Certains agents chimiothérapeutiques tels que les anthracyclines ou l'oxaliplatine induisent une mort cellulaire immunogène, ce qui implique que les cellules mourrantes du patient servent de vaccin thérapeutique en stimulant une réponse immunitaire antitumorale. La mort cellulaire immunogène est caractérisée par la libération de signaux d'alarme par la cellule tumorale mourante qui permettent l’activation du système immunitaire. En premier lieu, l'exposition de la calréticuline à la surface de la cellule tumorale mourante va agir comme un signal de type «eat-me» pour les cellules dendritiques. Une fois relâchée, la protéine nucléaire HMGB1 se lie au récepteur TLR4 afin de faciliter la présentation antigénique. Les cellules mourantes vont également libérer de l'ATP qui agit sur les récepteurs P2X7 et active l’inflammasomme NLRP3, conduisant à la libération d'IL-1β et ainsi à l’activation des cellules T CD8+ productrices d’IFN-γ. L’autophagie est un mécanisme cellulaire qui est activé en réponse à la chimiothérapie. L'autophagie signifie «self-ating», il s'agit d'un processus cellulaire activé par diverses conditions de stress, par lequel les cellules peuvent dégrader les protéines et les organites. Il peut aussi être induit par un stress du réticulum endoplasmique. Ce dernier étant également impliqué dans l'exposition de la calréticuline pendant la mort cellulaire immunogène, nous avons au cours de cette étude cherché à déterminer le rôle de l'autophagie dans la mort cellulaire immunogène. Nous avons constaté que l'autophagie est nécessaire pour la libération de l'ATP après un traitement par des chimiothérapies immunogènes, en observant que le nockdown de gènes essentiels de l'autophagie limitait la sécrétion d'ATP. Nous avons également observé que des cellules déficientes pour l'autophagie traitées par une chimiothérapie immunogène sont incapables d’immuniser des souris contre une injection de cellules vivantes. En outre, les tumeurs déficientes pour l’autophagie ne répondent pas à un traitement systémique immunogène dans des souris immunocompétentes et continuent à proliférer en comparaison à des tumeurs “wild-type”. De plus, nous avons montré que les cellules déficientes pour l'autophagie ne sont pas en mesure de recruter des cellules dendritiques dans le lit tumoral ou d'induire l’activation des cellules T CD8+. A l'inverse, l'inhibition des enzymes de dégradation de l’ATP extracellulaire accroit les concentrations d'ATP dans les tumeurs déficientes pour l'autophagie, ce qui rétablit le recrutement des cellules immunitaires dans le lit tumoral et restaure la réponse chimiothérapeutique des cancers déficients pour l'autophagie. Ainsi, cette étude a montré l'importance de l'autophagie dans la réponse anti-tumorale spécifique, après traitement par des chimiothérapies immunogènes. Ces résultats ouvrent de nouvelles perspectives dans le concept de la mort cellulaire immunogène. / In recent years it has been demonstrated that some chemotherapeutic agents such as anthracyclines or oxaliplatin can induce a type of tumor cell death that is immunogenic, implying that the patient’s dying cancer cells serve as a therapeuticvaccine that stimulates an antitumor immune response, which in turn can control or eradicate residual cancer cells. Immunogenic cell death is characterized by the emission of danger signals from the dying tumor cell, which activate the immune system. At first the exposure of calreticulin, acts as an «eat-me» signal for dendritic cells (DCs). Once released, the nuclear protein HMGB1 binds to TLR4 on DCs, facilitating antigen processing and presentation. The dying tumor cells also releases ATP, which acts on P2X7 receptors on DCs and activates the NLRP3 inflammasome, leading to IL-1β release, necessary for IFN-γ-producing CD8+ T cell activation. Autophagy literally ‘self-eating’ is a cellular process activated in response to various conditions of cellular stress, whereby cells can liberate energy resources via the degradation of proteins and organelles. Recently autophagy has been found activated in response to chemotherapy and in this project we aimed to determine the potential role of autophagy in immunogenic cell death. We found that autophagy isrequired for the release of ATP in response to immunochemotherapeutic treatment, as we observed that the knockdown of essential autophagy-related genes abolished its secretion. We observed that autophagy deficient cells treated with immunogenic cell death inducers failed to immunize mice against a re-challenge with living cells. Furthermore, autophagy deficient tumors growing on immunocompetent mice did not respond to systemic immunogenic treatment and continued proliferating in contrast to autophagy proficient tumors. We showed that autophagy deficient cells were neither able to recruit DCs into the tumor bed nor to activate CD8+ T cells. Conversely, the inhibition of extracellular ATP degrading enzymes increased extracellular ATP concentrations in autophagy deficient tumors, which reestablished the recruitment of immune cells into the tumor bed, and restored chemotherapeutic responses in autophagy-deficient cancers. Altogether, this study showed the importance of autophagy in tumor-specific immune response after treatment with chemotherapy, thus giving new insights into the concept of immunogenic cell death.

