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Endothelial cell apoptosis is an important process during vasculature remodelling, angiogenesis and inflammation. Medical interest in endothelial cell apoptosis as a target for arthritis and solid tumour treatment has prompted biochemical and pharmacological investigation into the mechanisms controlling endothelial cell survival and angiogenesis. In recent years it has been hypothesised that control of endothelial cell apoptosis and the induction of angiogenesis may in part be due to the enzyme cyclooxygenase (COX)-2. COX-2 is involved in the metabolism of arachidonic acid to prostaglandins in mammals. This pathway has been implicated in controlling inflammation and angiogenesis through prostaglandin (PG) production and more recently has been shown to inhibit endothelial cell death. It was the aim of this study to investigate endothelial cell apoptosis and angiogenesis focussing on the role of COX-2, prostaglandins and endogenous apoptotic inhibitors in these pathways. Endothelial cell apoptosis was assessed by chromatin condensation, DNA fragmentation and caspase activation. Angiogenesis was investigated by examining capillary-like tubule formation. Endothelial cell apoptosis induction and angiogenesis inhibition was observed using the selective COX-2 inhibitor 5-bromo-2-(4-fluorophenyl)-3- (methylsulfonyl) thiophene (DuP-697) at a concentration 100 times lower than has previously been reported. Apoptosis was confirmed by induction of caspases 8, 9 and 3 over 8 hr and DNA fragmentation and condensation over 24 hr. The effects observed may be due to a selective inhibition of COX-2 as apoptosis induction and angiogenic inhibition only occurred when COX-2 was inhibited by selective and non-selective COX inhibitors. Induction of endothelial cell death was induced by treatment with two natural products that inhibit COX-2, namely curcumin and 6-shogaol (from turmeric and ginger respectively) although only at concentrations higher than were required to inhibit COX- 2. Both compounds induced chromatin condensation in endothelial cells and lurkat E6.1 cells with no DNA laddering or caspase induction. Further examination of the mechanisms of endothelial cell survival were investigated by assessing the endogenous expression of the apoptosis repressor with a caspase recognition domain (ARC) protein through examining reverse transcriptase CRT) - peR, native protein expression and transgenic over-expression in the endothelial cells. Endogenous expression of ARC was found in endothelial cells. However this expression declined during in vitro culture. Transgenic expression of ARC was found to increase levels of ARC in vitro. However it had no effect on apoptosis inhibition after 24 hr. The underlying mechanisms of cell death induction may be compound dependent in endothelial cells. Pharmacological inhibition of COX-2 and possibly PGE2 generation has detrimental effects on angiogenesis and endothelial cell survival. However inhibition of COX-2 by natural products at low concentrations may be advantageous in preventing tumour angiogenesis with no apoptosis induction.

Apoptosis occurs extensively during the development of the mammalian nervous system, during which time neurons depend on neurotrophic factors, such as nerve growth factor (NGF), for survival. This process is tightly controlled and the BCL-2 family of proteins are key regulators of neuronal apoptosis. DP5, a member of the proapoptotic BH3-only subfamily with highly restricted expression in the nervous system, is upregulated in sympathetic neurons undergoing NGF deprivation-induced apoptosis. Sympathetic neurons from dp5-/- knockout mice die more slowly after NGF deprivation and dp5-/- motor neurons have increased resistance to axotomy-induced death. DP5 is regulated at the transcriptional level and the c-Jun N-terminal kinase (JNK) pathway contributes to the induction of dp5 after NGF withdrawal. The goal of my project was to determine the mechanism by which NGF deprivation induces dp5 transcription in sympathetic neurons and to identify the regions of the gene and signalling pathways involved. Firefly luciferase reporter constructs containing different regions of the dp5 gene were tested in a microinjection assay using sympathetic neurons cultured in vitro. Levels of luciferase activity after NGF deprivation or following treatment with chemical inhibitors were measured using immunofluorescence or a dual luciferase assay. Three regions important for the induction of dp5 after NGF withdrawal were identified: 1 kb of promoter, a 400 bp region of the intron conserved between the rat, mouse and human genes, and ~5 kb of 3'UTR. Sequences within the dp5 promoter and intron were shown to be regulated by the MLK/JNK pathway. In addition, a functionally important binding site for the transcription factor E4BP4 was identified in the dp5 promoter region. Overexpression of E4BP4 strongly repressed dp5 promoter activity, whereas mutation of this site reduced basal promoter activity suggesting that an activator, such as a related PAR family transcription factor, might bind to this site in sympathetic neurons.

