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The importance of Cytotoxic T-Cell (CTL) reponses during the course of intracellular infections has received a lot of attention during the past few decades. CTLs respond to epitopes presented by the Major Histocompatibility Complex (MHC) originating from intracellular proteins for which they have an appropriate T-Cell Receptor (TCR) for. This response is crucial for the control of pathogens such as Influenza, Hepatitis, HIV and others by destroying the cell in which the pathogen replicates. Due to the extreme polymorphism of MHC molecules, Computational Immunology techniques have been developed to detect potential MHC ligands and as a consequence, potential CTL epitopes. The polymorphism factor needs to be taken into account especially when concerning the design of vaccines with a CTL response component to maximize population coverage. Tools have been constructed that combine the predictions tools concerning major steps in this pathway, that is, proteasomal cleavage, Transporter associated with Antigen Presentation (TAP) affinity, Major Histocompatibility Complex (MHC) affinity and Immunogenicity. In this study, a novel method is developed to combine the different steps in the pathway, which includes the development of a novel TAP predictor. Furthermore, by using a BLOSUM-based score in conjunction with the epitope prediction results, a novel CTL epitopebased clustering method was developed. Two pathogens with major CTL epitope components, but vastly different mutation rates were chosen to infer whether the aforementioned methods can be used to detect potential CTL epitopes and group sequences together based on shared immunogenicity. / Dissertation (MSc)--University of Pretoria, 2011. / Bioinformatics and Computational Biology Unit / unrestricted

The prospect of utilizing the cichlid teleost tilapia (Oreochromis niloticus) as an alternative experimental model to mammals for immunotoxicity risk assessment is currently being proposed. As such, the National Toxicology Program's (NTP) standard battery of rodent immunotoxicity assays is being developed for use in this fish species. Included in the testing series are the hemolytic plaque forming cell (PFC) and the cytotoxic T-lymphocyte (CTL) assays, quantitative indicators of antibody production and cell-mediated activity, respectively. The assays were modified in consideration of specific tilapian immune parameters, then tested using fourteen environmental contaminants or drugs, ten of which are classified by the NTP as immunotoxic in rodents. Reduced antibody production via a decrease in plaque number was observed in response to exposure of tilapia to eight of the nine humoral immunotoxicants, and five of the five non-immunotoxicants. Under specific immunization circumstances, immunostimulation (also a response to immunotoxicity) was noted via an increase in plaque number in benzo[a]pyrene (B[a]P) exposed fish using the PFC assay, a result noted in rodents as well. Reduced T-cell recognition and lysis of allogeneic tilapian lymphocytes via a decrease in the percentage of specific 51Chromium (51Cr) release was observed in response to exposure of tilapia to the nine of the ten cell-mediated immunotoxicants, and four of the four non-immunotoxicants. Although the normal teleost immune responsiveness was slightly weaker than seen with mice under comparable conditions (presumably due to differences in antibody structure and decreased cells counts), tilapia were found to exhibit well-defined humoral and cell-mediated immune responses, and responses to immunotoxic and non-immunotoxic chemicals comparable to the rodent model. / Master of Science

