Programmed cell death (PCD) is a highly evolved mechanism of cellular suicide that is aberrantly activated following neural injury. Two fundamental PCD signaling pathways termed the extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, have been described. While each pathway is initiated by distinct cellular stimuli, both pathways culminate in the activation of downstream executioner caspases. Previous efforts to isolate the in vivo contribution of each pathway have been hindered by the embryonic lethality of casp8 and casp9 null mice. In the present study, I overcame this obstacle to directly assess the contribution of each pathway following two well-characterized forms of acute neural injury; excitotoxic destruction of CA1 pyramidal neurons, and the loss of motor neurons following facial nerve transection. To determine the role of caspase-8, I constructed several lines of mice in which caspase-8 was conditionally ablated within the relevant neuronal populations. The results obtained from these animals definitively demonstrate that caspase-8 is not required by either motor neurons or CA1 pyramidal neurons to undergo PCD following injury. Therefore, these findings have provided the first direct experimental evidence to counter the widely held dogma of caspase-8 as the central effector of death receptor-mediated signaling within neurons. With respect to the intrinsic pathway, several lines of evidence suggest that the apoptosome predominantly regulates the death of motor neurons. I tested this hypothesis by performing facial axotomies in mice containing a point mutation introduced (“knocked in”) into the genomic locus of cytochrome c which abolishes its ability to activate the intrinsic pathway. Homozygous cytochrome c knock-in mice displayed a significant enhancement in motor neuron survival in comparison to control littermates following injury. However, the level of motor neuron protection differed from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family. Therefore, the results of this study directly demonstrate the influence of the apoptosome on injury-induced neuronal PCD isolated from upstream Bcl-2 family-mediated effects. In addition, my results have provided the first evidence that activation of the apoptosome is required for the release of apoptosis inducing factor (AIF) from the mitochondria of injured motor neurons in vivo.

Recent studies have proposed that non-nitrogen-containing and nitrogen- containing bisphosphonate drugs inhibit osteoclastic bone resorption by different molecular mechanisms. The aim of this thesis was to investigate the molecular mechanisms of action of bisphosphonates in macrophages and osteoclasts and, in particular, the activation of caspase proteases and their role in apoptotic cell death. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates was found to involve the activation of caspase-3. By contrast, non-nitrogen- containing bisphosphonates did not cause caspase activation or J774 apoptosis, indicating that these bisphosphonates have different cellular effects. Further studies demonstrated that nitrogen-containing bisphosphonates induced J774 macrophage apoptosis by inhibiting the mevalonate pathway and preventing protein farnesylation and/or geranylgeranylation, since these compounds inhibited incorporation of [14 C] mevalonate into isoprenylated proteins, and addition of cell-permeable intermediates of the mevalonate pathway (FPP and GGPP) prevented bisphosphonate-induced apoptosis. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates or mevastatin (another inhibitor of the mevalonate pathway) was dependent on protein synthesis, since cycloheximide effectively prevented the activation of caspase-3 and prevented J774 cell apoptosis. Both nitrogen-containing bisphosphonates and non-nitrogen-containing bisphosphonates caused caspase-3 activation and apoptosis of rabbit and human osteoclasts in vitro. The active form of caspase-3 was detected in apoptotic osteoclasts by immunofluorescence staining, whilst caspase-3 activity was visualised in osteoclasts using a cell-permeable, fluorogenic substrate and detected in cell lysates using caspase-specific substrates. Bisphosphonate-induced osteoclast apoptosis involved loss of mitochondrial membrane potential and could be prevented by a specific inhibitor of caspase-3/-7. The ability of bisphosphonates to activate caspase-3 and cause apoptosis was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, suggesting that caspase activation and apoptosis in osteoclasts induced by bisphosphonates is the consequence of loss of geranylgeranylated proteins. Bisphosphonate-induced osteoclast apoptosis and inhibition of bone resorption in vitro was suppressed by RANK ligand. This did notappear to involve changes in Akt phosphorylation or increased expression of cIAP-1 or cIAP-2. These studies have helped to identify the molecular mechanisms of action of bisphosphonate drugs and have provided new insights into the involvement of caspases in osteoclast apoptosis.

