Transcription of meiotic-like-pathway genes in Giardia intestinalis

The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.

Role and regulation of islet-Brain 1 in pancreatic β-cells

Résumé : L'insuline est produite et sécrétée par la cellule ß-pancréatique. Son rôle est de régler le taux de sucre dans le sang. Si ces cellules meurent ou échouent à produire suffisamment de l'insuline, les sujets développent le diabète de type 2 (DT2), une des maladies les plus communes dans les pays développés. L'excès chronique des lipoprotéines LDL oxydés (oxLDL) et/ou des cytokines pro-inflammatoires comme l'interleukine-1ß (IL-1ß) participent au dérèglement et à la mort des cellules ß. Nous avons montré qu'une chute des niveaux d'expression de la protéine nommée «mitogen activated protein kinase 8 interacting protein 1» ou «islet brain 1 (IB 1)» est en partie responsable des effets provoqués par les oxLDL ou IL-1ß. IB1 régule l'expression de l'insuline et la survie cellulaire en inhibant la voie de signalisation « c-jun N-terminal Kinase (JNK)». La réduction des niveaux d'expression d'IB1 provoque l'activation de la voie JNK en réponse aux facteurs environnementaux, et ainsi initie la réduction de l'expression de l'insuline et l'induction du programme de mort cellulaire. Les mimétiques de l'hormone "Glucagon-like peptide 1", tel que l'exendin-4 (ex-4), sont une nouvelle classe d'agents hypoglycémiants utilisés dans le traitement du DT2. Les effets bénéfiques de l'ex-4 sont en partie accomplis en préservant l'expression de l'insuline et la survie des cellules ß contre les stress associés au DT2. La restauration des niveaux d'expression d'IB1 est un des mécanismes par lequel l'ex-4 prodigue son effet sur la cellule. En effet, cette molécule stimule l'activité du promoteur du gène et ainsi compense la réduction du contenu en IB1 causée par le stress. Outre ce rôle anti-apoptotique, dans ce travail de thèse nous avons mis en évidence une autre fonction d'IB1 dans la cellule ß. La réduction de l'activité ou des niveaux d'expression d'IB1 induisent une réduction importante de la sécrétion de l'insuline en réponse au glucose. Le mécanisme par lequel IB1 régule la sécrétion de l'insuline implique à la fois le métabolisme du glucose et éventuellement le transport vésiculaire en contrôlant l'expression de la protéine annexin A2. En résumé, IB 1 est une molécule clé à travers laquelle l'environnement du diabétique pourrait exercer un effet délétère sur la cellule ß. L'amélioration de l'activité d'IB1 et/ou de son expression devrait être considérée dans les approches thérapeutiques futures visant à limiter la perte des cellules ß dans le diabète. Abstract : ß-cells of the pancreatic islets of Langerhans produce and secrete insulin when blood glucose rises. In turn, insulin ensures that plasma glucose concentrations return within a relatively narrow physiological range. If ß-cells die or fail to produce enough insulin, individuals develop one of the most common diseases in Western countries, namely type 2 diabetes (T2D). Chronic excess of oxidized low density lipoproteins (oxLDL) and/or pro-inflammatory cytokines such as interleukin 1-ß (IL-1ß) contribute to decline of ß-cells and thereby are thought to accelerate progression of the disease overtime. We showed that profound reduction in the levels of the mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) causes ß-cell failure accomplished by oxLDL or IL-1 ß. IB1 regulates insulin expression and cell survivals by inhibiting the c-Jun N-terminal Kinase pathway. Diminution in IB 1 levels leads to an increase in activation of the JNK pathway induced by environmental stressors, and thus initiates loss of insulin expression and programmed cell death. The mimetic agents of the glucoincretin glucagon-like peptide 1 such as exendin-4 (ex-4) are new class of hypoglycaemic medicines for treatment of T2D. The beneficial property is in part achieved by preserving insulin expression and ß-cell survival against stressors related to diabetes. Restored levels in IB 1 account for the cytoprotective effect of the ex-4. In fact, the latter molecule .stimulates the promoter activity of the gene and thus compensates loss of IB1 content triggered by stress. Beside of the anti-apoptotic role, an additional leading function for IB 1 in ß-cells was highlighted in this thesis. Impairment in IB1 activity or silencing of the gene in ß-cells revealed a major reduction in insulin secretion elicited by glucose. The mechanisms whereby IB 1 couples glucose to insulin release involve glucose metabolism and potentially, vesicles trafficking by maintaining the levels of annexin A2. IB 1 is therefore a key molecule through which environmental factors related to diabetes may exert harmful effects on ß-cells. Improvement in IB 1 activity and/or expression should be considered as a target for therapeutic purpose.