La fonction de cytolyse est un mécanisme majeur des effecteurs du système immunitaire pour éliminer les cellules infectées ou tumorales. Cette fonction associe l'activité de la perforine, qui forme des pores dans la membrane d'une cellule cible, à la sécrétion de protéases: les granzymes. Ces dernières sont des molécules pro-apoptotiques qui induisent la mort de la cellule cible. Les granzymes et en particulier granzyme B ciblent plusieurs voies intracellulaires complémentaires pour assurer l'efficacité de la cytolyse. Or il est difficile d'observer directement la fonction de cytolyse au cours de réponse immunitaire in vivo dans des conditions physiologiques. Dans les travaux présentés dans cette thèse, nous avons développé un nouveau modèle qui permet de suivre la fonction de cytolyse en temps réel par l'expression d'une protéine de fusion fluorescente GZMB-tdTomato. Les résultats obtenus par expression rétrovirale ont montré que la protéine de fusion est correctement exprimée dans les vésicules cytolytiques qui deviennent fluorescentes. Dans un second temps, nous avons réalisé un nouveau modèle murin qui exprime GZMB-tdTomato de manière substituée au GZMB natif par recombinaison homologue (Knock In). Nous avons mis en évidence que la protéine de fusion conserve l'activité catalytique de la protéine native et ses caractéristiques (conditions d'expression, de maturation, de sécrétion et demeure active après le passage dans la cellule cible lors de la cytolyse). En utilisant un modèle murin exprimant un TCR transgénique nous avons pu suivre le déroulement de la fonction de cytolyse de lymphocytes cytotoxiques en temps réel par video microscopies. / Cytolysis is a major function used by the immune system's effectors to kill infected or tumor cells. Cytolysis depends on the pore forming protein perforin and the secretion of proteases of the granzyme family. Granzymes, including granzyme B (GZMB) have pro-apoptotic features and induce target cell death. Several complementary pathways are triggered by granzymes to ensure efficient cytolysis. It remains difficult to directly observe cytolysis during in vivo immune responses under physiological conditions. In this PhD we developed a new model to visualize cytolytic function in real time by expression of a fusion protein: GZMB-tdTomato. Results obtained from retroviral transduction showed that the fusion protein is correctly expressed in cytolytic vesicles, which became fluorescent. We then constructed a new mouse model by homologous recombination (Knock In) that express GZMB-tdTomato substituted for the native GZMB. The fusion protein conserves the catalytic activity of GZMB and its features (expression, maturation, secretion conditions) and remains active after its passage into target cells. Using TCR transgenic OTI cells, we followed the sequence of events of cytolysis from lymphocytes in real time by videomicroscopy. We also observed the cytolytic vesicles relocalization towards the cell contact zone and the death of target cell by cytolysis. Finally, we studied in vivo differentiation of naïve lymphocyte to cytolytic effector cells (the acquisition of cytolysis) and target cell death after bacterial infection.

Neste trabalho apresentamos uma implementação de revisão de crenças baseada em comparação de modelos (estados) em uma ferramenta de verificação automática de sistemas de estados finitos. Dada uma fórmula (na lógica CTL) inconsistente com o modelo do sistema, revisamos esse modelo de tal maneira que essa fórmula temporal se torne verdadeira. Como temos oito operadores temporais (AG, AF, AX, AU, EG, EF, EX e EU), foram criados algoritmos especícos para cada um deles. Como o modelo do sistema deriva do seu código na linguagem SMV, a sua revisão passa obrigatoriamente por mudanças na sua descrição. A nossa implementação contempla três tipos de mudanças: acréscimo de linhas, eliminação de linhas e mudança no estado inicial, sendo que as duas primeiras provocam modicações nas transições entre os estados que compõe o modelo. Alguns testes foram aplicados para comprovar a contribuição da revisão de crenças (revisão de modelos) como ferramenta de auxílio ao usuário durante o processo de modelagem de sistemas. / In this work we present an implementation of belief revision based on comparison of models (states) in a tool for automatic verication of nite state systems. Given a formula (in the language of CTL) inconsistent with the model of the system, we revise this model in such way that the temporal formula becomes valid. As we have eight temporal operators (AG, AF, AX, AU, EG, EF, EX and EU), specic algorithms for each one of them have been created. As the model of the system is related with its code in SMV language, its revision forces changes in its description. Our implementation contemplates three types of change: addition of lines, elimination of lines and change in the initial state, where the rst two cause modications in the transitions between the states of the model. Some tests were applied to prove the contribution of the belief revision (model revision) as aid-tool to the user during the process of systems modeling.

Uma importante etapa do desenvolvimento de software é o de levantamento e análise dos requisitos. Porém, durante esta etapa podem ocorrer inconsistências que prejudicarão o andamento do projeto. Além disso, após finalizada a especificação, o cliente pode querer acrescentar ou modificar as funcionalidades do sistema. Tudo isso requer que a especificação do software seja revista, mas isso é altamente custoso, tornando necessário um processo automatizado para simplificar tal revisão. Para lidar com este problema, uma das abordagens utilizadas tem sido o processo de Revisão de Crenças, juntamente com o processo de Verificação de Modelos. O objetivo deste trabalho é utilizar o processo de revisão de crenças e verificação de modelos para avaliar especificações de um projeto procurando inconsistências, utilizando o fragmento universal da Computation Tree Logic (CTL), conhecido como ACTL, e revisá-las gerando sugestões de mudanças na especificação. A nossa proposta é traduzir para lógica clássica tanto o modelo (especificação do software) quanto a propriedade a ser revisada, e então aplicar um resolvedor SAT para verificar a satisfazibilidade da fórmula gerada. A partir da resposta do resolvedor SAT, iremos gerar sugestões válidas de mudanças para a especificação, fazendo o processo de tradução reversa da lógica clássica para o modelo original. / The objective of this work is to join the proccess of belief revision and model checking to evaluate project specifications looking for inconsistences, using the universal fragment of Computation Tree Logic (CTL), known as ACTL, and revise them generating changes suggestions in the specification. Our approach will translate the model (software specification) and the property to be revised to classical logic. Then we will apply a SAT solver to verify the generated formulas satsifability. From the SAT solver answer, we will create changes valid suggestions to the specification making the translation back from classical logic to the original model. To generate the changes suggestions, we proposed a framework based on heuristics where different approaches and decisions can be implemented, aiming a better application for each project scope. We implemented a basic heuristic as an example and used it to test the implementation to analise the proposed algorithm