Introduction : l’infection par le cytomégalovirus (CMV) humain est la plus fréquente des infections après greffe d'organe. Des effets indirects à long terme sont fortement suspectés mais restent encore largement incompris. Notre travail de thèse s’est intéressé à mieux comprendre les conséquences de l’infection du donneur par le CMV sur la réponse immunitaire du receveur et sur le devenir de son greffon. Résultat : nous avons initialement rapporté que l'infection du donneur (D+) par le CMV est un facteur de risque indépendant de perte de fonction du greffon rénal particulièrement si le receveur est également séropositif avant la greffe (D+R+ comparé aux D+R-). Le risque est fortement majoré en cas de mésappariement complet en HLA de classe I entre le receveur et son donneur. Puis nous avons analysé le rôle du greffon infecté dans le développement de la réponse lymphocytaire anti-CMV. Nous avons rapporté pour la première fois que la superinfection CMV entraine une augmentation du nombre de LT CD8 répondeurs spécifiques du CMV à distance de la transplantation, à condition que le donneur et le receveur partagent des identités HLA-I. De plus nous avons montré chez le sujet D+R- que l'expansion des lymphocytes T CD8 anti CMV restreints par le HLA-A2 nécessite l'expression de ce HLA par le donneur. Ces résultats ensemble indiquent le rôle des cellules du donneur dans l’inflation des LT CD8 anti-CMV à distance de la greffe. Dans un troisième travail, nous avons montré qu’un polymorphisme du gène de Programmed Cell Death 1 (PD-1.3) influe sur la survie des greffons rénaux et pulmonaires D+, les patients porteurs de l’allèle variant A ayant un meilleur pronostic que les patients homozygotes GG. Nos données indiquent aussi que les patients homozygotes AA ont un plus grand nombre de lymphocytes anti-CMV producteurs d'IFN-ɣ, suggérant que ce polymorphisme pourrait être associé à une dysfonction de la réponse immunitaire spécifique anti-CMV. Conclusion : ensemble ces données suggèrent pour la première fois que la qualité de la réponse lymphocytaire cytotoxique anti-CMV pourrait être importante pour contrôler la réplication virale dans le greffon et les lésions induites par cette dernière. Ainsi nous proposons deux mécanismes à l’origine du développement des lésions liées à l'infection à CMV dans le rein: défaut de reconnaissance des cellules allogéniques infectées en cas de mésappariement complet en HLA de classe I et une dysfonction LT CD8 anti-CMV. / Background: cytomegalovirus (CMV) is the leading cause of viral infection after solid organ transplantation. Despite a large body of literature, the effects of chronic cytomegalovirus (CMV) infection on graft outcome remain controversial.Results: we first reported that donor CMV infection (D+) was an independent risk factor of kidney graft loss, especially in pretransplant infected recipients (R+). In addition, we observed that full HLA-I mismatching was an important determinant of this risk. In a second study, we focused on effect of donor CMV infection on anti-CMV specific immune response. We reported that CMV superinfection greatly increased the number of anti-CMV IFN-ɣ-producing T cells, provided that donor and recipient shared at least one HLA-I identity. Then in D+R- HLA-A2-expressing recipients, we compared the number of anti-CMVpp65 CD8+T cells restricted by HLA-A2 depending on whether the donor expressed or not HLA-A2. Patients who received non-HLA-A2 kidneys developed very few anti-CMVpp65 T-cells restricted by HLA-A2 as compared to those who received an HLA-A2-expressing kidney. This result indicated that presentation of CMV peptides by donor cells was crucial to stimulate the expansion of pp65-specific memory CD8 T cells. Finally, we established that a SNP in the Programmed Cell Death 1 gene (PD-1.3) influenced D+ kidney and lung transplants survival, while it was also associated with the level of anti-CMV specific T-cell response. Conclusion: taken together, these data suggest that anti-CMV specific immune response is pivotal to control infection within the graft and prevent subsequent organ damages. We propose two mechanisms to explain effect of donor CMV infection on graft outcome: (1) inability of anti-CMV CD8 T cells to recognize donor-infected cells in case of full HLA-I mismatching, (2) dysfunction of anti-CMV CD8 T cells after transplantation in some patients, highlighted by our genetic study.

