Sha Ou. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 167-189). / Abstracts in English and Chinese. / abstract --- p.i / 中文摘要 --- p.iv / acknowledgements --- p.vii / Chapter chapter 1. --- introduction --- p.1 / Chapter 1.1 --- Overview --- p.1 / Chapter 1.2 --- Ribosome-inactivating proteins (RIPs) --- p.1 / Chapter 1.2.1 --- Classification --- p.2 / Chapter 1.2.2 --- Structure --- p.3 / Chapter 1.2.3 --- Enzymatic activities --- p.3 / Chapter 1.3 --- Type II RIPs --- p.5 / Chapter 1.3.1 --- Ricin --- p.5 / Chapter 1.3.2 --- Ricinus communis agglutinin (RCA) --- p.6 / Chapter 1.3.3 --- Intracellular mechanism --- p.7 / Chapter 1.3.4 --- Application of RIPs in neuroscience research: suicide axonal transport --- p.10 / Chapter 1.4 --- Type I RIPs --- p.12 / Chapter 1.4.1 --- Trichosanthin (TCS) --- p.12 / Chapter 1.4.2 --- Ricin A chain (RTA) --- p.15 / Chapter 1.4.3 --- Medical applications: immunolesioning and immunotherapy --- p.16 / Chapter 1.5 --- The types of Cell death --- p.17 / Chapter 1.5.1 --- Necrosis --- p.18 / Chapter 1.5.2 --- Apoptosis --- p.18 / Chapter 1.6 --- Inflammations --- p.21 / Chapter 1.6.1 --- Acute inflammation --- p.21 / Chapter 1.6.2 --- Chronic inflammation --- p.22 / Chapter 1.6.3 --- Retinitis --- p.22 / Chapter 1.7 --- Eye model for neurotoxicity studies in CNS --- p.23 / Chapter 1.8 --- Objective of present study --- p.24 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Plan of this chapter --- p.25 / Chapter 2.2 --- Toxins and methods used --- p.25 / Chapter 2.3 --- Animals --- p.26 / Chapter 2.4 --- Preparation of toxin solutions --- p.27 / Chapter 2.4.1 --- RIP solutions --- p.27 / Chapter 2.4.2 --- Labeling type I RIPs with fluorescence --- p.27 / Chapter 2.4.3 --- Control solutions --- p.29 / Chapter 2.5 --- Administrations of solutions --- p.30 / Chapter 2.5.1 --- Basic procedures of vitreous chamber injection --- p.30 / Chapter 2.5.2. --- Injection of trichosanthin (TCS) --- p.31 / Chapter 2.5.3 --- Injection of ricin A chain (RTA) --- p.31 / Chapter 2.5.4 --- Injection of ricinus communis agglutinin (RCA) --- p.32 / Chapter 2.5.5 --- Administration of FITC-TCS --- p.33 / Chapter 2.5.6 --- Administration of FITC-RTA --- p.33 / Chapter 2.6 --- Retinal tissue processing --- p.33 / Chapter 2.6.1 --- Paraffin method --- p.34 / Chapter 2.6.2 --- Cryostatic method --- p.35 / Chapter 2.6.3 --- Electron microscopic method --- p.35 / Chapter 2.7 --- General effects of RIPs on rat retinas --- p.36 / Chapter 2.7.1 --- Hematoxylin-and-eosin staining --- p.36 / Chapter 2.7.2 --- Retinal thickness --- p.37 / Chapter 2.7.3 --- Pathological changes --- p.38 / Chapter 2.7.4 --- Dosage study on TCS --- p.39 / Chapter 2.7.5 --- Statistics --- p.40 / Chapter 2.8 --- Mechanisms of cell death --- p.40 / Chapter 2.8.1 --- Terminal dUTP nick-end labeling (TUNEL) --- p.40 / Chapter 2.8.2 --- Immunohistochemistry for caspase-3 --- p.42 / Chapter 2.8.3 --- Double staining of cleaved caspase-3 and TUNEL --- p.42 / Chapter 2.8.4 --- Electronic microscope observation --- p.43 / Chapter 2.9 --- Entry of type I RIPs into cells --- p.43 / Chapter 2.9.1 --- Propidium iodide staining --- p.43 / Chapter 2.9.2 --- Immunohistochemical localization of Muller cells --- p.44 / Chapter 2.9.3 --- Double staining of Muller cells and TUNEL --- p.44 / Chapter 2.9.4 --- Confocal microscope --- p.44 / Chapter 2.10 --- Reactions of glial cells --- p.45 / Chapter CHAPTER 3. --- RESULTS --- p.47 / Chapter 3.1 --- Preparation of fluorescein-type I RIP conjugates --- p.47 / Chapter 3.1.1 --- Conjugate of FITC-TCS --- p.47 / Chapter 3.1.2 --- Conjugate of FITC-RTA --- p.47 / Chapter 3.2 --- Effects of TCS on retina --- p.47 / Chapter 3.2.1 --- Retina cell count - a dose-dependence study --- p.48 / Chapter 3.2.2 --- Retinal thickness measurement - a time-course study --- p.49 / Chapter 3.2.3 --- Pathological changes --- p.50 / Chapter 3.3 --- Effects of RTA on retina --- p.51 / Chapter 3.3.1 --- Retinal thickness measurement - a time-course study --- p.51 / Chapter 3.3.2 --- Pathological changes --- p.53 / Chapter 3.4 --- Effects of RCA on retina --- p.54 / Chapter 3.4.1 --- Retinal thickness measurement --- p.54 / Chapter 