No / Genotoxic agents can interact with DNA in germ cells possibly resulting in a heritable trait (germline mutation). Thus, in vitro male germ cell tests, which can detect phase specificity of such agents, could be used by regulatory agencies to help evaluate the potential risk of mutation. The male germ cell system now has a well-established model for studying phase specificity using the STA-PUT velocity sedimentation. On treatment with genotoxic agents, differences in chemical structure and metabolic differences in types of male germ cell lead to differing susceptibilities to genotoxicity, so careful investigation is required for phase specificity. This can yield valuable information about the potential mechanisms involved in the genotoxicity responses and thus increase the significance of the findings. This is especially important because mutations induced in the germline could also affect future generations. In this chapter, we briefly review the field of the male germ cell DNA damage response.

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.

No description available.

En France, le cancer de sein héréditaire représente environ 2500 nouveaux cas par an, dont prés de la moitié est attribuée à la mutation du gène BRCA1.La recherche de la mutation par biologie moléculaire est un travail fastidieux, coûteux et long (8 mois d’attente environ actuellement).Pour trouver une solution à ce délai, nous avons étudié en immunohistochimie une série initiale de 21 anticorps répartis en 5 groupes : anticorps antiBrca1 du commerce, liés à la perte de l’inactivation de l’X, liés à la signature basale ou myoépithéliale, anticorps dits classiques du cancer de sein et finalement dérivés de signatures établies par cDNAarray.Nous avons utilisé la technique de’ tissue microarrays’ en utilisant de manière comparative une population de 27 cas de cancer de sein présentant une mutation germinale de BRCA1, et 81 cas témoins de cancer de sein sporadiques appariés à l’âge, ainsi qu’à des lignées cellulaires d’origine mammaires. Dans une deuxième série indépendante de validation nous avons appliqué les résultats obtenus de la première série sur 28 cas de cancer mammaire muté, et 28 cas du cancer mammaire sporadique dans les mêmes conditions initiales.Nos résultats montrent pour la première fois sur des tissus tumoraux une probabilité forte d’une association entre la mutation Brca1 et la perte de l’inactivation de l’X ; confirment la valeur de MS110 comme un bon anticorps prédictif d’une mutation de Brca1 ; apportent un argument pour une participation myoépithéliale dans l’oncogenèse de cancer mammaire Brca1 muté; appuient la relation entre ce dernier et les récepteurs RE,RP ainsi que P53 , Bcl2,Ki67 et valident en protéomique la valeur discriminant de CDC47 correspondant à un des gènes de la signature génomique.Après confirmation des mêmes résultats dans la série de validation, nous soutenons en analyses multivariés un modèle qui comprend seulement Grade 3, MS110, Lys27H3 négative, Vimentine et KI67 positive. Cette équation correspond à une sensibilité de 82% et spécificité de 81% et propose une approche rapide économique de pré- ciblage de la mutation Brca1 ; ce qui améliorait la prise en charge préventive, thérapeutique et globale des patients et leurs familles. / Family structure, lack of reliable information, cost and delay are usual concerns faced with when deciding to perform BRCA analyses. Testing the breast cancer tissues with four antibodies (MS110, lys27H3, Vimentin, KI67) in addition to grade evaluation enabled to rapidly select patients to carry out genetic testing identification. We constituted an initial breast cancer tissue micro-array, considered as a learning set comprising 27 BRCA1 and 81 sporadic tumours. A second independent validation set of 28 BRCA1 tumours was matched to 28 sporadic tumours using the same original conditions.We have investigated morphological parameters and 21 markers by immunohistochemistry.A logistic regression model was used to select the minimal number of markers providing the best model to predict BRCA1 status. The model was applied to the validation set to estimate specificity and sensibility.In the initial set, the univariate analysis identified 11 markers significantly associated with BRCA1 status. Then the best multivariate model comprised only Grade 3, MS110, Lys27H3, Vimentin and KI67. When applied to the validation set, BRCA1 tumours were correctly classified with a sensitivity of 83% and a specificity of 81%. The performance of this model was superior when compared to other profiles.This work offers a new rapid and economic method for the pre-screening of patients at high risk of being BRCA1mutation carriers, then to guide genetic testing, and finally to provide appropriate preventive measure, advices and treatments including targeted therapy to patients and their families.