by Suen Yick-keung. / "July 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 255-273). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Chan Yee Man Elaine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 130-143). / Abstract --- p.I / Acknowledgements --- p.III / Abbreviations --- p.IV / Objectives of the study --- p.VII / Table of Contents --- p.VIII / Chapter Section 1 --- Introduction --- p.1 / Chapter 1.1 --- Necrosis vs Apoptosis --- p.2 / Chapter 1.2 --- Cell Death by Apoptosis --- p.4 / Chapter 1.3 --- The Biochemistry of Nitric Oxide --- p.9 / Chapter 1.4 --- Mechanisms of NO Action --- p.14 / Chapter 1.5 --- Signal Transduction Pathways to Apoptosis --- p.17 / Chapter 1.5.1 --- Regulation by Ca2+ --- p.17 / Chapter 1.5.2 --- Protein Kinase C --- p.20 / Chapter 1.5.3 --- cAMP --- p.21 / Chapter 1.5.4 --- Protein tyrosine kinase --- p.21 / Chapter 1.5.5 --- Ceramide --- p.22 / Chapter 1.5.6 --- pH --- p.23 / Chapter 1.5.7 --- Oxygen Radicals --- p.23 / Chapter 1.5.8 --- Anchorage Dependence and Extracellular Matrix --- p.24 / Chapter Section 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Animal --- p.28 / Chapter 2.1.2 --- Cell line --- p.28 / Chapter 2.1.3 --- "Culture media, buffers and chemicals" --- p.28 / Chapter 2.1.4 --- Dye solutions --- p.30 / Chapter 2.1.5 --- Reagents and buffers for polyacrylamide gel electrophoresis (PAGE) --- p.31 / Chapter 2.1.6 --- Reagents and buffers for Western blotting --- p.32 / Chapter 2.1.7 --- Reagents and buffers for agarose gel electrophoresis --- p.33 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Cell culture --- p.35 / Chapter 2.2.2 --- [3H]-Thymidine incorporation --- p.35 / Chapter 2.2.3 --- MTT assay --- p.36 / Chapter 2.2.4 --- Determination of NO by Griess assay --- p.36 / Chapter 2.2.5 --- Observation of apoptotic morphology of cells by confocal laser scanning microscopy (CLSM) --- p.37 / Chapter 2.2.6 --- Determination of cell death induced by NO producing drugs --- p.38 / Chapter 2.2.7 --- Determination of cell death induced by Concanavalin A --- p.38 / Chapter 2.2.8 --- Determination of effect of nitric oxide synthase (NOS) inhibitor on cell death induced by Con A --- p.39 / Chapter 2.2.9 --- Determination of the requirement of Ca2+ in cell death induced by NO producing drugs --- p.39 / Chapter 2.2.10 --- Determination of the requirement of cGMP in cell death induced by NO producing drugs --- p.40 / Chapter 2.2.11 --- Determination of cell death induced by PKC activation and depletion --- p.40 / Chapter 2.2.12 --- Determination of effect of PKC depletion on cell death induced by NO producing drugs and Con A --- p.41 / Chapter 2.2.13 --- Observation of immunofluorescence by confocal laser scanning microscopy --- p.41 / Chapter 2.2.14 --- Preparation of protein samples for PAGE --- p.42 / Chapter 2.2.15 --- Polyacrylamide gel electrophoresis --- p.43 / Chapter 2.2.16 --- Western blotting of PKC --- p.44 / Chapter 2.2.17 --- Preparation of DNA samples from cells --- p.46 / Chapter 2.2.18 --- Agarose gel electrophoresis of DNA --- p.47 / Chapter 2.2.19 --- Statistical analysis --- p.48 / Chapter Section 3 --- Results --- p.49 / Chapter 3.1 --- Induction of apoptosis in macrophages by NO producing drugs --- p.50 / Chapter 3.2 --- Apoptosis induced by NO producing drugs was caused by NO --- p.54 / Chapter 3.3 --- Signal transduction pathways to the NO-induced cell death in macrophages --- p.66 / Chapter 3.3.1 --- Calcium ion --- p.66 / Chapter 3.3.2 --- cGMP --- p.68 / Chapter 3.3.3 --- Protein kinase C --- p.71 / Chapter 3.4 --- Induction of apoptosis by Con A --- p.89 / Chapter 3.5 --- Tubulin structure in Con A-treated cells --- p.94 / Chapter 3.6 --- Nitric oxide and Con A-induced cell death in macrophages --- p.96 / Chapter 3.7 --- Effect ofNOS inhibitor on cell death induced by Con A --- p.102 / Chapter 3.8 --- Involvement of PKC in the Con A-induced cell death in macrophages --- p.106 / Chapter Section 4 --- Discussion --- p.114 / Chapter 4.1 --- Induction of apoptosis in macrophages by NO producing drugs --- p.118 / Chapter 4.2 --- Signal transduction pathways to the NO-induced cell death in macrophages --- p.120 / Chapter 4.2.1 --- Ca2+ ion --- p.120 / Chapter 4.2.2 --- cGMP --- p.120 / Chapter 4.2.3 --- Protein kinase C --- p.121 / Chapter 4.3 --- Induction of apoptosis by Con A --- p.124 / Chapter 4.4 --- Tubulin structure in Con A-treated cells --- p.124 / Chapter 4.5 --- Nitric oxide and Con A-induced cell death --- p.125 / Chapter 4.6 --- Effect ofNOS inhibitor on cell death induced by Con A --- p.125 / Chapter 4.7 --- Involvement of PKC in the Con A-induced cell death in macrophages --- p.127 / Chapter Section 5 --- Bibliography --- p.129 / References --- p.130

Macroautophagy (‘autophagy’ hereafter) mediates the capture of aberrant cytoplasmic material into vesicles called autophagosomes, which then shuttle to lysosomes for degradation. Autophagy is implicated in numerous diseases, largely in a pro-survival role. However, autophagy has also been suggested as a form of programmed cell death (PCD), from cases of dying cells showing autophagosome accumulations. Debate occurs between whether these vesicles drive the lethality, or are instead a failing rescue attempt. This study aimed to provide clarity on this issue. Via the use of chemical and genetic strategies of inducing autophagosome accumulations, we found combining stimulators of autophagosome biogenesis with lysosomal degradation inhibitors gave rise to toxicity. Notably, this effect was dependent on the autophagy machinery and independent of other PCD routes. Research into the underlying mechanisms revealed an energy deficit under these conditions. Since autophagosomes cannot be recycled at lysosomes here, their continued synthesis affords no survival benefits, and instead just serves to deplete cellular energy further. For this reason, we designate this event ‘Futile Autophagosome Synthesis’ (FAS) toxicity. Other contributors to this toxicity include the persistence of harmful agents like Reactive Oxygen Species (ROS). Having established our FAS model, we explored its relevance in both cancer and neurodegeneration. Importantly, we found FAS inducing strategies to be effective in tumour treatment. Also, inhibiting FAS reduced the toxicity seen in neurodegenerative disease. Therefore, not only does this study improve our knowledge of autophagy in PCD, but also indicates it may have important medical implications.