3.4.2 --- Pathological changes --- p.55 / Chapter 3.5 --- Summary of results: general effects of RIPs --- p.56 / Chapter 3.6 --- Cell death - TUNEL method --- p.56 / Chapter 3.6.1 --- TCS experiment --- p.57 / Chapter 3.6.2 --- RTA experiment --- p.58 / Chapter 3.6.3 --- RCA experiment --- p.58 / Chapter 3.7 --- Cell death 一 cleaved caspase-3 immunohistochemistry --- p.58 / Chapter 3.7.1 --- TCS experiment --- p.59 / Chapter 3.7.2 --- RTA experiment --- p.59 / Chapter 3.8 --- EM observation --- p.59 / Chapter 3.8.1 --- TCS experiment --- p.59 / Chapter 3.8.2 --- RTA experiment --- p.60 / Chapter 3.9 --- Summary of results: mode of cell death --- p.60 / Chapter 3.10 --- Localisation of type I RIPs --- p.61 / Chapter 3.10.1 --- FITC-TCS --- p.62 / Chapter 3.10.2 --- FITC-TCS and Muller cell double staining --- p.63 / Chapter 3.10.3 --- Muller cell and TUNEL double staining --- p.64 / Chapter 3.10.4 --- FITC-RTA --- p.64 / Chapter 3.10.5 --- Summary of results: route of intoxication --- p.65 / Chapter 3.11 --- Glial cell reactions after RIP treatment --- p.65 / Chapter 3.11.1 --- TCS experiment --- p.65 / Chapter 3.11.2 --- RTA experiment --- p.66 / Chapter 3.11.3 --- RCA experiment --- p.67 / Chapter 3.11.4 --- Summary of results: glial reactions --- p.67 / Chapter CHAPTER 4. --- DISCUSSION --- p.69 / Chapter 4.1 --- General effects of RIPs on rat retinas --- p.69 / Chapter 4.1.1 --- Effects of trichosanthin (TCS) --- p.69 / Chapter 4.1.2 --- Effects of ricin A chain (RTA) --- p.71 / Chapter 4.1.3 --- Effects of ricinus communis agglutinin (RCA) --- p.73 / Chapter 4.2 --- The mechanisms of cell death --- p.74 / Chapter 4.2.1 --- Cell death caused by TCS --- p.75 / Chapter 4.2.2 --- Caspase-3 and the retina of RCS rat --- p.77 / Chapter 4.2.3 --- Cell death caused by RTA --- p.78 / Chapter 4.2.4 --- Cell death caused by RCA --- p.80 / Chapter 4.2.5 --- Mechanism of RTA - induced necrosis --- p.81 / Chapter 4.3 --- The mechanisms of type I RIPs entering cells --- p.82 / Chapter 4.3.1 --- Transport of TCS in retinal cells --- p.82 / Chapter 4.3.2 --- The uptake of Pure FITC by rat retina --- p.85 / Chapter 4.4 --- Reactions of glial cells --- p.85 / Chapter 4.4.1 --- Glial cell reactions in TCS experiment --- p.86 / Chapter 4.4.2 --- Glial cell reactions in RTA and RCA experiments --- p.87 / Chapter 4.5 --- Possible applications of RIPs on retinal studies --- p.88 / Chapter 4.5.1 --- Potential applications of TCS --- p.88 / Chapter 4.5.2 --- Possible uses of RTA and RCA --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS --- p.91 / "FIGURES, TABLES, GRAPHS, AND LEGENDS" --- p.93 / APPENDICES --- p.154 / Appendix A Source of materials --- p.154 / Appendix B Dosages for vitreous chamber injection --- p.156 / Appendix C Protocol of conjugate fluorescein to proteins --- p.157 / Appendix D Electronic Microscope methods --- p.160 / Appendix E Histological methods --- p.162 / Appendix F Protocols of TUNEL --- p.163 / Appendix G Protocols of Immunohistochemistry staining --- p.165 / REFERENCES --- p.167

Turnip crinkle virus (TCV) has been implicated in the suppression of the hypersensitive response (HR), a type of programmed cell death induced during active resistance in Arabidopsis thaliana. In order to investigate the involvement of individual viral components in mediating suppression, TCV genes were cloned for use in an Agrobacterium tumefaciens mediated transient expression in Nicotiana benthamiana. Agroinfiltration of the HR-inducing avrPto/Pto system in conjunction with individual TCV genes has identified the p38 gene, which encodes the viral coat protein, as the gene responsible for the cell death suppression phenotype. The extent of cell death suppression by coat protein was quantified and found to be equal to the level of suppression by the whole virus and AvrPtoB, another cell death inhibitor from bacteria. Thus, the coat protein alone is sufficient to inhibit the HR in plants. Further, the effect of TCV on HR initiation by an avirulence factor from an unrelated bacterial pathogen was investigated. The presence of TCV does not affect the production, secretion or cellular processing of the bacterial avirulence factor.