Genetic variability of the mTOR pathway and prostate cancer risk in the European Prospective Investigation on Cancer (EPIC)

The mTOR (mammalian target of rapamycin) signal transduction pathway integrates various signals, regulating ribosome biogenesis and protein synthesis as a function of available energy and amino acids, and assuring an appropriate coupling of cellular proliferation with increases in cell size. In addition, recent evidence has pointed to an interplay between the mTOR and p53 pathways. We investigated the genetic variability of 67 key genes in the mTOR pathway and in genes of the p53 pathway which interact with mTOR. We tested the association of 1,084 tagging SNPs with prostate cancer risk in a study of 815 prostate cancer cases and 1,266 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC). We chose the SNPs (n = 11) with the strongest association with risk (p<0.01) and sought to replicate their association in an additional series of 838 prostate cancer cases and 943 controls from EPIC. In the joint analysis of first and second phase two SNPs of the PRKCI gene showed an association with risk of prostate cancer (ORallele = 0.85, 95% CI 0.78–0.94, p = 1.3×10−3 for rs546950 and ORallele = 0.84, 95% CI 0.76–0.93, p = 5.6×10−4 for rs4955720). We confirmed this in a meta-analysis using as replication set the data from the second phase of our study jointly with the first phase of the Cancer Genetic Markers of Susceptibility (CGEMS) project. In conclusion, we found an association with prostate cancer risk for two SNPs belonging to PRKCI, a gene which is frequently overexpressed in various neoplasms, including prostate cancer.

Phylogeny and evolution of the aspartyl protease family from clinically relevant Candida species

Aspartyl proteases are a class of enzymes that include the yeast aspartyl proteases and secreted aspartyl protease (Sap) superfamilies. Several Sap superfamily members have been demonstrated or suggested as virulence factors in opportunistic pathogens of the genus Candida. Candida albicans, Candida tropicalis, Candida dubliniensis and Candida parapsilosis harbour 10, four, eight and three SAP genes, respectively. In this work, genome mining and phylogenetic analyses revealed the presence of new members of the Sap superfamily in C. tropicalis (8), Candida guilliermondii (8), C. parapsilosis(11) and Candida lusitaniae (3). A total of 12 Sap families, containing proteins with at least 50% similarity, were discovered in opportunistic, pathogenic Candida spp. In several Sap families, at least two subfamilies or orthologous groups were identified, each defined by > 90% sequence similitude, functional similarity and synteny among its members. No new members of previously described Sap families were found in a Candida spp. clinical strain collection; however, the universality of SAPT gene distribution among C. tropicalis strains was demonstrated. In addition, several features of opportunistic pathogenic Candida species, such as gene duplications and inversions, similitude, synteny, putative transcription factor binding sites and genome traits of SAP gene superfamily were described in a molecular evolutionary context.