The model angiosperm Arabidopsis thaliana produces viable seed through coordinated growth of three constituents; seed coat, embryo and endosperm. During development the embryo grows to fill the space defined by the seed coat. The growing embryo is surrounded by endosperm, an ephemeral, nutritive structure. The process of programmed cell death (PCD) is essential for endosperm consumption by the embryo however very little is known about developmental PCD in the endosperm. ZOU is a transcription factor expressed specifically in endosperm adjacent to the growing embryo in the Embryo Surrounding Region (ESR) (Yang et al., 2008). zou seed likely have reduced PCD resulting in abnormally persistent endosperm and a small embryo at seed maturity which results in seed shriveling. A second zou phenotype is an impairment of cuticle development in the embryonic leaves (cotyledons), suggesting that ZOU may mediate a signal from endosperm to embryo. The ESR expressed gene ALE1 is down-regulated in zou. When ALE1 is artificially expressed in zou ESR by the construct pSUC5::ALE1 the zou epidermal phenotype is rescued but not the seed shriveling phenotype of zou (Xing et al., 2013). Fixed and resin-embedded sections of zou and pSUC5::ALE1 lines herein confirm that zou-like endosperm is exhibited in pSUC5::ALE1 lines. This confirms that the two phenotypes of zou are genetically separable. The involvement of ZOU in epidermal processes is further confirmed through genetic studies showing that ZOU acts in the same pathway to impart embryonic cuticle as the embryo-expressed Receptor Like Kinases GSO1 and GSO2. In order to quantify PCD in the endosperm of wild-type and zou seed, PCD expression marker and TUNEL analysis were conducted. One PCD marker, pCEP:H2A-YFP is shown to be expressed in wild-type ESR, it is not clear if expression is lost in zou. To identify candidate genes under the control of ZOU active in endosperm PCD the results from several transcriptional profiling experiments were analysed and validated; this detailed gene expression in wild-type, ale1 and zou siliques which allowed for the identification of targets of ZOU but not of ALE1, targets predicted to be PCD effectors. In silico expression and ontology analysis confirmed likely roles for some candidates in endosperm PCD processes (particularly cell wall modification). Selected targets were cloned under pSUC5 and expressed in the ESR of zou seed as part of a molecular screen for the rescue of the zou endosperm phenotype. The ZOU target FRINGE-Like, a Glycosyl Transferase which shows strong endosperm expression is shown to partially rescue the zou phenotype but does not rescue the epidermal phenotype, suggesting that it may mediate PCD processes under ZOU control. The initial discovery that a Glycosyl Transferase may be active in a developmental PCD process in plants is exciting and novel and benefits understanding of developmental PCD and endosperm breakdown, two poorly characterized processes in plants.

Programmed cell death and cell corpse clearance are an essential part of an organism’s overall health and development. Cell corpses are often engulfed by professional phagocytes such as macrophages. However, in certain tissues, neighboring non-professional cells can also carry out phagocytic functions. Here, we use the Drosophila melanogaster ovary to investigate novel genes required for engulfment by non-professional phagocytes. In the Drosophila ovary, neighboring epithelial cells facilitate the clearance of dying germline cells. We performed an unbiased kinase screen to identify novel proteins and pathways involved in cell clearance in the nurse cell. Several genes identified in this screen were members of the phosphoinositide 3-kinase (PI3K) family. The class II and III PI3Ks are required for nurse cell clearance and acidification during late-stage oogenesis. Class I PI3K is required for progression during engulfment in mid-stage oogenesis. This kinase screen has revealed novel genes for further exploration and investigation. / 2019-02-28T00:00:00Z