Le développement d’un cancer chez un individu immunocompétent témoigne, en partie, d’un échappement tumoral au système d’immunosurveillance. Par conséquent, la restauration ou l’induction de ces mécanismes de défense anti-tumorale est une des stratégies thérapeutiques actuelles. Un des principes de l’immunothérapie est basé sur l’injection d’anticorps ayant pour cible la cellule tumorale ou les cellules effectrices de l’immunité. L’efficacité anti-tumorale de ces anticorps a été considérablement améliorée par une meilleure compréhension des modes d’action et des effets modulateurs de ces anticorps. Ainsi, afin d’optimiser l’action des effecteurs immunitaires sur les cellules tumorales, un anticorps bispécifique, trifonctionnel, le catumaxomab, capable de se lier à la molécule d'adhérence des cellules épithéliales (EpCAM) exprimée par les cellules tumorales et à l'antigène CD3 des lymphocytes T, a été développé, essentiellement en traitement intra-péritonéal des ascites néoplasiques réfractaires.L’objectif de cette étude était de déterminer les effets immunomodulateurs du catumaxomab sur des cellules néoplasiques exprimant EpCAM, à partir de deux modèles expérimentaux (allogénique et autologue), de rechercher une cytotoxicité induite par la catumaxomab, et de la caractériser, notamment en analysant la présence ou non de signaux de stress inducteurs d’une mort immunogène tels que l’exposition membranaire de la calréticuline par les cellules tumorales pré-apoptotiques, la libération d’HMGB1 et d’adénosine triphosphate (ATP) dans le milieu extra-cellulaire, responsables d’une activation des lymphocytes T.En présence de cellules EpCAM+, le catumaxomab entrainait une activation majeure des lymphocytes T (expression de CD69, CD107a, HLA-DR et PD1), stimulait une réponse inflammatoire de type Thelper 1(Th1), et provoquait la synthèse d’interféron-gamma par les lymphocytes T CD8. Le catumaxomab engageait le CD16 (FcR) des cellules monocytaires et NK. De plus, sur des modèles allogéniques, le catumaxomab, provoquait une mort cellulaire associée à la libération d’ATP et induisait une mort immunogène après pré-incubation dans de l’oxaliplatine.Par conséquent, le catumaxomab permet de moduler l’environnement immunitaire dans les ascites néoplasiques, et de convertir une inflammation chronique et immunosuppressive (Th2) en une inflammation aigüe et immunogène (Th1). En revanche, dans ces conditions, l’administration seule de catumaxomab ne semble pas déclencher de mort immunogène.Différents moyens pourraient permettre d’améliorer la cytotoxicité de cet anticorps bispécifique : (1) le combiner avec un agent anti-néoplasique tel que l’oxaliplatine afin de promouvoir une mort immunogène, (2) affiner son action sur le CD3 des lymphocytes en modifiant sa configuration spatiale (anticorps BiTE), (3) amplifier son affinité pour le récepteur Fcdes cellules accessoires (Fc défucosylé), (4) augmenter sa cytotoxicité en modifiant la cible dirigée contre la molécule du système immunitaire (anti-PD-1…). Enfin, l’utilisation clinique pourrait être facilitée en humanisant cet anticorps chimérique murin afin d’éviter la formation d’anticorps anti-murins, dirigés contre le catumaxomab.Un essai thérapeutique de phase II dont le but est d’évaluer l’efficacité du catumaxomab intrapéritonéal après chirurgie de cytoréduction complète d’une carcinose gastrique, chez des patients ayant reçu en préopératoire une chimiothérapie systémique à base d’oxaliplatine vient de débuter. Au cours de cette étude, nous allons valider la capacité du catumaxomab 1) à induire un stress cellulaire immunogène et la mort des cellules cancéreuses, 2) à modifier la polarisation des cellules effectrices vers une maladie inflammatoire Th1, 3) à promouvoir l'expression des molécules de costimulation et TRAIL sur les cellules NK et monocytes, et corréler ces biomarqueurs immunitaires à l’efficacité du traitement. / The development of cancer in an immunocompetent individual reflects, in part, a tumor escape from the immunosurveillance. The tumor escape is a complex, multifactorial, in which tumor cells will evade the defense mechanisms of the host by changing their microenvironment. Therefore, restoration or induction of these defense mechanisms is one of the therapeutic strategies against cancer. One of the principles of immunotherapy is based on the injection of antibodies that target tumor cells or effector cells of immunity. The anti-tumor efficacy of these antibodies has been greatly improved by a better understanding of modes of action and modulatory effects of these antibodies.Thus, to optimize the action of immune effectors to tumor cells, a bispecific antibody, trifunctional: catumaxomab, capable of binding to the adhesion molecule of the epithelial cells (EpCAM), expressed by tumor cells and the CD3 antigen of T cells, has been developed mainly in intraperitoneal treatment of refractory malignant ascites.The objective of this study was to determine the immunomodulatory effects