Safety and Effectiveness of two treatment regimes with tranexamic acid to minimize inflammatory response in elective cardiopulmonary bypass patients: a randomized double-blind, dose-dependent, phase IV clinical trial

Background. In cardiopulmonary bypass (CPB) patients, fibrinolysis may enhance postoperative inflammatory response. We aimed to determine whether an additional postoperative dose of antifibrinolytic tranexamic acid (TA) reduced CPB-mediated inflammatory response (IR). Methods. We performed a randomized, double-blind, dose-dependent, parallel-groups study of elective CPB patients receiving TA. Patients were randomly assigned to either the single-dose group (40 mg/Kg TA before CPB and placebo after CPB) or the double-dose group (40 mg/Kg TA before and after CPB). Results. 160 patients were included, 80 in each group. The incident rate of IR was significantly lower in the double-dose-group TA2 (7.5% vs. 18.8% in the single-dose group TA1; P = 0.030). After adjusting for hypertension, total protamine dose and temperature after CPB, TA2 showed a lower risk of IR compared with TA1 [OR: 0.29 (95% CI: 0.10-0.83), (P = 0.013)]. Relative risk for IR was 2.5 for TA1 (95% CI: 1.02 to 6.12). The double-dose group had significantly lower chest tube bleeding at 24 hours [671 (95% CI 549-793 vs. 826 (95% CI 704-949) mL; P = 0.01 corrected-P significant] and lower D-dimer levels at 24 hours [489 (95% CI 437-540) vs. 621(95% CI: 563-679) ng/mL; P = 0.01 corrected-P significant]. TA2 required lower levels of norepinephrine at 24 h [0.06 (95% CI: 0.03-0.09) vs. 0.20(95 CI: 0.05-0.35) after adjusting for dobutamine [F = 6.6; P = 0.014 corrected-P significant]. We found a significant direct relationship between IL-6 and temperature (rho = 0.26; P < 0.01), D-dimer (rho = 0.24; P < 0.01), norepinephrine (rho = 0.33; P < 0.01), troponin I (rho = 0.37; P < 0.01), Creatine-Kinase (rho = 0.37; P < 0.01), Creatine Kinase-MB (rho = 0.33; P < 0.01) and lactic acid (rho = 0.46; P < 0.01) at ICU arrival. Two patients (1.3%) had seizure, 3 patients (1.9%) had stroke, 14 (8.8%) had acute kidney failure, 7 (4.4%) needed dialysis, 3 (1.9%) suffered myocardial infarction and 9 (5.6%) patients died. We found no significant differences between groups regarding these events. Conclusions. Prolonged inhibition of fibrinolysis, using an additional postoperative dose of tranexamic acid reduces inflammatory response and postoperative bleeding (but not transfusion requirements) in CPB patients. A question which remains unanswered is whether the dose used was ideal in terms of safety, but not in terms of effectiveness.

Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Current standards and progress in understanding and treatment of GIST.

Gastrointestinal stromal tumours (GIST), despite being rare, pose a relevant medical problem from the viewpoint of diagnosis and management. GIST are fragile, liable to metastasize and often located in delicate structures. Surgical options, therefore, are limited. In the last decade an improved understanding of the molecular mechanisms of the disease has resulted in novel modes of treatment. The introduction of systemic tyrosine kinase inhibitor therapy with imatinib has significantly improved the outcome of the disease and prolonged the survival of GIST patients. For many patients the acute threat of a deadly cancer has been transformed into a manageable chronic condition. Drug safety, tolerability and compliance, subjects of concern in all long-term therapies, have proven to be acceptable for the tyrosine kinase inhibitor imatinib. The present paper provides a compact overview of the epidemiology, pathophysiology and morphology of GIST, with special reference to the underlying molecular biology. Relevant aspects of diagnosis, therapy and monitoring of the disease are reviewed with particular emphasis on the available clinical evidence and recent guidelines.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Role of ALK gene and mutations in initiation and progression of neuroblastoma