Ferroptosis is a novel non-apoptotic, oxidative form of regulated cell death that can be triggered by diverse small-molecule ferroptosis inducers (FINs) and genetic perturbations. Current lack of insights into the cellular contexts governing sensitivity to ferroptosis has hindered both translation of FINs as anti-cancer agents for specific indications and the discovery of physiological contexts where ferroptosis may function as a form of programmed cell death. This dissertation describes the identification of cellular features predicting susceptibility to ferroptosis from data generated through a large-scale profiling experiment that screened four FINs against a panel of 860 omically-characterized cancer cell lines (Cancer Therapeutics Response Portal Version 2; CTRPv2 at http://www.broadinstitute.org/ctrp/). Using correlative approaches incorporating transcriptomic, metabolomic, proteomic, and gene-dependency feature types, I uncover both pan-lineage and lineage-specific features mediating cell-line response to FINs. The first key finding from these analyses implicates high expression of sulfur and selenium metabolic pathways in conferring resistance to FINs across lineages. In contrast, the transsulfuration pathway, which enables de novo cysteine synthesis, appears to plays a role in ferroptosis resistance in a subset of lineages. The second key finding from these studies identifies cancer cells in a high mesenchymal state as being uniquely primed to undergo ferroptosis. This susceptibility stems from a specific dependency of high mesenchymal-state cancer cells on the lipid hydroperoxide quenching mechanisms inhibited by FINs and is conserved across cancer cell lines of mesenchymal origin, epithelial-derived cancer cell lines that have undergone an epithelial-to-mesenchymal-transition, and patient-derived cancer cells exhibiting mesenchymal state-mediated resistance to anti-cancer therapies. The work presented herein formalizes frameworks for studying small molecule inducers of cell death through cell-line profiling. The results advance current mechanistic understanding of the cellular circuitry underlying ferroptosis sensitivity and lay the foundation for a novel therapeutic approach using ferroptosis inducers to target high mesenchymal-state cancer cells.

Red blood cell (RBC) transfusions are associated with adverse effects, such as an increased risk of bacterial infection. In preparing RBCs for transfusion, donor RBCs are refrigerator stored for extended periods of time, during which they undergo oxidative damage, ultimately leading to their rapid post-transfusion clearance from the circulation. Macrophages play important roles in recycling iron derived from the clearance of RBCs. They are also a critically important component of host defense, protecting against invading pathogens. However, the effects on macrophage biology of acutely ingesting large numbers of RBCs are not completely understood. To investigate this issue, we used a mouse model of RBC transfusion and clearance, which mimics the clinical setting. In this model, transfusions of refrigerator storage-damaged (i.e., “old”) RBCs led to increased erythrophagocytosis by splenic red pulp macrophages (RPMs). This robust erythrophagocytosis induced ferroptosis, an iron-dependent form of cell death, in RPMs. This was accompanied by increases in reactive oxygen species and lipid peroxidation in vivo, which were reduced by treatment in vitro with ferrostatin-1, a ferroptosis inhibitor. Old RBC transfusions also induced RPM-dependent chemokine expression by splenic Ly6Chi monocytes, which signaled Ly6Chi monocyte migration from bone marrow to spleen, where these cells subsequently differentiated into RPMs. The combination of cell division among remaining splenic RPMs, along with the influx of bone marrow-derived Ly6Chi monocytes, suggests that, following RPM depletion induced by robust erythrophagocytosis, there is a coordinated effort to restore homeostasis of the RPM population by local self-maintenance and contributions from circulating monocytes. However, the effects on the overall functioning of the splenic Ly6Chi monocytes and remaining RPMs are unclear, especially their responses to subsequent immune challenges. In a mouse model of RBC storage and transfusion, we found that, following a transfusion of old RBCs, macrophages were less capable of phagocytosing a subsequent particle stimulus, such as bacteria (i.e., Escherichia coli and Staphylococcus aureus) or additional old RBCs. However, splenic Ly6Chi monocytes became activated in a specific timeframe following the initial old RBC transfusion, thereby increasing their phagocytic capacity. Nonetheless, despite contributions from activated splenic Ly6Chi monocytes, RPM function was indispensable for clearing S. aureus; this functional impairment may make the transfusion recipient susceptible to S. aureus sepsis. In conclusion, these findings may be clinically relevant to pathological conditions that can arise as a result of increased erythrophagocytosis, such as transfusion-related immunomodulation and impaired host immunity.