of catumaxomab on tumoral cells expressing EpCAM, from two experimental models (allogeneic and autologous), evaluate and characterize cytotoxicity induced by catumaxomab, and analyze the presence of stress signals inducing immunogenic cell death such as membrane exposure of calreticulin by pre-apoptotic tumor cells, release of HMGB1 and of adenosine triphosphate (ATP) in the extracellular medium, inducing a T cell activation.In the presence of EpCAM + cells, catumaxomab induced a major the activation of T cells (expression of CD69, CD107a, HLA-DR and PD1), stimulated an inflammatory response Thelper type 1 (Th1) and the synthesis of interferon-gamma by CD8 T cells. Catumaxomab committed CD16 NK cells and monocytes. More, in models allogeneic catumaxomab, caused cell death associated with ATP release and induced an immunogenic cell death after pre-incubation of oxaliplatin.Therefore, catumaxomab modulates the immune environment in malignant ascites, and convert chronic and immunosuppressive inflammation (Th2) in acute and immunogenic inflammation (Th1). However, in these conditions, catumaxomab alone does not seem to trigger immunogenic cell death.the cytotoxicity of this bispecific antibody could be enhance by different techniques: (1) combining with chemotherapy such as oxaliplatin to promote immunogenic cell death, (2) refining its action on CD3 lymphocytes by changing its spatial configuration (BiTE antibody), (3) increasing its affinity for the FcR of accessory cells (Fc aglycosylated), (4) increasing its cytotoxicity by changing the target directed against the immune molecule (anti-PD-1 ...). Finally, the clinical use could be facilitated by this humanizing murine chimeric antibody to prevent the formation of anti-murine antibodies directed against catumaxomab.A phase II clinical trial aimed to evaluate the efficacy of intraperitoneal catumaxomab after complete cytoreductive surgery of gastric carcinomatosis in patients who received preoperative systemic chemotherapy with oxaliplatin have just started. In this study, we will validate the ability of catumaxomab 1) to induce immunogenic cell stress and death of cancer cells, 2) to change the polarization of effector cells to Th1 inflammatory disease, 3) to promote the expression of costimulatory molecules and TRAIL on NK cells and monocytes, and we will correlate these immune biomarkers to treatment efficacy.

Understanding of cell death mechanisms is important to identifying therapeutic approaches to treat excess cell growth, as seen in tumors, or to inhibit excess cell death, as seen in neurodegenerative disease and ischemia. In the first part of this work, we aim to extend the understanding of a non-apoptotic cell death phenotype, ferroptosis, through use of a genome-wide siRNA screen. We identified knockdown of CARS, or cysteinyl-tRNA synthetase, as an inhibitor of erastin-induced ferroptosis. Loss of CARS led to upregulation of the transsulfuration pathway, where methionine is used as the source of sulfur for cysteine synthesis, as a suppressive mechanism. Upregulation of the transsulfuration pathway may serve as a biomarker to identify tumor types that may be insensitive to ferroptosis-inducing therapeutics. On the other hand, induction of the transsulfuration pathway may be beneficial in disease contexts that involve excess cell death. In the second part of this work, we elucidate the mechanism of action of a small molecule Mdm2 inhibitor, or MEL. Mdm2 is a negative inhibitor of p53; therefor, an inhibitor of Mdm2 may be useful in treating tumors driven by Mdm2 overexpression. We found MEL to inhibit the E3-ligase activity of Mdm2/MdmX heterocomplex, proving to be a useful tool to probe the importance of the heterocomplex in normal physiology and disease development. We also explored the structural scaffold of MEL compounds, an indole, and identified a novel ferroptosis inducer, increasing the chemical toolbox available to study ferroptosis.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-31Bitstream added on 2014-06-13T20:01:10Z : No. of bitstreams: 1 berger_b_dr_rcla.pdf: 1800024 bytes, checksum: b803c12973aa74cd00fe939959c8d1f1 (MD5) / Os ovários das rainhas diferem dos de operárias de Apis mellifera quanto ao número e comprimento dos ovaríolos. Tanto o número, como o comprimento destes, é muito maior na rainha que nas operárias. No entanto, em ambos os casos os ovários são funcionais, isto é, capazes de produzir óvulos maduros. Apesar disso, operárias e rainhas diferem muito quanto à fertilidade e aos mecanismos controladores/estimuladores da vitelogênese, ou seja, da maturação dos óvulos. Em condições normais da colônia, nas rainhas a vitelogênese é desencadeada pelo acasalamento e nas operárias, pela ausência da rainha ou do recebimento de informações sobre sua presença na colônia. Passada a ocasião própria para o acasalamento no caso da rainha, e em idade avançada das operárias, os ovários entram em degeneração. O objetivo do presente trabalho foi