dependent manner. This suggests that a bacterial-encoded protein is binding to and recruiting Rab10. In this study, we determined that A. phagocytophilum hijacks two very nutrient-rich sources in the secretory pathway - trans-Golgi- and endoplasmic reticulum-derived vesicles. A. phagocytophilum localizes perinuclearly adjacent to the Golgi apparatus during infection. A. phagocytophilum and Anaplasma marginale, an intravacuolar bovine pathogen, also localize near the smooth ER and rough ER in both mammalian and tick host cells. These results are supported by transmission electron microscopy analyses of infected cells. Membrane markers for the rough ER label the peripheries of A. marginale and A. phagocytophilum organisms in both mammalian and tick host cells, which suggests that they are translocated into the pathogen vacuole. Furthermore, membrane markers for trans-Golgi-derived vesicles, including endogenous Rab10, label the periphery of intravacuolar A. phagocytophilum organisms. Markers for the trans-Golgi and the ER co-fractionate with A. phagocytophilum in density gradient centrifugation studies. siRNA knockdown of Rab10 pronouncedly reduces delivery of trans-Golgi markers into the pathogen-occupied vacuole, significantly reduces infection, and impedes bacterial conversion to the bacterium’s dense-cored form. These results suggest that trans-Golgi recruitment is Rab10 dependent and is critical for bacterial development. We identified an outer membrane A. phagocytophilum moonlighting protein, uridine monophosphate kinase that specifically binds GST-Rab10 in affinity chromatography assays and interacts with Rab10 in vivo. We hypothesize that this surface protein is mediating the interaction of the bacteria with intravacuolar trans-Golgi derived vesicles. This interaction could be critical for the delivery of essential nutrients. Taken together, these data suggest that nutritional virulence mechanisms of A. phagocytophilum and A. marginale target the host secretory pathway. Additionally, they suggest a novel mechanism whereby pathogens translocate nutrient rich vesicles into the pathogen vacuole, thus delivering essential nutrients right to their front door.

L’adaptation cellulaire au stress dépend en partie de changements dans l’expression de gènes de réponse au stress, souvent accompagnés par des modifications dans la structure chromatinienne. Des facteurs chromatiniens pourraient être à l’origine de ces modifications mais leurs mécanismes d’action restent mal connus au cours du développement. La réponse aux protéines malconformées (UPR) est une réponse à des conditions de stress physiologique qui ciblent le réticulum endoplasmique (RE) ; l’UPR a été impliquée dans de nombreuses maladies humaines incluant le cancer et différents composants de cette réponse pourraient être de potentielles cibles pharmaceutiques. Nous avons démontré que HPL-2, l’homologue de la protéine HP1 chez Caenorhabditis elegans, est nécessaire pour la réponse au stress du RE. L’inactivation d’HPL-2 montre une résistance accrue au stress du RE qui dépend d’une part de la voie XBP-1 de l’UPR et d’autre part d’un flux autophagique augmenté. La résistance accrue des vers dépourvu d’HPL-2 est associée avec une augmentation de l’activation d’XBP-1 et de chaperonnes du RE en conditions physiologiques. L’expression d’HPL-2 est ubiquitaire et nous avons déterminé qu’HPL-2 joue un rôle antagoniste dans les cellules neuronales et intestinales pour influencer la réponse au stress du RE. Nous avons également montré qu’une modulation de l’état de la chromatine par une inhibition chimique d’histones déacétylases donnait le même phénotype que l’absence d’HPL-2. De plus, l’augmentation ou la diminution de la méthylation de la lysine 4 de l’histone 3 (H3K4me) joue également un rôle dans la réponse au stress du RE. Ces travaux contribuent ainsi à une meilleure compréhension du lien entre l’UPR, le stress du RE et la structure chromatinienne aussi bien dans un processus normal que dans certaines pathologies. / Cellular adaptation to environmental changes and stress relies on a wide range of regulatory mechanisms which are tightly controlled at several