Introduction Le neuroblastome (NB) est la tumeur maligne solide extra-crânienne la plus fréquente chez l'enfant. Sa présentation clinique est très hétérogène, allant d'une tumeur localisée à une atteinte métastatique sévère. Malgré des traitements agressifs, environ 55% des NB de hauts risques sont actuellement résistants aux thérapies. L'espoir réside dans le développement de traitements ciblant les mécanismes moléculaires responsables du développement et de la progression du NB. Le gène Anaplastic Lymphoma Kinase (ALK) codant pour un récepteur tyrosine kinase a été particulièrement étudié ces dernières années car il est muté, amplifié ou surexprimé dans une majorité des NBs. Le but de ce projet était d'investiguer le rôle de ALK-wt, ainsi que de ces deux plus fréquentes mutations, ALK- F1174L et ALK-R1245Q, dans l'oncogenèse du NB. Le NB étant originaire des cellules de la crête neurale, nous avons analysé le potentiel oncogénique de ces différentes formes de ALK dans des cellules progénitrices de la crête neurale (NCPC). Méthode Des NCPC de souris (JoMal), possédant un c-MycER inductible pour leur maintien en culture in vitro, ont été transduites par un rétrovirus permettant l'expression stable de ALK-wt, ALK-F1174L et ALK-R1245Q. Des tests in vitro ont d'abord été effectués pour tester le système c-MycER, la stabilité de nos cellules transduites, leur phénotype, leur capacité de croissance et leur tumorigénicité. Les cellules transduites ont ensuite été injectées dans des souris immunosupprimées en sous-cutané, puis en orthotopique, c'est-à-dire dans leur glande surrénale, afin de mesurer leur tumorigénicité in vivo. Résultats La transduction et l'expression stable de ALK n'ont pas modifié le phénotype indifférencié des JoMal, ni de manière significative la capacité de croissance des cellules in vitro en absence d'activation de c-MycER. Par contre, lorsque c-MycER est actif, les cellules porteuses des mutations Fl 174L et R1245Q ont montré une meilleure capacité de prolifération et de formation de colonies, par rapport aux JoMal-ALK-wt et aux cellules contrôles en culture 3D dans de la méthylcellulose et dans un test de formation de neurosphères. In vivo, les souris injectées avec les cellules JoMal-ALK- F1174L en sous-cutané ou dans la glande surrénale ont rapidement développé des tumeurs, suivies par le groupe JoMal-ALK-R1245Q et le groupe JoMal-ALK-wt, alors que les groupes de souris contrôles n'ont présenté aucune tumeur. En orthotopique, nous avons obtenu 5/6 tumeurs ALK-F1174L, 7/7 tumeurs ALK-R1245Q et 6/7 tumeurs ALK-wt. Les tumeurs sous-cutanées ne présentaient pas de différences morphologiques et histologiques entre les différents groupes et montraient une histologie compatible avec un NB. Les tumeurs orthotopiques restent encore à analyser. Conclusion Cette étude a permis de démontrer que les mutations activatrices Fl 174L et R1245Q ont des propriétés tumorigéniques in vitro dans des NCPC et in vivo tandis que la forme sauvage de ALK montre une capacité oncogénique uniquement in vivo. Bien que la caractérisation des tumeurs orthotopiques n'a pas encore été effectuée, l'analyse des tumeurs sous-cutanées nous suggère que l'expression de ALK- wt ou muté est suffisante pour induire la formation de NB à partir des cellules progénitrices de la crête neurale. Le gène ALK semble donc jouer un rôle important dans l'oncogénèse du NB, aussi bien par la présence de mutations activatrices que par sa fréquente surexpression.

Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus

Human adenoviruses (HAdV) are a major cause of acute respiratory diseases (ARD), gastroenteritis, conjunctivitis and urinary infections. Between November 2000-April 2007, a total of 468 nasopharyngeal aspirate samples were collected from children with ARD at the Clinics Hospital of Uberlândia. These samples were tested by immunofluorescence assay (IFA) and 3% (14/468) tested positive for the presence of HAdV. By performing polymerase chain reaction (PCR) to detect HAdV DNA in samples that tested negative or inconclusive for all viruses identifiable by IFA (respiratory syncytial virus, parainfluenza viruses 1, 2 and 3, influenza viruses A and B and HAdV), as well as negative for rhinoviruses by reverse transcription-PCR, additional 19 cases were detected, for a total of 33 (7.1%) HAdV-positive samples. Nucleotide sequences of 13 HAdV samples were analyzed, revealing that they belonged to species B, C and E. Further analyses showed that species C (HAdV-2) was the most prevalent among the sequenced samples. To our knowledge, this is the first report describing the presence of HAdV-4 in Brazil. We also detected an isolate that was 100% identical to a part of the feline adenovirus hexon gene sequence.

Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs) and praziquantel (PZQ) kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW) mice of both genders were infected (100 cercariae) on postnatal day 10 and killed on post-infection days (PIDs) 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD)], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD)], 2E1 [p-nitrophenol-hydroxylase (PNPH)] and 3A11 [erythromycin N-demethylase (END)] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction). On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally) levels were measured (high-performance liquid chromatography) at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

Synthesis and transport of creatine in the CNS: importance for cerebral functions.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06935.x Abstract Apart of its well known function of 'energetic buffer' through the creatine/phosphocreatine/creatine kinase system allowing the regeneration of ATP, creatine has been recently suggested as a potential neuromodulator of even true neurotransmitter. Moreover, the recent discovery of primary creatine deficiency syndromes, due to deficiencies in l-arginine : glycine amidinotransferase or guanidinoacetate methyltransferase (the two enzymes allowing creatine synthesis) or in the creatine transporter, has shed new light on creatine synthesis, metabolism and transport, in particular in CNS which appears as the main tissue affected by these creatine deficiencies. Recent data suggest that creatine can cross blood-brain barrier but only with a poor efficiency, and that the brain must ensure parts of its needs in creatine by its own endogenous synthesis. Finally, the recent years have demonstrated the interest to use creatine as a neuroprotective agent in a growing number of neurodegenerative diseases, including Parkinson's and Huntington's diseases. This article aims at reviewing the latest data on creatine metabolism and transport in the brain, in relation to creatine deficiencies and to the potential use of creatine as neuroprotective molecule. Emphasis is also given to the importance of creatine for cerebral function.

Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period.

PPARs are a family of nuclear hormone receptors involved in various processes that could influence ovarian function. We investigated the cellular localization and expression of PPARs during follicular development in ovarian tissue collected from rats 0, 6, 12, 24, and 48 h post-PMSG. A second group of animals received human CG (hCG) 48 h post-PMSG. Their ovaries were removed 0, 4, 8, 12, and 24 h post-hCG to study the periovulatory period. mRNAs corresponding to the PPAR isotypes (alpha, delta, and gamma) were localized by in situ hybridization. Changes in the levels of mRNA for the PPARs were determined by ribonuclease protection assays. PPAR gamma mRNA was localized primarily to granulosa cells, and levels of expression did not change during follicular development. Four hours post-hCG, levels of mRNA for PPAR gamma decreased (P &lt; 0.05) but not uniformly in all follicles. At 24 h post-hCG, levels of PPAR gamma mRNA were reduced 64%, but some follicles maintained high expression. In contrast, mRNAs for PPAR alpha and delta were located primarily in theca and stroma, and their levels did not change during the intervals studied. To investigate the physiologic significance of PPAR gamma in the ovary, granulosa cells from PMSG-primed rats were cultured for 48 h with prostaglandin J(2) (PGJ(2)) and ciglitazone, PPAR gamma activators. Both compounds increased progesterone and E2 secretion (P &lt; 0.05). These data suggest that PPAR gamma is involved in follicular development, has a negative influence on the luteinization of granulosa cells, and/or regulates the periovulatory shift in steroid production. The more general and steady expression of PPARs alpha and delta indicate that they may play a role in basal ovarian function.