o de avaliar como se comporta o desenvolvimento do ovário em rainhas e operárias mantidas fora dos condicionamentos da colônia e o efeito do tratamento destas com CO2, prática corrente na apicultura. Para tanto, operárias e rainhas foram mantidas aprisionadas em caixas com candy e água durante 15 dias e seguida a seqüência de desenvolvimento de seus ovários. O efeito do não acasalamento na época própria e do tratamento com CO2 foi feito estudando a morfologia do desenvolvimento da ovogênese, usando TUNEL e reação de fosfatase acida para caracterizar possíveis alterações celulares. As células do filamento terminal apresentaram-se empilhadas em fila única. Na transição para o germário as células tornam-se piramidais com a base apoiada sobre a lâmina própria e o ápice voltado para o centro do ovaríolo. São encontradas células esféricas, provavelmente ovogônias. No germário estão presentes células somáticas e germinativas, sendo da linhagem germinativa, os cistoblastos, os cistócitos, os ovócitos e as futuras... / The ovaries of queens and workers of Apis mellifera differs in number and length of the ovarioles. Length and number of ovarioles are larger in queen than in workers. However, in both cases, the ovaries are functional, i.e., it is able of produce mature eggs. Despite of that, workers and queens differ very in fertility and mechanisms of controlling/inducing vitellogenesis. In colony conditions, queen’s vitellogenesis is triggered by the matting and in workers by the absence of the queen or of the receipt of information about its presence in the colony. After the age proper to mate or in workers advanced age, the ovaries enter in degeneration. The objective of the present work was the evaluating of the ovary development in queens and workers maintained caged outside of the colony conditionings and the effect of the narcosis with CO2, practice current in the beekeeping. Newly emerged queens and workers were caged with candy and water during 15 days. For the queens the effect of the mate delay and CO2 narcosis were studied using TUNEL and acid fosfatase reaction to evaluate cell damages. The cells of the terminal filament appear as rows of one single cell, with a rectangular shape, poor in organelles and with a big central nucleus. In the transition for the germarium the cells present a pyramidal form with their base widened resting on tunica propria and the apex directed to center of the ovariole. Below the region of transition to the germarium are spherical cells, probably the oogonia. In the germarium are found somatic (pre-follicular cells) and germinative (cystoblasts, the cystocists, the oocytes and the future nurse cells) cells. The queen’s ovaries develop normally until the mating age, 5 days old queens. About the 10 days, the virgin queen beginning to presents an ovariolar disorganization with big incidence of injured cells with characteristics of apoptosis and autofagic death... (Complete abstract click electronic access below)

Au cours de ce travail de thèse, les deux approches permettant la caractérisation de nouveaux agents antileishmaniens ont été suivies : d’une part identification de molécules de synthèse originales et actives, par des méthodes de criblage, et d’autre part recherche de nouvelles cibles thérapeutiques leishmaniennes, faisant appel à des outils de biologie moléculaire, et de protéomique.Dans une première partie consacrée à l’optimisation du criblage antileishmanien de molécules de synthèse, nous avons notamment mis au point et validé une nouvelle méthode applicable aux formes promastigotes. Cette méthode reposant sur le principe de la bioluminescence, s’est avérée précise, rapide, répétable, sensible, automatisable et applicable à des isolats cliniques. Nous nous sommes également intéressés à la recherche d’une nouvelle méthode de criblage sur les formes amastigotes intracellulaires remplissant ces mêmes critères.Dans une seconde partie consacrée au lien entre la mort cellulaire programmée et les cystéines peptidases (métacaspase et Raptor) chez Leishmania, nous avons mis en évidence un lien possible entre métacaspase et autophagie chez L. infantum. De plus, nous avons identifié, in vivo, plusieurs substrats protéiques potentiels de ces peptidases : HSP70, ARN-Hélicase ATP-Dépendante et Lmjf09.1010 pour la métacaspase ; NDPKb et la protéine associée à l’ADN du kinétoplaste pour Raptor. Ces différents résultats ont permis de proposer un modèle conciliant les rôles possibles des cystéine-Peptidases métacaspase et Raptor au cours de l’autophagie et de l’apoptose chez Leishmania, rôles potentiellement dus au clivage de substrats protéiques spécifiques. / During this thesis work, two approaches affording the characterization of new antileishmanial agents were followed: on the one hand, the identification of new original antileishmanial synthetic drugs, through screening assays and on the other hand, research of new parasitic therapeutic targets by using molecular biology and proteomic tools. In