Recently a new inhibitor of apoptosis, Golgi anti-apoptotic protein (GAAP), was discovered in camelpox virus and in some vaccinia virus (VACV) strains. GAAP is non-essential for VACV replication but affects virus virulence, is located in the Golgi, inhibits apoptosis and modulates Ca2+ fluxes. The VACV GAAP (vGAAP) shows high sequence identity with a previously uncharacterized human protein, named human GAAP (hGAAP). GAAPs belong to the Bax inhibitor-1 family and are highly conserved among eukaryotes, sharing a similar sequence, length and hydrophobicity profile. A yeast system was used to express and purify vGAAP and Arabidopsis thaliana GAAP3 (AtGAAP3). Data from patch clamp experiments using reconstituted purified protein in artificial lipid bilayers showed that vGAAP and AtGAAP3 allow the passage of ions, suggesting that GAAPs might form ion channels or ion exchangers. Amino acid sequence alignments of GAAP with other known ion channels or ion exchangers allowed the identification of amino acid residues in GAAP that might be involved in channel gating or ion selection. These residues were mutated in vGAAP and the mutants screened for anti-apoptotic and Ca2+ modulation activity, and purified to confirm and further characterize GAAP’s ion channel-like activity. Using chemical crosslinking, FRET and cysteine mutagenesis it was shown that GAAPs are able to form oligomers in a cysteine-dependent (vGAAP) or cysteine-independent (hGAAP) manner. Using detachment and attachment assays it was shown that hGAAP overexpression is able to increase cell adhesion and, conversely hGAAP knock down caused decreased adhesion and an elongated cell phenotype. Using a focal adhesion (FA) marker it was observed that the FA turnover after GAAP knockdown is reduced. Taken together, these results suggest that GAAP modulation of apoptosis and cell adhesion may be via its ion channel activity and subsequent modulation of Ca2+ fluxes.

La senescence est un état d’arrêt prolifératif mis en place par les cellules en réponse à différents stress (raccourcissement des télomères, stress oxydant, ou activation d’oncogènes). Bien que la sénescence soit considérée comme irréversible, nous avons récemment montré, en utilisant des kératinocytes humains normaux d’épiderme, que certaines cellules sénescentes réactivent spontanément le processus mitotique pour générer des cellules proliférantes, baptisées émergentes post-sénescence, qui sont transformées et tumorigènes en souris nude. Nous avons montré dans la première partie de ce travail que les cellules sénescentes qui ne génèrent pas de cellules émergentes meurent. La mort engagée à la sénescence n’est ni apoptotique ni nécrotique, mais implique l’élimination par macroautophagie de nombreux composés cellulaires vitaux. Nous avons ensuite démontré que le stress oxydant, via les dommages qu’il crée, notamment aux niveaux nucléaire et mitochondrial, est responsable de l’activation de la mort cellulaire programmée par macroautophagie. Les cellules sénescentes progénitrices des cellules néoplasiques génèrent quant à elle moins d’espèces réactives de l’oxygène (ROS) que le reste des cellules sénescentes, ce qui leur permet d’échapper à la mort. Cependant, pour générer les cellules émergentes, elles doivent maintenir une activité macroautophagique de ménage. L’ensemble de ces travaux démontre donc que le devenir des kératinocytes sénescents dépend de leur niveau de ROS. Un haut niveau de ROS induirait une activité macroautophagique élevée et létale, alors qu’un niveau plus bas induirait une activité trop faible pour induire la mort, mais suffisante pour éliminer les composés cellulaires oxydés. Dans cette situation, les cellules deviendraient permissives à l’évolution néoplasique si les dommages oxydants touchent l’ADN et affectent des oncogènes, suppresseurs de tumeurs, ou d’autres régulateurs fondamentaux. / Senescence is a non proliferative state that occurs in response to telomere shortening, oxidative stress or oncogenic activation. Whereas senescence is generally considered as an irreversible growth arrest, we recently reported, using Normal Human Epidermal Keratinocytes (NHEKs), that few senescent cells can spontaneously