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria. / Dissertation

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Endogenous retrovirus ERVWE1 is an integral part of the human genome. In the course of evolution, a protein encoded by the env gene of this retrovirus - Syncytin-1 - has gained unique function in human development. It mediates cell-to-cell fusion of placental cytotrophoblasts. Receptor that binds to Syncytin-1 is expressed in different cell types. Syncytin-1-mediated fusion is essential in placenta, but it can cause disruption of tissue integrity in other cell types. ERVWE1 expression is regulated by promoter DNA methylation, transcription factor GCM1 and efficient mRNA splicing. This thesis concerns the ERVWE1 expression and its regulation in non-placental tissues. It was found out that the moderate GCM1 overexpression was not sufficient to induce Syncytin-1 expression. Neither treatment with DNA demethylation agent 5-azacytidine nor with Syncytin-1 activator forskolin was able to manage Syncytin-1 expression. This thesis extends previous findings concerning high syncytin-1 expression in seminomas. In same tissues, there was found elevated TET1 expression on mRNA level in comparison with controls. The presence of the TET1 demethylation enzyme can influence ERVWE1 promoter DNA methylation. Previously unreported splicing variant of TET1 has been found during the construction of human TET1 expression...

Patienten mit metastasierten Keimzelltumoren, die einen Progress oder ein Rezidiv ihrer Erkrankung nach einer cisplatinhaltigen Vortherapie erleiden, haben eine schlechte Prognose. Unter Verwendung einer erneuten konventionellen Chemotherapie können maximal 15-30% dieser Patienten geheilt werden, so dass die Mehrzahl der Patienten an ihrer Erkrankung verstirbt. Aus diesem Grund ist die Optimierung der therapeutischen Möglichkeiten ein wesentliches Ziel. Unsere Daten zeigen, dass die Hochdosischemotherapie (HDCT) eine wesentliche therapeutische Verbesserung darstellt und mittels dieser Therapie mit einem ereignisfreien Überleben von 30-60% zu rechnen ist. Eine "matched-pair" Analyse konnte im Hinblick auf das ereignisefreie und das Gesamtüberleben einen Vorteil von mehr als 10% zu Gunsten der HDCT feststellen. Darüber hinaus hat die zunehmende Erfahrung und die Verwendung von peripheren Blutstammzellen und hämatopoetischen Wachstumsfaktoren, den Einsatz der HDCT deutlich sicherer gemacht. Aus den genannten Gründen sollte alle Patienten mit Rezidiv oder Progress eines Keimzelltumors der HDCT zugeführt werden. Die operative Entfernung von residuellen Tumormanifestationen (RTR) nach primärere Chemotherapie ist heute Standard bei Patienten mit metastasierten Keimzelltumoren. Zwar findet sich in der histologischen Aufarbeitung bei den meisten Patienten ausschließlich nekrotisches Gewebe, doch werden bei einem Teil der Patienten auch Anteile von reifem Teratom und vitalen differenzierten und undifferenzierten Karzinomen gefunden. Während die Resektion von Nekrose keinen therapeutischen Benefit für den Patienten darstellt, ist die komplette Entfernung von reifem Teratom oder Zellen eines Karzinoms für die Prognose entscheidend. In Bezug auf die HDCT konnten bisher keine vergleichbaren Daten erhoben werden. Zur Evaluierung des Stellenwertes der RTR nach HDCT analysierten wir unser eigenes Patientenkollektiv und fanden, dass vergleichbar zur Primärtherapie alle Patienten nach Salvage-HDCT, die eine partielle markernegative oder markerpositive Remission erreicht haben, einer RTR zugeführt werden sollten. Bis auf intrazerebrale Reste sollten alle residuellen Tumormanifestationen komplett reseziert werden. Neben der Optimierung der therapeutischen Möglichkeiten ist auch die Minimierung der chemotherapieassoziierten Toxizitäten ein wesentlicher Bestandteil meiner wissenschaftlichen Arbeit. Aus diesem Grund evaluierten wir die Wirksamkeit der Substanz Amifostin im Hinblick auf die Verringerung von Toxizitäten, die Wirkung auf die Mobilisierung von peripheren Blutstammzellen und den Einfluß auf die Rekonstitution des Immunstatus bei Patienten mit rezidivierten oder progredienten Keimzelltumoren, die mittels einer konventionellen Chemotherapie und anschließender HDCT behandelt wurden. Der Einsatz von Amifostin erbrachte in diesem Zusammenhang und in diesem Patientenkollektiv keinen therapeutischen oder prophylaktischen Nutzen, so dass dessen Verwendung bei Patienten mit Keimzelltumoren nicht generell empfohlen werden kann. / Overall, patients with relapsed or progressive germ cell tumors (GCT) after cisplatin-based chemotherapy have a low chance of cure. Using conventional-dose chemotherapy as salvage treatment only 15-30% of the patients will become long-term survivors. It is well known that the majority of these patients will ultimately die of their disease. Therefore, improvment of standard treatment is clearly desirable. Our data has been established high-dose chemotherapy (HDCT) as an effective salvage modality with an event-free survival of 30-60%. A matched-pair analysis showed an advantage for HDCT compared with conventional-dose chemotherapy with improvement in event-free and overall survival of more than 10%. Furthermore, due to increasing clinical experience in the management of side-effects, the use of peripheral blood progenitor cells, and the availability of hematopoietic growth factors, HDCT has become relatively safe. In GCT patients with relapsed or rogressive disease HDCT has been demonstrated as a feasible and safe treatment concept which will be curative for a substantial proportion of these patients. Therefore, HDCT should be administered in patients with first relapse and unfavorable prognostic factors and as second or subsequent salvage treatment. Surgical resection of residual tumors (RTR) after first-line chemotherapy is recommended in patients with metastatic GCT. Necrosis will be the only histological finding in the majority of these patients. However, in others mature teratoma, viable cancer consisting of residual GCT, non germ-cell tumors, undifferentiated cancer or a combination of these histologies may be found. Whereas the resection of necrosis offers no therapeutic benefit, resection of mature teratoma or viable cancer adds to long-term event-free and overall survival in these patients. However, limited data exist on the results of surgery and the respective histologies in patients after first or subsequent salvage treatment with HDCT. To assess the contribution of RTR in this setting, we retrospectively analyzed a cohort of patients who had been treated with HDCT for relapsed or refractory GCT. Our data show that RTR contributes to the overall treatment outcome and should be offered to all patients with a partial remission after HDCT. Complete resections of all residual tumors outside the CNS should be attempted. Furthermore, we assessed the efficacy of amifostine for protection from chemotherapy-induced toxicities, for peripheral blood progenitor cell mobilization and for immune-reconstitution in patients treated with conventional-dose paclitaxel, ifosfamide, cisplatin (TIP) and high-dose carboplatin, etoposide and thiotepa (CET) followed by PBPC rescue. In conclusion, amifostine additional to conventional-dose chemotherapy or HDCT showed no unequivocal advantage in protection from treatment-related toxicities and had no effect neither on PBPC mobilization nor on immune-reconstitution.