Abstract not available

Doctor of Philosophy / Rice (Oryza sativa L.) is very susceptible to water deficit at any time during its life cycle as a semi-aquatic cereal crop. However, the consequential damage is particularly severe if water deficit occurs during reproductive phases. The conspicuous injury often observed in rice plants exposed to water stress during meiosis of the pollen mother cell is the reduction of grain set, which is attributed to the decline of male fertility. In spite of much research on drought-induced male sterility in rice, the underlying mechanisms of the problem are poorly understood. This project was therefore conducted to investigate the molecular mechanisms of water deficit-induced pollen sterility in rice. In this study three consecutive days of water deficit treatment at -0.5 MPa osmotic potential during anther development effectively reduced the leaf water potential (leaf) and the number of viable pollen which later led to a decrease in grain set. Moreover, this thesis demonstrates that the immediate deleterious effects of water deficit to plant fertility could be estimated using a young microspore viability index, which showed a strong correlation with mature viable pollen and grain set. The present work has also illustrated that oxidative stress appears to be a plausible cause for the decline of male fertility and grain set. Water deficit has induced the excessive production of reactive oxygen species (ROS) above the redox balance, which in turn caused detrimental effects to cellular DNA and might result in programmed cell death (PCD) in the anthers. Moreover, ROS accumulation effectively influenced ATP synthesis leading to a decrease in the level of ATP in the anthers. Excessive ROS accumulation after drought could be the consequence of insufficient activity of the antioxidant system, which has been illustrated by qRT-PCR expression analysis of major antioxidant genes. Down-regulation of those genes would increase the incidence of oxidative damage. In contrast, stable or up-regulated expression of these genes resulted in less oxidative damage. Detailed investigations of sugar metabolism in anthers has provided supplemental data to develop a model of sugar unloading and transport within anther using in situ hybridisation to mRNA techniques. Analysis of sugar transportation within the cellular compartments of anther has unveiled the role of sugar metabolism on pollen sterility in rice. qRT-PCR assays of genes associated with the sugar metabolic pathway has demonstrated that the supply of both sucrose and hexoses from the anther walls to the locules was not restricted after water deficit stress. The results indicate that water deficit might not cause sugar starvation for developing microspores as previously thought, nor inhibit the initial steps of sugar utilisation such as glycolysis. This thesis has suggested new ideas regarding the role of rising sugar levels to cope with oxidative stress in anthers. Sugar accumulation might have provided protection against oxidant damage by strengthening the antioxidant system. However, the interplay between sugar and oxidative stress is not straightforward and needs to be further characterised. In-depth investigations on the interaction between sugar signalling and oxidative stress responses may help indentify the role of sugars in protecting anthers under water deficit. Although many studies on drought and chilling stresses in rice anthers have been performed, the causal mechanism of male sterility still remains to be elucidated. Findings presented in this thesis may contribute to understanding molecular mechanisms of male sterility in rice as a response to drought stress. A more detailed investigation of mitochondrial respiration in rice anthers is required to further examine this problem. Finally, this thesis suggests that signalling molecules such as 14-3-3 proteins and abscisic acid (ABA) might act upstream of ROS production and antioxidant defence in plants. Further work on these molecules might therefore further illustrate how they influence plant fertility under water shortage conditions.

[Truncated abstract] Clinical therapeutic agents to directly inhibit ischaemic neuronal death are presently unavailable. One approach to developing therapeutics is based upon the identification of proteins up-regulated by 'preconditioning', a natural adaptive response utilised by the neural cells to counter damaging insults, such as ischaemia. Thus, my project aimed to firstly identify proteins differentially expressed following erythropoietin (EPO) mediated neuronal preconditioning and secondly to assess whether any of these proteins possessed neuroprotective activity using in vitro ischaemia like models. To achieve the first aim, it was shown that in vitro neuronal EPO preconditioning could: (i) induce cell signal changes in neuronal cultures, (ii) protect neurons against in vitro ischaemia and (iii) induce differential protein expression. Overall, 40 differentially expressed proteins were identified in cortical neuronal cultures following EPO preconditioning. In order to investigate the neuroprotective or neurodamaging activity of proteins induced by EPO preconditioning I developed an adenoviral expression system for use in neuronal cultures. To this end, I assessed the suitability of four promoters (cytomegalovirus [CMV], rous sarcoma virus [RSV], human synapsin 1 [hSYN1], rat synapsin 1 [rSYN1]) previously used to express proteins in neuronal cultures and demonstrated the superiority of the RSV promoter for this purpose. ... Finally, in order to validate this adenoviral expression system, I over-expressed the anti-apoptotic protein Bcl-XL in neuronal cultures and subsequently confirmed its neuroprotective activity in the in vitro ischaemia and oxidative stress models used in my project. Using this adenoviral vector system and the in vitro oxidative stress model I assessed a number of proteins up-regulated by EPO preconditioning. The results of this preliminary study indicated that cyclophilin A (CyPA), peroxiredoxin 2 (PRDX2) and superoxide dismutase 1 (SOD1) over-expression were neuroprotective. It was subsequently verified that adenoviral mediated over-expression of CyPA and PRDX2, v but not SOD1 in cortical neuronal cultures could protect neurons from in vitro ischaemia. I also confirmed that CyPA mRNA increased in the rat hippocampus in response to 3 minutes of global cerebral ischaemia. Interestingly, an increase in CyPA, PRDX2 or SOD1 protein was not observed in the same experimental paradigm. To investigate CyPA's mode of action I confirmed that cultured neurons, but not astrocytes, express the CyPA receptor, CD147. It was also demonstrated that administration of exogenous CyPA protein to neuronal cultures could protect neurons against oxidative and ischaemic injury. I further demonstrated that exogenous administration of CyPA induces a rapid and transient activation of the extracellular signal-regulated kinase (ERK) 1/2 pathway in neuronal cultures. From this observation, I have proposed that the extracellular mediated neuroprotective activity of CyPA occurs via CD147 receptor signalling and activation of ERK1/2 pro-survival pathways. Based on the findings reported in this thesis, the neuroprotective activities of PRDX2 and CyPA warrant further investigation as targets for the development of new therapies to treat cerebral ischaemia.