reactivate a mitotic process to generate so-called post-senescence emergent cells which are transformed and able to form skin hyperplasia in nude mice. In the first part of this work, we have investigated the outcome of the majority of senescent cells that do not generate emergent cells. We highlighted that senescent cells massively die during the growth arrest. Interestingly, the death is not associated with apoptotic or necrotic features but involves the elimination of numerous vital cells components by macroautophagy. We next investigated the mechanism that activates the autophagic programmed cell death in senescent keratinocytes. We show that oxidative stress occuring during senescence causes numerous cellular damages, notably to nucleus and mitochondria, that activate the macroautophagic process to ultimately lead to the death. In the last part of this work, we have investigated the relationship between oxidative stress and macroautophagy during the generation of post-senescence emergent cells. We show that progenitors of these neoplastic cells display less reactive oxygen species (ROS) production than other senescent keratinocytes, and hence escape autophagic cell death. However, in order to generate PS emergent cells, they have to maintain an housekeeping autophagic activity. Taken together, these results indicate that the outcome of a senescent cell is driven by its ROS level. A high ROS level induces a high and lethal activation of autophagy. At a lower ROS level, the cell activates a moderated autophagy that fails to induce death but favors the elimination of oxidized proteins and organelles. By this way, this cell becomes permissive to neoplastic evolution consecutively to the putative oxidative alteration of oncogenes, tumor suppressor genes or other crucial cell regulators.

Apoptosis can be distinguished from necrosis, the classical form of cell death, by several morphological and biochemical criteria. Apoptotic cells, but not necrotic cells, show early condensation of chromatin as well as endonuclease activation resulting in cleavage of the nuclear DNA into oligonucleosomal fragments. Both physiological and low level cytotoxic stimuli have been shown to induce apoptosis, which in some cell models can be suppressed by inhibitors of protein and RNA synthesis. The concept of the cell being actively involved in its own death, combined with the demonstration that factors which alter the rate of cell death, such as the proto-oncogene bcl-2, can directly affect the number of cells within a population, has resulted in the identification of cell death alongside proliferation and differentiation as a means for controlling celi population growth. The purpose of this study was to determine if bcl-2 and the Epstein-Barr virus gene BHRF1, which share 25% primary amino acid sequence homology, could suppress apoptosis in response to a variety of anti-cancer treatments. After demonstrating apoptotic cell death on treatment with chemotherapeutic agents in an IL-3 dependent cell line (FDCP-1) and three different EBV genome-positive Burkitt's lymphoma cell lines, the survival of EBV-BL cell lines expressing either exogenous bcl-2 or BHRF1 was examined. Suppression of apoptosis in response to treatment with chemotherapeutic drugs or y radiation was clearly shown in EBVBL cells expressing bcl-2 or BHRF1 when compared to control transfectants. This study has further confirmed that BHRF1 is functionally homologous to bcl-2, suggesting that BHRF1 may act to prevent apoptosis during EBV infection. Suppression of chemotherapeutic drug induced cell death by either bcl-2 or BHRF1 also represents a novel form of drug resistance and may form an alternative mechanism by which multidrug resistance may arise during chemotherapy. The identification and investigation of other genes which produce suppression of apoptosis is also important in order to determine the extent of involvement of apoptotic suppression in the transformation to the malignant state and in the acquisition of multidrug resistance. A protocol to screen for 'apoptosis-suppressed cells' in the FDCP-1 E -3 dependent cell line was developed to identify new genes involved in the pathway(s) of apoptosis.