Dans le contexte de la physiologie comparée de la reproduction, les amphibiens sont peu étudiés. Le travail réalisé durant cette thèse visait à analyser des marqueurs de différenciation testiculaire chez l'urodèle Pleurodeles waltl, dont le déterminisme génétique du sexe (ZZ/ZW) peut être influencé par la température. Nos études ont d'abord porté sur le gène sox9 marqueur de la différenciation testiculaire chez les vertébrés supérieurs. Le gène cloné chez le pleurodèle montre une bonne conservation par rapport aux autres vertébrés. Son expression plus élevée dans la gonade mâle n'apparaît que tardivement suggérant qu'il n'est probablement pas impliqué dans les stades précoces de la différenciation testiculaire. En outre, son expression dans le mésonéphros rend difficile son utilisation comme marqueur de différenciation testiculaire. Nous avons ensuite étudié l'Amh, hormone testiculaire impliquée dans la régression des canaux de Müller chez de nombreux vertébrés. Son expression spécifique de la gonade, précocement plus élevée chez les larves ZZ que les ZW en font un excellent marqueur de la différenciation testiculaire. Le fait que les pleurodèles mâles voient les canaux de Müller persister malgré la présence d'Amh suggère que la fonction primaire de cette hormone était en relation avec la différenciation gonadique et que la fonction de régression des canaux de Müller n'est apparue que secondairement au cours de l'évolution. Ces marqueurs ont été mis à profit pour caractériser le phénotype gonadique lors d'inversions sexuelles ou lors de développements en absence de cellules germinales. Ils ont permis de montrer que les cellules germinales ne semblent pas jouer de rôle dans la différenciation gonadique du pleurodèle / In the context of comparative physiology of reproduction, amphibians are poorly studied. This work was dedicated to the analysis of testis differentiation markers in the newt Pleurodeles waltl, which shows a ZZ/ZW genetic mode of sex determination that can be affected by temperature. First, we studied sox9, a testis differentiation marker well characterized in many higher vertebrates. The gene cloned in Pleurodeles shows a good level of identity with other vertebrates. The testis-enriched expression appears late during the testis differentiation process indicating that it is probably not involved in the early steps of testis differentiation. Its use as a marker of testicular differentiation proved difficult since it is expressed not only in the gonads but also in the mesonephros. Then, we studied amh, a testis hormone responsible for müllerian duct regression in many vertebrates. Its early expression in the gonad, significantly higher in male than in female larvae makes it an excellent marker for testis differentiation. Since in Pleurodeles waltl, Müllerian ducts persist in males, it suggests that during the course of evolution, the function of Amh on the regression of Müllerian ducts appeared secondarily after its role in gonadal differentiation. These markers have been used to characterize the gonadal phenotype during sex reversal, or in gonads developed in the absence of germ cells. They showed that these cells do not seem to play a role in gonadal differentiation of Pleurodeles waltl