Le but de cette thèse était d’identifier les mécanismes moléculaires responsables des propriétés particulières de la couche G et les propriétés mécaniques remarquables du bois de tension (BT). Trois acteurs moléculaires potentiels (des protéines à arabinogalactane avec domaine fasciclin-like (FLA), une protéine chitinase-like (CTL) et une β-galactosidase (BGAL)) ont été choisis et étudiés dans: analyse phylogénétique, analyses d'expression et caractérisation de peupliers transgéniques affectés dans l’expression de chacun de ces acteurs. La caractérisation fine de ce matériel a révélé que CTL2 et les FLA jouent un rôle dans la régulation de la cristallinité de la cellulose dans le BT. CTL2 apparaît également important dans l'organisation de la paroi cellulaire et des propriétés mécaniques des tiges. BGAL a été avant proposé pour une fonction dans modification de pectine RG-I potentiellement important pour des propriétés mécaniques de BT. Le bois de tension exhibe une activité BGAL plus élevée que dans le bois opposé. L’inhibition par RNAi de l’expression de BGAL7, spécifiquement exprimée dans le BT, n’est pas responsable à lui seul de la forte activité BGAL présente dans le BT. En contrepoint à l’étude menée sur le peuplier, nous avons également évalué la présence d’acteurs moléculaires potentiellement responsables des propriétés mécaniques du BT chez le simarouba qui développe dans leur BT des fibres ayant leurs sous-couches de la paroi intermédiaire entre la G et la S2. Des protéines à arabinogalactanes ainsi que des pectines du type RG-I sont présentes dans les fibres de BT de peuplier et de simarouba et pourraient avoir une fonction dans un mécanisme commun de génération des contraintes dans le BT. Finalement, un modèle est proposé sur le rôle présumé des différents acteurs moléculaires étudié dans la régulation des propriétés de la couche G et la génération des fortes contraintes du bois de tension. / The aim of this thesis was to approach the underlying molecular mechanisms responsible for the particular properties of the G-layer and the outstanding mechanical properties of tension wood (TW). Accordingly, three potential molecular players (fasciclin-like arabinogalactan protein (FLA), chitinase-like protein (CTL) and β-galactosidase (BGAL)) were chosen and studied through a phylogenetic analysis, expression analyses and most importantly characterization of RNAi transgenic poplars. This multilevel characterization revealed that CTL2 and FLAs have function in the regulation of cellulose crystallinity in TW. CTL2 was also shown to be important both for the cell wall organization and stem mechanical properties. BGAL was studied in a light of the previously reported modifications of RG-I pectin, potentially important for the mechanical properties of TW. Study of BGAL revealed that the enzyme has higher activity in TW than in opposite wood. BGAL7, whose gene was expressed specifically in TW, does not seem to be responsible for the higher BGAL activity in TW. In comparison to poplar, we analyzed the occurrence of molecular players potentially responsible for TW mechanical properties in simarouba, a tropical species, which develops different TW fiber. Arabinogalactan proteins and RG-I pectin potentially targeted by BGAL were localized in TW fibers both in poplar and simarouba and therefore may be involved in a common mechanism of tensile stress generation in different TW types. A model was finally proposed to elucidate a potential function of the studied molecular players in the regulation of G-layer properties and tensile stress generation.