La cellule vivante est en permanence exposée à divers types de stress pouvant endommager ses composants. Quand les dommages induits sont détectés, des mécanismes de défense sont activés pour les maîtriser tout en gérant au mieux les ressources énergétiques disponibles et nécessaires au bon fonctionnement cellulaire. Si le stress est trop sévère et que le système ne peut se défendre, la mort sera inévitable. La réponse cellulaire au stress est orchestrée par des réseaux de signalisation intracellulaires qui présentent une complexité extraordinaire. Les espèces moléculaires constituant ces réseaux assurent des tâches diverses à travers des réactions biochimiques, formant des machineries de processus biologiques synchronisées. Notre approche dans cette thèse pour l’étude de ces réseaux consiste à les modéliser mathématiquement pour reproduire un phénomène observé et repérer ses acteurs clés, analyser leurs réactions en réponse à différents signaux, et éventuellement réaliser des prédictions assez précises et expérimentalement vérifiables qui peuvent se montrer d’une extrême utilité pour les applications thérapeutiques. Dans nos travaux, nous nous focalisons sur le stress thermique et sur la réponse cellulaire qui en résulte en termes de dynamique des espèces moléculaires impliquées, mais également de destin cellulaire (mort ou survie) à l’issue de l’exposition, nous abordons cette problématique par des modèles dynamiques décrivant la cinétique biochimique des variables du système en conséquence d’une variation de la température. Dans un premier temps, nous démontrons à travers des simulations, suivies par une validation expérimentale, que la forme temporelle du stress thermique impacte considérablement la survie cellulaire. Ce premier résultat met en évidence un mécanisme de saturation des espèces réparatrices en conséquence d’une exposition à des températures élevées. Dans un second temps, nous étudions la potentielle corrélation entre une variabilité introduite sur les niveaux de deux protéines du réseau de réponse au choc thermique et le phénomène de mort fractionnelle. Selon les prédictions de notre modèle, la variabilité des protéines chaperons (espèces réparatrices) mesurée expérimentalement ne suffit pas à elle seule à expliquer la mort fractionnelle, qui doit impliquer d’autres sources de variabilité. Enfin, une analyse des courbes iso-effet générées à l’aide d’un modèle générique de la réponse cellulaire aux stress transitoires nous montre l’existence de quatre régimes de sensibilité en fonction des paramètres durée-intensité du stress ainsi que des paramètres du réseau et de ses échelles de temps. Nos travaux soulignent le potentiel et l’utilité des modèles de réseaux dynamiques dans la caractérisation des courbes dose-effet. / The living cell is constantly exposed to various types of stress that can damage its components. When the induced damages are detected, defense mechanisms are activated to repair them while optimally managing the energy resources available and necessary for cell function. If the stress is too severe and the system can not defend itself, death will be inevitable. The cellular response to stress is orchestrated by intracellular signaling networks that are extraordinarily complex. The molecular species constituting these networks perform various tasks through biochemical reactions, forming synchronized biological process machineries. Our approach in this thesis for the study of these networks is to model them mathematically to reproduce an observed phenomenon and identify its key players, analyze their reactions in response to different signals, and possibly make precise enough and experimentally verifiable predictions that can be of an extreme utility for therapeutic applications. In our studies, we focus on thermal stress and on the resulting cellular response in terms of the dynamics of the molecular species involved, but also of cell fate (death or survival) at the end of the exposure, we adress those questions by dynamic models describing the biochemical kinetics of system variables as a consequence of temperature variation. In a first step, we demonstrate through simulations, followed by experimental validation, that the temporal form of heat stress significantly impacts cell survival. This first result highlights a mechanism of saturation of the repair species as a consequence of exposure to high temperatures. In a second step, we study the potential correlation between a variability introduced on the levels of two proteins in the heat shock response network and the phenomenon of fractional killing. According to our model predictions, experimentally measured chaperone proteins (repair species) variability alone is not sufficient to explain fractional killing, which must involve other sources of variability. Finally, an analysis of the isoeffect curves generated by a generic model of the cellular response to transient stress shows the existence of four sensitivity regimes depending on the duration-intensity parameters of the stress as well as on the parameters of the response network and its time scales. Our work highlights the potential and utility of dynamic network models in the characterization of dose-response curves.