the first part, dedicated to the optimization of the antileishmanial screening of synthetic compounds, we set up and validated a new bioluminescence-Based screening method for studying anti-Promastigote compounds. This method appears accurate, rapid, repeatable, sensitive and is also transposable to automats and usable on clinical isolates. In parallel, we investigated new screening protocols aiming at improving the screening in intracellular amastigotes which could meet the same criteria. In the second part focusing on the link between programmed cell death and cystein peptidases (metacaspase and Raptor) in Leishmania, our study first highlighted a possible link between metacaspase and autophagy in L. infantum. Moreover, we identified in vivo several potential subtrates for both peptidases: HSP70, ATP-Dependent RNA-Helicase and Lmjf09.1010 for the metacaspase; NDPKb and kinetoplast-DNA-Associated-Protein for Raptor. These results allowed us to propose a model reconciling the possible roles of cystein peptidases metacaspase and Raptor during autophagy and apoptosis in Leishmania, roles potentially due to the cleavage of specific proteic subtrates.

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No. of bitstreams: 2 Dissertação - Thaís Rosa Marques dos Santos - 2016.pdf: 1811805 bytes, checksum: 381d90956275aee309bb1d2948f724de (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-05-20 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Although the efforts employed by scientific community to discover new anticancer therapies suitable to the increasing cancer incidence and multidrug resistance, its necessary to develop more selective and target driven drugs. Therefore, in this work we have done a screening with LQFM030 analogues, which is a Nutlin-1 analogue, aiming to evaluate their cytotoxic potential. Furthermore, we have evaluated the cytotoxicity, the morphological alterations and the cell death induction mechanisms of the compound LQFM166 in leukemia cell line K562. In parallel, we have investigated the security profile of the compound upon 3T3 basal cell line to estimate its LD50 and the Selectivity Index. The cytotoxicity assays included the tetrazolium salt (MTT) reduction and the Neutral Red Uptake assays, to assess the cytotoxicity of LQFM166 in K562 and 3T3 cell lines, respectively. The investigation of cell death induction mechanisms was carried out using flow cytometry, whereby we have evaluated the cells biochemistry parameters, including cell cycle progression, phosphatidylserine externalization, caspases 3/7, 8 and 9 activity, cytochrome c release from mitochondria, p21, p27, Bax, Bcl-2, cyclin-B1 and NFkB expression, using specific labeling for each assay. Data were analyzed by t test and the difference between control and treated groups averages was considered statistically significant when p<0,05. Regarding leukemia cell line K562, the compound LQFM166 was cytotoxic, showing a dose-time-dependent profile. Morphological alterations were observed after treatment with the compound at cellular and nuclear levels, which corroborate with apoptotic cell death. Additionally, treatment with the IC50 for 48 hours has promoted cellular and molecular changes that characterize this process, including phosphatidylserine externalization, increase of caspases 3/7, 8 and 9 activity, expression of pro-apoptotic proteins Bax, p21and p27, as well as diminution of Bcl-2 and cyclin-B1. We have also observed increase of cytochrome-c release and NFkB expression. Concerning the security profile, the compound was considered relatively selective, once the IC50 found to basal cell line (185,3 µM) was the double of the obtained to leukemia cell line regarding the same time of exposure (56,76 µM). The outcomes allow us to conclude that LQFM166 was cytotoxic upon leukemia cells K562, promoting morphological and biochemical alterations that indicate apoptotic cell death induction. / Mesmo com os esforços da comunidade científica em descobrir ou desenvolver medicamentos adequados a crescente incidência do câncer e ainda adequados à suas formas multirresistentes, é necessário o desenvolvimento de medicamentos que sejam seletivos e alvo-dirigidos. Assim, no trabalho proposto, foi realizada uma triagem com compostos análogos ao LQFM030, análogo estrutural do Nutlin-1, visando determinar o potencial citotóxico dos mesmos. Além disso, foram investigados a citotoxicidade, as alterações morfológicas e os mecanismos de indução de morte celular do composto escolhido LQFM166 em linhagem de células leucêmicas K562. Paralelamente, foi investigado o perfil de segurança do composto sobre a linhagem basal 3T3, a fim de estimar a DL50 e o Índice de Seletividade do mesmo. Os ensaios de citotoxicidade incluíram o método de redução do sal de tetrazólio (MTT) e o método de incorporação do corante vermelho neutro, para a avaliação do potencial citotóxico sobre as linhagens K562 e 3T3, respectivamente. Para a investigação dos mecanismos de