<p>Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.</p><p>We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of <em>E. coli</em> decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.</p><p>Type 1 fimbria-expressing <em>E. coli </em>adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of <em>E. coli</em>, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production <em>per se</em> is important.</p><p>Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.</p>

<p>Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide.</p><p>Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL).</p><p>CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. </p><p>Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity.</p><p>In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.</p>

The steroid hormone ecdysone plays vital roles during Drosophila development. Pulses of 20E during Drosophila life cycle function as temporal cues, signaling the onset of metamorphic processes, including the stage specific programmed cell death of larval tissues. Ecdysone is the critical developmental cue orchestrating the metamorphic reformation of CNS, resulting in the formation of adult-specific neural circuitry. Ecdysone signaling is transduced by a heterodimeric receptor complex formed between two nuclear receptors: EcR and Ultraspiracle (USP). There are 18 nuclear receptors known in Drosophila and EcR is the only receptor whose functions in neuronal PCD have been well recognized. Therefore, the current study is aimed to define the role of nuclear receptors in neuronal cell death mechanisms in Drosophila. Here, I examine the function of nuclear receptors in PCD of two groups of peptidergic neurons: vCrz and CCAP. EcR and USP receptor complex on activation results in the coordinated transcriptional regulation of a host of transcription factors regulating genes essential for PCD. USP plays a dual role in ecdysone response, as its function is necessary for both activation and repression of ecdysone primary response genes. I have developed a possible dominant-negative mutant USP (usp3), and expressed it in flies using the GAL4-UAS system to illustrate the role of USP in ecdysone mediated PCD of vCrz neurons. Targeted expression of usp3 in corazonin neurons results in a complete blockage of PCD pathway. Another interacting partner of USP, Drosophila Hormone Receptor 38, however shows no involvement in PCD of vCrz neurons. I have also designed an ecdysone sensor to monitor the developmental timing of EcR activation in vCrz neurons. Further, I investigate the survival factors required for preventing the untimely PCD of these two groups of neurons. The study reveals that DIAP1 is required for the survival of larval vCrz and CCAP neurons. Also, the nuclear receptor E75 is shown to be critical for preventing premature PCD of CCAP neurons.

Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide. Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL). CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity. In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.

Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process. We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of E. coli decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis. Type 1 fimbria-expressing E. coli adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of E. coli, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production per se is important. Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.

Programmed cell death, or apoptosis, has during the last decade received a lot of attention due to its involvement in a large number of pathological conditions. Since death is always irreversible, it is important for cells to fully control the initiation and execution of this process. One of many apoptosis-regulatory proteins is XIAP, which blocks the action of caspases, a family of proteases that are important during apoptosis. However, apoptosis inhibitors have to be tightly controlled since too little cell death can lead to the development of tumors and other diseases. This thesis is the result of an aspiration to fully understand the function and regulation of XIAP. By using the yeast-2-hybrid system, we identified two novel binding partners of XIAP. The first, GPS2, was found to bind XIAP and inhibit its ability to block caspase-activity. In addition, GPS2 induced caspase-mediated cell death in two different tumour cell lines and XIAP inhibited this effect. The second binding partner, Nulp1, preferentially bound XIAP in the presence of the apoptosis-inducer staurosporine. Nulp1 induced or sensitized cell lines to cell death when overexpressed, but this was not blocked by caspase-inhibitors or XIAP, suggesting a different reason for binding than apoptosis regulation. With the aim to understand the Nulp1-XIAP interaction, we continued to study Nulp1 in vivo and in vitro. We studied three different splice variants of Nulp1 and found that they were regulated by poly-ubiquitination and nuclear shuttling. Also, Nulp1 was expressed in embryonic mice, especially in the cortical plate, hippocampal neurons and cerebellar granular neurons. Expression of Nulp1 decreased with age but was still present in cerebellar deep nuclei and Purkinje cells of adult mice. To summarize, we have identified GPS2 as an apoptosis-inducing factor and an inhibitor of XIAP in vitro, and Nulp1 as a XIAP-interacting protein during staurosporine-induced apoptosis.