levels, including transcription. Chromatin structure and chromatin binding proteins are important factors contributing to the transcriptional response to stress. However, it remains largely unknown to what extent specific chromatin factors influence these distinct responses in a developmental context. One of the best characterized stress response pathways is the unfolded protein response (UPR), which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). Here, we show that Caenorhabditis elegans HPL-2, the homologue of the HP1 chromatin associated protein, is required for the ER stress response. Inactivation of HPL-2 results in enhanced resistance to ER stress dependent on the XBP-1 branch of the UPR and the closely related process of autophagy. Increased resistance to ER stress in animals lacking HPL-2 is associated with increased basal levels of XBP-1 activation and ER chaperones under physiological conditions. Using tissue specific rescue experiments, we find that HPL-2 plays antagonistic roles in intestinal and neuronal cells to influence the ER stress response. We further show that chemical inhibition of histone deacetylase activity mimics the HPL-2 loss of function phenotype, and that increasing or decreasing histone H3 lysine 4 methylation (H3K4me) has antagonistic effects on animal survival in response to ER stress. Altogether our results point to an important function for specific chromatin factors and chromatin modifications in maintaining ER homeostasis in a developmental context.

<p>mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.</p> / Dissertation

Photodynamic therapy (PDT) is a treatment modality for cancer. It combines selective accumulation of chemical compounds, called photosensitizers (PS), with light to irreversibly damage cancer cells via oxidative stress. The main goal of this thesis was to study photosensitizers represented by a unique group of newly synthesized porphyrin derivatives with glycol chain substitution. Glycol-functionalized porphyrins containing one to four low molecular weight glycol chains that are linked via ether bonds to the meta-phenyl positions of meso-tetraphenylporphyrin (mTPP(EG)1-4) were compared with fluorinated (pTPPF(EG)4) and nonfluorinated (TPP(EG)4) derivatives having glycol chains in para-phenyl positions. The cellular uptake and photodynamic activity was significantly dependent on terminal groups of the glycol substituent. Hydroxy glycol porphyrins, in contrast with methoxy glycol porphyrins, exhibited efficient intracellular transport and high induction of apoptosis in tumor cell lines in vitro. After initial testing effective prototype hydroxy ethylene glycol derivatives were selected and analyzed in detail. Para derivatives pTPP(EG)4 and pTPPF(EG)4 accumulated mainly in lysosomes whereas meta derivatives mTPP(EG)1-4 in the endoplasmic reticulum (ER). Position of ethylene glycol chain on the...

Este estudo descreve um novo método para a produção da Ca2+-ATPase do retículo sarcoplasmático de coelho em levedura utilizando um vetor de expressão regulado por choque térmico. Após solubilização das membranas de levedura com lisofosfatidilcolina, a introdução de um \"tag\" de 6 histidinas na extremidade amino-terminal da Ca2+-ATPase permitiu a sua purificação por cromatografia de afinidade utilizando uma resina carregada com níquel. Utilizando essa estratégia, foi possível obter frações enriquecidas em até 75% de Ca2+-ATPase recombinante, algo não descrito ainda na literatura. A 6xHis Ca2+-ATPase solubilizada em LPC e purificada em coluna de níquel se mantém estável desde que seja introduzido DOPC juntamente com o detergente nas etapas de lavagem e eluição. Nessas condições, a enzima purificada possui elevada atividade ATPásica cálcio-dependente (1.5-2.0 &#181;mol/mg proteína.min) durante vários minutos de reação. A titulação da atividade ATPásica em função do cálcio livre demonstrou que a 6xHis Ca2+-ATPase purificada possui alta afinidade para o íon (K0.5= 0.15 &#181;lM) e manteve uma forte cooperatividade na ativação por cálcio (nH = 2.07). A quantidade e o grau de pureza obtidos são suficientes para permitir a caracterização bioquímica e espectroscópica de