Stimulating the Immune system of fish by oral administration of immunomodulatory substances can prevent disease outbreaks in aquaculture. Yeast p( l ,3/1 ,6)-glucan, the active ingredient of the commercially available feed supplement MacroGard®, has been associated with production of microbicical and cytocidal oxygen radicals and the induction of apoptosis in human cancer cells. Hence it was hypothesized that the immunosuppressive effects of this substance, which were observed by some authors, could be caused by induction of apoptosis in immune cells due to oxidative stress. Utilizing molecular and immunohistochemical staining techniques it has been shown that although MacroGard® can induce apoptosis il1 vitro it is not associated with this form of cell death il1 vivo. However dietary MacroGard® influences the expression of apoptosisrelated genes in a time and organ dependent manner. Apoptosis is also associated with disease and can be modulated by both the host as a means of controlling infection, and by pathogens in an attempt to avoid the host immune system. It was thus hypothesized that bacteria (Aeromol1as saimol1icida) and vilUses (koi herpes vilUs (KHV) and spring viremia of earp virus (SVCV)) ean modulate apoptosis in carp and that this can be affected by oral immunostimulation. In this thesis it was established that the bacterial pathogen A. saimol1icida and the SVC vilUS induce apoptosis and that this is associated with changes of apoptosis-related gene expression. KHV in contrast appeared to supress apoptosis during early stages of the infection but induced it during the later stages possibly as a means to dissen'linate the vilUs. MacroGard® enhanced gene expression in response to SVCV infection and exposure to vilUs- and bacteriaassociated molecular patterns (i.e. Poly(I:C) and LPS). In conclusion, MacroGard® can influence apoptosis-related gene expression but does not appear to induce apoptosis on its own.

Η δοξορουβικίνη αποτελεί μια ευρέως χρησιμοποιούμενη αντικαρκινική ένωση σε διάφορους τύπους νεοπλασιών και ανήκει στην οικογένεια των ανθρακυκλινών. Για την κυτταροστατική της δράση έχουν προταθεί διάφοροι μηχανισμοί, μεταξύ των οποίων επαγωγή απόπτωσης και γενετικές αλλαγές. Η απόπτωση αποτελεί μια φυσιολογική διαδικασία, η οποία ελέγχει τον αριθμό των κυττάρων σ’ έναν οργανισμό. Λειτουργεί ως αμυντικός μηχανισμός σε περιπτώσεις κυττάρων που έχουν υποστεί βλάβη και παρεμποδίζει την εξάπλωση μεταλλάξεων συμπεριλαμβανομένων και εκείνων που οδηγούν σε καρκινογένεση. Η απόπτωση μπορεί να ενεργοποιηθεί κυρίως μέσω της εξωγενούς και της ενδογενούς οδούς. Στην παρούσα ερευνητική εργασία μελετήθηκε η σχέση επαγωγής απόπτωσης και γενετικών βλαβών που προκαλούνται από τη δοξορουβικίνη σε κυτταρική σειρά ποντικού C2C12. Για τη διερεύνηση των γενετικών βλαβών μελετήθηκε η συχνότητα των μικροπυρήνων (ΜΝ) με χρώση DAPI. H επαγωγή απόπτωσης εξετάστηκε με τη μέθοδο διπλής χρώσης αννεξίνη V/ιωδιούχο προπίδιο. Η μελέτη του μηχανισμού προέλευσης των μικροπυρήνων πραγματοποιήθηκε με σήμανση πρωτεϊνών του κινητοχώρου, την ανοσοχημική μέθοδο CREST σε συνδυασμό με ανοσοσήμανση της α-τουμπουλίνης για την εμφάνιση του κυτταροπλάσματος. Παρατηρήθηκε ότι η δοξορουβικίνη στις μελετηθείσες συγκεντρώσεις (0.05 μg/ml, 0.10 μg/ml, 0.20 μg/ml, 1.00 μg/ml, 2.50 μg/ml και 5.00 μg/ml,) προκαλεί αυξημένες συχνότητες αποπτωτικών κυττάρων και κυττάρων που περιέχουν μικροπυρήνες. Διαπιστώθηκε ότι η αύξηση της απόπτωσης σχετίζεται με μείωση της συχνότητας των μικροπυρήνων, υποδεικνύοντας πιθανή συμμετοχή της απόπτωσης στον περιορισμό των κυττάρων που φέρουν γενετική βλάβη. Η μελέτη του μηχανισμού προέλευσης των μικροπυρήνων έδειξε ότι η δοξορουβικίνη επάγει τη χρωμοσωματική θραύση και σε μικρότερο βαθμό τη χρωμοσωματική καθυστέρηση. Η τελευταία παρατήρηση μας οδήγησε στη μελέτη της οργάνωσης της μιτωτικής συσκευής μετά την επίδραση με δοξορουβικίνη. Η μιτωτική συσκευή αποτελεί στόχο χημικών ενώσεων που επηρεάζουν το χρωμοσωματικό αποχωρισμό και η