Identification and characterization of a caspase-2 activating complex

Résumé Les caspases sont des protéases essentielles lors de l'induction de l'apoptose ou pour la maturation de certaines cytokines. Elles peuvent être divisées en deux groupes: les caspases initiatrices, qui sont les premières activées lors d'un signal pro-apoptotique, et les caspases effectrices, qui sont activées par les caspases initiatrices et sont responsables du clivage et de la dégradation des substrats cellulaires. Les caspases initiatrices sont activées dans des complexes de haut poids moléculaire: l'apoptosome pour la caspase-9 et le DISC pour la caspase-8. La caspase-2 est également une caspase initiatrice qui contient un domaine CARD. Cependant son mécanisme d'activation n'est pas encore connu. Lors de cette étude, nous avons découvert et caractérisé le complexe qui permet l'activation de la caspase-2. Ce complexe, appelé le PIDDosome, est composé de PIDD/LRDD, de la protéine adaptatrice RAIDD et de la protéase caspase-2. L'expression forcée de PIDD induit l'activation constitutive de la caspase-2. Cela entraîne la mort ou la sensibilisation à la mort des cellules selon la lignée étudiée. Cet effet est expliqué par une perte du potentiel de membrane de la mitochondrie, certainement dû à un effet direct de la caspase-2. Peu de choses sont connues sur PIDD: c'est une protéine contenant un domaine DD qui peut être induite par p53. Nous avons caractérisé PIDD et montré qu'elle est exprimée de façon ubiquitaire. PIDD est constitutivement auto-clivée environ au milieu de la protéine, ce qui génère deux fragments qui restent liés l'un à l'autre. Le fragment N-terminal a une activité régulatrice et le C-terminal une activité effectrice. De plus, PIDD peut se déplacer entre le cytoplasme et le noyau. Enfin, nous avons découvert que PIDD est également impliquée dans l'induction de NF¬ -κB en réponse à des dommages à l'ADN. PIDD est responsable de la modification par sumo de NEMO, étape nécessaire à l'induction de NF-κB après des dommages à l'ADN. Ainsi PIDD semble être à l'intersection de la décision que prend la cellule entre survivre et réparer les dommages, ou entrer en apoptose. Summary Caspases are a family of proteases that fulfill varied and often critical roles in mammalian apoptosis or proteolytic activation of cytokines. Caspases can be divided into two sub-groups: initiator caspases, which are the first activated after a pro-apoptotic signal, and effector caspases, which are activated by initiator caspases and that are responsible for the cleavage and degradation of cellular components. Initiator caspases are activated in high molecular weight platforms such as the apoptosome for caspase-9 or the DISC for caspase-8. Caspase-2 is a CARD-containing initiator caspase whose mechanism of activation was not yet known. In this study we have identified an activating platform for caspase-2. This high molecular weight complex, called the PIDDosome, is composed of PIDD/LRDD, the adaptor protein RAIDD and caspase-2. Constitutive expression of PIDD led to constitutive activation of caspase-2, which in some cell lines was sufficient to induce cell death while in others it merely sensitizes. Active caspase-2 was found to disturb directly the mitochondria by inducing a partial loss of the transmembrane potential. Very little was known on PIDD. It can be induce by p53 and inhibition of its expression by antisense oligonucleotides diminishes p53-dependent apoptosis. We decided to further characterize PIDD function and expression. PIDD possesses seven LRR, two Zu5 domains and one DD. It is ubiquitously expressed and appears to be constitutively cleaved by auto- processing into two main fragments equal in size. The two fragments remain bound to one another and constitute a regulatory N-terminal fragment and an active C-terminal fragment. In addition, PIDD can shuttle between the cytoplasm and the nucleus. Finally, investigating the possible relevance of new interaction partners, we found that PIDD is implicated in DNA damage-induced NF- κB. PIDD binds to RIP1 and to NEMO. In response to DNA damage, PIDD translocates to the nucleus and mediates sumo- modification of NEMO, a necessary step in DNA damage-induced NF-κB. All together these results raise the possibility that PIDD acts as a molecular switch between proliferation and repair, and apoptosis following DNA damage.