mutantes pontuais da Ca2+-ATPase já construídos e expressos em levedura. A conversão da energia presente em ligações químicas em gradiente eletroquímico é um tema central da bioenergética. Espera-se que o estudo dos mutantes pontuais de triptofano da Ca2+-ATPase gerados nesse trabalho contribua para uma melhor compreensão do mecanismo de acoplamento entre a hidrólise de ATP e o transporte vetorial de íons nesse modêlo de estudo de proteínas de transporte. / We describe in this work a new method for the production of SERCA-l Ca2+-ATPase in yeast using a heat-shock regulated expression vector. Following solubilization of yeast membranes with lysophospholipids, the presence of an hexahistidine tag introduced at the Nterminal end of the Ca2+-ATPase allowed its purification by metal chelating affinity chromatography using a nickel-NTA resin. Using this procedure highly enriched ftactions (75% oftotal protein in the ftaction) of yeast-expressed rabbit Ca2+-ATPase were obtained. Detergent-solubilized 6xHis-Ca2+-ATPase retained highly active (1.5 - 2 &#181;mol/mg protein .min) calcium-dependent, vanadate inhibitable ATPase activity as determined by 32P-&#947;-ATP hydrolysis. Titration of ATPase activity as a function of ftee calcium revealed high Ca2+ affinity (K0.5 =~ 0.15 &#181;M) and the persistence of a strong cooperative pattem of calcium activation (Hill number of 2.07). The yield and purity of 6xHis Ca2+-ATPase fractions produced with this method allows the biochemical and spectroscopic characterization of Ca2+-ATPase mutants produced in the course of this work. Conversion of the energy present in chemical bonds to electrochemical gradient is a central theme of bioenergetics. It is hoped that the study of the Ca2+-ATPase tryptophan mutants generated in this work will contribute to a better understanding of the coupling mechanism between ATP hydrolysis and the vectorial transport of ions across membranes that occur in this model system.

A fecundação resulta no aumento intracelular de cálcio que é necessário para a transição do oócito até o estádio de zigoto. Os eventos que ocorrem durante esta transição são caracterizados como ativação, sendo estes dependentes de cálcio. Entretanto, os eventos bioquímicos que ocorrem durante a ativação ainda não estão completamente elucidados. A proteína quinase C (PKC) e a proteína quinase dependente de cálcio/calmodulina (CaMKII), por apresentarem atividade durante a fecundação e por serem ativadas por cálcio são implicadas na regulação dos eventos da ativação. Entretanto, existem muitas dúvidas sobre o real papel destas proteínas na ativação do oócito. Deste modo, o objetivo do presente trabalho foi avaliar o papel da PKC e da CaMKII na ativação de oócitos bovinos. Para tal, oócitos bovinos maturados in vitro foram ativados partenogeneticamente (AP) com cálcio ionóforo A23187 (5&mu;M) por 5 minutos, sendo a retomada da meiose, a organização do citoesqueleto e do retículo endoplasmático (RE) avaliada 1 hora após a ativação. No experimento 1 foi avaliado o papel da CaMKII nestes eventos. Os oócitos foram AP na presença ou ausência de 100M do inibidor de CaMKII (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). A inibição da CaMKII não afetou a retomada da meiose e nem a distribuição dos RE, após a AP. Entretanto, não ocorreu a rotação do fuso meiótico no estádio de telófase II quando a CaMKII foi inibidada. Estes resultados demonstram que embora a CaMKII não tenha efeito na retomada da meiose, esta proteína participa na progressão do ciclo celular de oócitos bovinos, após a AP. No experimento 2 foi avaliado o papel da PKC em oócitos bovinos AP. Os oócitos foram ativados partenogeneticamente na presença ou ausência de 10&mu;M do inibidor de PKC (Bisindolymaleimide I). A inibição da PKC não afetou a retomada da meiose e nem a progressão pelo ciclo celular até o estádio de telófase II. Entretanto, a organização do RE foi afetada pela inibição da PKC. Resultado semelhante foi obtido quando os oócitos foram ativados na presença de citocalasina C, um despolimerizador de filamentos de actina. O presente experimento demonstra a participação da via PKC-actina na organização do RE