indução de morte celular foi utilizada a técnica de citometria de fluxo, por meio da qual foi realizada a avaliação de parâmetros bioquímicos incluindo progressão ciclo celular, externalização da fosfatidilserina, atividade das caspases 3/7, 8 e 9, liberação do citocromo c, expressão das proteínas p21, p27, Bax, Bcl-2, ciclina-B1 e NFkB, empregando-se técnicas de marcação específicas para cada ensaio. Os dados foram analisados pelo teste t e a diferença entre as médias dos grupos controle e tratado foram consideradas estatisticamente significativas quando p<0,05. Em relação à linhagem leucêmica K562, o composto LQFM166 foi citotóxico apresentando um perfil dose e tempo dependentes. Foram observadas alterações morfológicas a níveis celular e nuclear, após o tratamento com composto, condizentes com o processo de morte celular por apoptose. Adicionalmente, externalização da fosfatidilserina, aumento da atividade das caspases 3/7, 8 e 9, aumento da expressão das proteínas pró-apoptóticas Bax, p21 e p27, bem como diminuição da expressão das proteínas Bcl-2 e ciclina-B1, após tratamento com a CI50 por 48 horas, desencadeou alterações celulares e moleculares que reforçam a sugestão de processo de morte celular por apoptose. Observouse ainda aumento da liberação do citocromo-c e da expressão da proteína NFkB. Já em relação ao perfil de segurança, o composto mostrou-se relativamente seletivo e com menor toxicidade, uma vez que a CI50 encontrada para a linhagem basal (185,3 μM) foi cerca de duas vezes maior ao encontrado para a linhagem tumoral para o mesmo tempo de exposição (56,76 μM). Considerando os resultados obtidos, pode-se concluir que o composto LQFM166 foi citotóxico sobre a linhagem leucêmica K562, desencadeando alterações morfológicas e bioquímicas características de morte celular por apoptose.

Dans de nombreuses maladies neurodégénératives dont la Maladie de Parkinson, mais également dans le processus de vieillissement normal, il a été observé une accumulation excessive des niveaux de fer associée à une production accrue d‘espèces réactives de l’oxygène (ROS).Il est bien connu que le fer (i) participe à la réaction de Fenton pour produire le ROS le plus toxique, le radical hydroxyle, (ii) accélère l’auto-oxidation de la dopamine, augmentant le stress oxydant et (iii) augmente la peroxydation lipidique qui va déclencher l'agrégation des protéines, dont l’&#945;-synucléine, une caractéristique de la maladie de Parkinson. Tous ces phénomènes rendent les neurones dopaminergiques très sensible au stress oxydant.Récemment, une nouvelle forme de mort cellulaire a été découverte sur des cellules cancéreuses. Cette mort cellulaire est appelée « ferroptose » car elle dépend essentiellement du fer intracellulaire. De plus, elle est liée à une très grande augmentation du stress lipidique.Le principal objectif de ce travail de thèse a donc porté sur la possible implication de la ferroptose dans la mort des cellules dopaminergiques.Dans un premier temps, nous avons montré que la lignée de neurones dopaminergiques (LUHMES) est un modèle particulièrement adapté à l’étude de mort cellulaire induite par le fer comparativement à d’autres modèles plus classique.La seconde partie du travail a donc été consacrée à l'étude de l’implication de la ferroptose dans les neurones dopaminergiques. D’abord par une approche cellulaire avec les cellules LUHMES, nous avons pu observer (i) que l’inducteur spécifique de cette mort, l’érastine, est très efficace pour induire la mort des cellules dopaminergiques, (ii) que l'inhibition de la ferroptose protégeait de la mort induite par des agents oxydants classiquement utilisés dans les études sur la maladie de Parkinson comme le MPP+ et (iii) que la PKC joue un rôle majeur dans la mort par ferroptose. Tous ces résultats ont ensuite été confirmés dans un modèle de culture organotypique, à l’interface entre la culture cellulaire et le modèle animal.Enfin, nous avons montré que l’utilisation des inhibiteurs de la ferroptose ainsi que celui de la PKC, confère une protection des neurones dopaminergiques dans un modèle de souris MPTP, modèle d’étude de la maladie de Parkinson.En conclusion ce travail de thèse montre pour la première fois l'implication de la ferroptose dans une maladie neurodégénérative comme la maladie de Parkinson et suggèrent que le développement d'inhibiteurs spécifiques de cette mort cellulaire pourrait être des futures cibles thérapeutiques possibles. / In many neurodegenerative diseases including Parkinson's disease, as well as in the regular aging process, an excessive accumulation of iron levels associated with increased production of oxygen species (ROS) have been observed.It is well known that iron (i) participates in the Fenton reaction to produce the most toxic ROS, the hydroxyl radical, (ii) accelerates auto-oxidation of dopamine, increasing the oxidative stress and (iii) increases lipid peroxidation that will trigger the aggregation of proteins, including &#945;-synuclein, a hallmark