Bei Säugerkeimzellen, Zygoten und Embryos in frühen Stadien kommt der epigenetischen Neuprogammierung eine außergewöhnlich wichtige Rolle in der Regulation der Genomfunktionen in entscheidenden Entwicklungsstadien zu. Die epigenetische Neuprogrammierung in Keimzellen löscht zuerst die Imprinting-Markierungen und Epi-Mutationen und stellt dann geschlechtsspezifische Markierungen (genomische Prägung) wieder her. Die vorliegende Arbeit bezieht sich auf das Löschen epigenetischer Modifikationen in primordialen Mauskeimzellen (primordial germ cells (PGCs)) zwischen dem 10.5 bis 13.5 Tag nach der Befruchtung. Entgegen früheren Annahmen zeigen unsere Ergebnisse, daß primordiale Mauskeimzellen (PGCs) beim Eintritt in die embryonalen Keimdrüsen noch immer DNS Methylierungsmarker besitzen, die ähnlich dem Marker in somatischen Zellen sind. Kurz nach dem Eintritt in die Keimdrüsen werden die DNS Methylierungsmarker, die in Verbindung mit geprägten und nicht geprägten Genen stehen, gelöscht. Für die Mehrzahl der Gene beginnt die Löschung der Marker in männlichen und weiblichen Embryos gleichzeitig und ist innerhalb eines Entwicklungstages abgeschlossen. Diese Kinetik deutet auf einen aktiven Demethylierungsprozess hin, initiiert durch ein somatisches Signal, ausgehend von der embryonalen Keimdrüse. Der Zeitpunkt der Neuprogrammierung in den primordialen Keimzellen ist entscheidend, da er sicherstellt, daß Keimzellen beiden Geschlechts einen epigenetisch äquivalenten Status erhalten, bevor sie geschlechtsspezifisch ausdifferenzieren und anschließend neu elterlich geprägt werden. Vollständiges Verständnis des Prozesses der Neuprogrammierung der Keimzellen ist nicht nur im Hinblick auf genomisches Imprinting wichtig, sondern auch für die Erforschung von Mechanismen für die Wiederherstellung von omnipotenten Zellen bei Klonierung und Stammzellenerhaltung. / Epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. Germ line epigenetic reprogramming assures erasure of all the imprinting marks and epi-mutations and establishment of new sex-specific gametic imprints. The presented work focuses on the erasure of epigenetic modifications that occur in mouse primordial germ cells (PGCs) between day 10.5 to 13.5 post coitum (dpc). Contrary to previous assumptions, our results show that as they enter the genital ridge the PGCs still possess DNA methylation marks comparable to those found in somatic cells. Shortly after the entry of PGCs into the gonadal anlagen the DNA methylation marks associated with imprinted and non-imprinted genes are erased. For most genes the erasure commences simultaneously in PGCs of both male and female embryos and is completed within only one day of development. The kinetics of this process indicates that is an active demethylation process initiated by a somatic signal emanating from the stroma of the genital ridge. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which, new parental imprints are established subsequently. Complete understanding of the germline reprogramming processes is important not only in the light of genomic imprinting but also for resolving other mechanisms connected with restoring cellular totipotency, such as cloning and stem cell derivation.