na ativação de oócitos bovinos. / The intracellular calcium increase resulting from fertilization is necessary for oocyte transition to zygote. The events that occur during this transition are characterized as activation, which are dependent on calcium. However the biochemical events that occur during this activation are still not fully elucidated. The protein kinase C (PKC) and the calcium/calmodulin-dependent protein kinase II (CaMKII), are involved in regulating the events of activation, since these proteins have activity during fertilization and are activated by calcium. However there are many doubts about the real role of these proteins in the oocyte activation. Thus, the objective of this study was to evaluate the role of PKC and CaMKII in bovine oocyte activation. For this purpose, in vitro matured bovines oocytes were parthenogenetically activated (PA) by using calcium ionophore A23187 (5&mu;M) for five minutes, and the resumption of meiosis, the cytoskeleton organization and the endoplasmic reticulum (ER) organization were evaluated 1 hour post-activation. In experiment 1, were evaluated the role of CaMKII in these events. The oocytes were PA in the presence or absence of 100M of CaMKII inhibitor (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). The inhibition of CaMKII did not affect the meiosis resumption and the ER after the PA. However, there was no spindle rotation at telophase II stage when the CaMKII was inhibited. These results showed that although the CamKII has no effect on resumption of meiosis, it participates in the regulation of cell cycle progression after PA of bovine oocytes. In experiment 2, was evaluated the role of PKC on PA bovine oocytes. The oocytes were parthenogenetically activated in the presence or absence of 10&mu;M of PKC inhibitor (Bisindolymaleimide I). The PKC inhibition did not affected the resumption of meiosis and the progression through the cell cycle until the stage of telophase II. However, the ER organization was affected by PKC inhibition. A similar result was obtained when the oocytes were activated in the presence of cytochalasin C, which promotes the depolymerization of the actin filaments. The current experiment showed the participation of the PKC-actin pathway at the ER organization in the bovine oocytes activation.

A lesão de isquemia e reperfusão (IRI) continua a ser um problema clínico e o estresse do retículo endoplasmático (ERS) parece ser um importante mediador desse processo. A presença da heme oxigenase-1 (HO-1) ou do monóxido de carbono (CO), parece proteger da IRI. O objetivo do nosso trabalho foi avaliar a papel da HO-1 e CO na IRI renal. A indução da HO-1 em camundongos promoveu uma proteção na IRI renal, com melhora da função renal, menos inflamação e atenuação do ERS. Ao avaliarmos o papel do CO, verificamos que há também uma proteção, mediada por p38, vias purinérgicas, estabilização de HIF-1a e eritropoietina. Há ainda uma melhora do metabolismo energético celular após o tratamento com CO. Enfim, podemos concluir que, na presença da HO-1 ou do CO, há uma melhora da lesão isquêmica, através de uma maior ativação de vias citoprotetoras, com atenuação do ERS, redução da inflamação e consequente melhora da função renal. / Ischemia-reperfusion injury (IRI) remains a clinical problem and endoplasmic reticulum stress (ERS) seems to be an important mediator of this process. The presence of heme oxygenase-1 (HO-1) or carbon monoxide (CO) appears to protect from IRI. The aim of our study was to evaluate the role of HO-1 and CO in renal IRI. The induction of HO-1 in mice promoted protection in renal IRI with improved renal function, less inflammation and attenuation of ERS. When evaluating the role of CO, we found that there is also a protection mediated by p38, purinergic signaling, HIF-1a stabilization and erythropoietin. There is still an improvement of cellular energy metabolism after treatment with CO. Finally, we conclude that, in the presence of HO-1 or CO, there is an improvement of the ischemic lesion, through greater activation of cytoprotective pathways, with reduced ERS, reducing inflammation and consequent improvement in renal function.