of Parkinson's disease. All theses factors make dopamine neurons very sensitive to oxidative stress.Recently, a new form of cell death was found on cancer cells. This cell death is called ferroptosis because it essentially depends on the intracellular iron. In addition, it is linked to a very large increase in lipid stress.The main objective of this work has focused on the possible involvement of ferroptosis in the death of dopamine cells.First, we showed that the line of dopaminergic neurons (LUHMES) is a model particularly suited to the study of cell death induced by iron compared to other more conventional models.The second part of the work has been devoted to the study of the involvement of ferroptose in dopaminergic neurons. First by a cellular approach with LUHMES cells, we observed that (i) the specific inducer of this death, erastin, is very effective to induce the death of dopaminergic cells, (ii) inhibition of ferroptosis protected from death induced by oxidizing agents conventionally used in studies of Parkinson's disease like MPP+ and (iii) that PKC plays a major role in death by ferroptosis. These results were then confirmed in an organotypic culture model, at the interface between cell culture and animal models.Finally, we have shown that the use of ferroptosis inhibitors and PKC inhibitor, provides protection of dopaminergic neurons in MPTP mice model, animal model for studying of Parkinson's disease.In conclusion this study demonstrates for the first time the involvement of ferroptosis in a neurodegenerative disease such as Parkinson's disease and suggest that the development of specific inhibitors of this cell death could be possible future therapeutic targets.

Phylloquinone is a natural analog of vitamin K that has been shown to both inhibit cancer cell growth and induce apoptosis in several cancer cell lines. This study examined these effects in a non-Hodgkin lymphoma cell line, known as U937. Cell growth inhibition and apoptosis were assessed through the quantification of cell density and area, following treatment with several concentrations of phylloquinone. In addition, apoptosis was detected and quantified using immunofluorescent markers of apoptosis (i.e. annexin V, APO-BrdU). Treatment with phylloquinone resulted in reduced overall cell density, increased overall cell area, and an increased frequency of apoptosis in U937 cells. Increasing both phylloquinone concentration and treatment time enhanced these effects. These results are significant because they document the anti-cancer effects of this analog of vitamin K, as well as provide insight into the morphological changes that occur during apoptosis in U937 cells.

Ovarian cancer is a deadly disease that kills an estimated 15,000 women annually in the United States. It is estimated that approximately 10% of ovarian cancers are due to familial inheritance. The most commonly mutated genes in familial ovarian cancer are BRCA1 and BRCA2. It has been reported that cells carrying the BRCA1 185delAG mutation undergo an enhanced caspase-3 mediated apoptotic response. Here, we report on the transfection of cDNA coding for the putative truncated protein product of the BRCA1 185delAG mutant gene into BRCA1 wild-type human immortalized ovarian surface epithelial (IOSE) cells and ovarian cancer cells. Cells transfected with the BRCA1 185delAG truncation protein (BRAt) showed increased levels of active caspase 3, increased cleavage of caspase 3 substrates, PARP and DFF45, and decreased XIAP and cIAP1 following staurosporine (STS) treatment. BRAt also reduced Akt phosphorylation and over expression of activated Akt in BRAt cells restored caspase-3 activity to that seen in wild type cells. Further, BRAt expression increased chemosensitivity in platinum resistant ovarian cancer cells. Similarly, maspin protein has been shown to sensitize breast carcinoma cells to STS-induced apoptosis. We provide the first evidence that BRAt is sufficient to induce maspin protein in IOSE cells. IOSE cell lines carrying the BRCA1 185delAG mutation showed higher maspin levels than wild-type BRCA1 IOSE cell lines. BRCA1 wild-type IOSE cells were transfected with BRAt protein and showed increased maspin mRNA levels and increased nuclear maspin protein levels as compared to control cells. Additionally, both heterozygous carriers of the BRCA1 185delAG mutation and cells transfected with BRAt protein show an increased ability to activate the maspin promoter as compared to control cells. The transcription factor AP1 is at least partially required for full activation of the maspin promoter in BRAt cells, as siRNA directed towards c-jun decreased activation of the full-length maspin promoter. Taken together, our data demonstrate that truncated proteins arising from BRCA1 185delAG mutation increase Akt-mediated apoptosis by increasing nuclear maspin expression, suggesting a possible mechanism by which ovarian cancer patients with germline BRCA1 mutations may respond better to chemotherapy.