μελέτη της πραγματοποιήθηκε με διπλή ανοσοσήμανση για τους μικροσωληνίσκους (β-τουμπουλίνη) και το κεντρόσωμα (γ-τουμπουλίνη). Παρατηρήθηκαν επίσης, ανώμαλες μεταφάσεις και τροποποιημένος αριθμός κεντροσωμάτων, φαινόμενα που σχετίζονται με λανθασμένο χρωμοσωματικό αποχωρισμό. Τέλος σε προκαταρκτικά πειράματα κατά τα οποία πραγματοποιήθηκε αναστολή της απόπτωσης που προκαλείται από τη δοξορουβικίνη με το γενικό αναστολέα Z-VAD-fmk, παρατηρήθηκε αύξηση της συχνότητας των κυττάρων με μικροπυρήνες που προκαλούνται από την ίδια ένωση. Η παρατήρηση αυτή αποτελεί μια ακόμη ένδειξη για την πιθανή συμμετοχή της απόπτωσης στον περιορισμό των κυττάρων με γενετικές αλλαγές. Επίσης παρατηρήθηκε ότι η αναστολή της απόπτωσης τροποποιεί τη συχνότητα των κυττάρων με ανώμαλο αριθμό κεντροσωμάτων που επάγονται από τη δράση της δοξορουβικίνης, αυξάνοντας τα κύτταρα με ένα κεντρόσωμα και μειώνοντας τα κύτταρα με πολλαπλά κεντροσώματα, υποδεικνύοντας πιθανή εμπλοκή της απόπτωσης στο σχηματισμό ασύμμετρων κυτταρικών διαιρέσεων. Οι τελευταίες παρατηρήσεις χρειάζονται περαιτέρω διερεύνηση. / Doxorubicin is one of the most widely used anticancer drugs due to its broad spectrum of antitumor activity and belongs to the family of anthracyclines. Various mechanisms have been proposed for its cytostatic activity, including induction of apoptosis and genetic alterations. Apoptosis is a normal procedure which provides a cell control mechanism. It is a defense mechanism for the elimination of mutated cells including those that might cause carcinogenesis. Apoptosis is carried out by intrinsic as well as extrinsic pathways. In the present thesis we investigated the relationship between apoptosis and genetic damage induced by doxorubicin in C2C12 mouse cell line. Genetic damage was evaluated by studying the micronucleus (MN) frequency by DAPI staining.The induction of apoptosis was examined by double fluorescence staining of ANNEXIN V/PI. The analysis of the mechanism by which the micronuclei are induced was further investigated by kinetochore labeling using the immunocytochemical method CREST with simultaneous labeling of α- tubulin, for the detection of cytoplasm. It was observed that doxorubicin at the studied concentrations (0.05 μg/ml, 0.10 μg/ml, 0.20 μg/ml, 1.00 μg/ml, 2.50 μg/ml and 5.00 μg/ml) increased frequencies of apoptotic and micronucleated cells. It was shown that the induction of apoptosis was related with decreased micronucleus frequency, indicating a possible involvement of apoptosis in the elimination of the micronucleated cells. The analysis of the mechanism of micronucleus formation showed that doxorubicin induced chromosome breakage and in a lesser extent chromosome loss. The latter ability was further investigated by analyzing the integrity of mitotic apparatus after exposure to doxorubicin. The mitotic apparatus consists one of the main targets of chemical compounds which induce chromosome missegregation. The analysis was achieved by double immunofluorescence staining for microtubules (β-tubulin) and for centrosome (γ- tubulin). It was shown that doxorubicin provoked high abnormal metaphase figures and abnormal centrosome numbers, phenomena that are related with abnormal chromosome segregation. Finally, preliminary experiments, where doxorubicin-induced apoptosis was inhibited by the pancaspase inhibitor Z-VAD-fmk, resulted to an increase of the frequency of micronucleated cells generated by doxorubicin. This observation is an additional evidence for the involvement of apoptosis in the elimination of cells with genetic damage. Moreover, it was observed that inhibition of apoptosis altered the frequency of cells with abnormal centrosome number, which are formed after doxorubicin treatment, by increasing cells with only one centrosome and decreasing cells with multiple centrosomes, indicating a possible participation of apoptosis in the formation of asymmetric divisions. Further investigation is warranted to clarify the last observations.