Uma importante etapa do desenvolvimento de software é o de levantamento e análise dos requisitos. Porém, durante esta etapa podem ocorrer inconsistências que prejudicarão o andamento do projeto. Além disso, após finalizada a especificação, o cliente pode querer acrescentar ou modificar as funcionalidades do sistema. Tudo isso requer que a especificação do software seja revista, mas isso é altamente custoso, tornando necessário um processo automatizado para simplificar tal revisão. Para lidar com este problema, uma das abordagens utilizadas tem sido o processo de Revisão de Crenças, juntamente com o processo de Verificação de Modelos. O objetivo deste trabalho é utilizar o processo de revisão de crenças e verificação de modelos para avaliar especificações de um projeto procurando inconsistências, utilizando o fragmento universal da Computation Tree Logic (CTL), conhecido como ACTL, e revisá-las gerando sugestões de mudanças na especificação. A nossa proposta é traduzir para lógica clássica tanto o modelo (especificação do software) quanto a propriedade a ser revisada, e então aplicar um resolvedor SAT para verificar a satisfazibilidade da fórmula gerada. A partir da resposta do resolvedor SAT, iremos gerar sugestões válidas de mudanças para a especificação, fazendo o processo de tradução reversa da lógica clássica para o modelo original. / The objective of this work is to join the proccess of belief revision and model checking to evaluate project specifications looking for inconsistences, using the universal fragment of Computation Tree Logic (CTL), known as ACTL, and revise them generating changes suggestions in the specification. Our approach will translate the model (software specification) and the property to be revised to classical logic. Then we will apply a SAT solver to verify the generated formulas satsifability. From the SAT solver answer, we will create changes valid suggestions to the specification making the translation back from classical logic to the original model. To generate the changes suggestions, we proposed a framework based on heuristics where different approaches and decisions can be implemented, aiming a better application for each project scope. We implemented a basic heuristic as an example and used it to test the implementation to analise the proposed algorithm

Neste trabalho apresentamos uma implementação de revisão de crenças baseada em comparação de modelos (estados) em uma ferramenta de verificação automática de sistemas de estados finitos. Dada uma fórmula (na lógica CTL) inconsistente com o modelo do sistema, revisamos esse modelo de tal maneira que essa fórmula temporal se torne verdadeira. Como temos oito operadores temporais (AG, AF, AX, AU, EG, EF, EX e EU), foram criados algoritmos especícos para cada um deles. Como o modelo do sistema deriva do seu código na linguagem SMV, a sua revisão passa obrigatoriamente por mudanças na sua descrição. A nossa implementação contempla três tipos de mudanças: acréscimo de linhas, eliminação de linhas e mudança no estado inicial, sendo que as duas primeiras provocam modicações nas transições entre os estados que compõe o modelo. Alguns testes foram aplicados para comprovar a contribuição da revisão de crenças (revisão de modelos) como ferramenta de auxílio ao usuário durante o processo de modelagem de sistemas. / In this work we present an implementation of belief revision based on comparison of models (states) in a tool for automatic verication of nite state systems. Given a formula (in the language of CTL) inconsistent with the model of the system, we revise this model in such way that the temporal formula becomes valid. As we have eight temporal operators (AG, AF, AX, AU, EG, EF, EX and EU), specic algorithms for each one of them have been created. As the model of the system is related with its code in SMV language, its revision forces changes in its description. Our implementation contemplates three types of change: addition of lines, elimination of lines and change in the initial state, where the rst two cause modications in the transitions between the states of the model. Some tests were applied to prove the contribution of the belief revision (model revision) as aid-tool to the user during the process of systems modeling.

Les lymphocytes T cytotoxiques (CTL) et les cellules tueuses naturelles (NK) éliminent leurs cellules cibles tumorales grâce à l’exocytose et à la libération du contenu des granules cytotoxiques contenant une protéine formant des pores, appelée perforine (PFN), et une famille de serine-protéases induisant la mort cellulaire, appelés granzymes (Gzms). Ces Gzms, pénètrent dans les cellules cibles de manière dépendante de la PFN et activent diverses voies de signalisation apoptotiques aboutissant à la mort de la cellule cible. Au cours de ce travail, nous avons étudié le rôle de la protéine suppresseur de tumeurs p53 dans la cascade moléculaire conduisant à l’apoptose induite par les effecteurs cytotoxiques via la voie PFN/Granzyme B (GzmB). Nous avons ainsi pu montrer qu’en réponse au GzmB ou à des effecteurs cytotoxiques, la forme sauvage de p53 s’accumule dans les cellules cibles au niveau des mitochondries afin d’interagir avec la protéine anti-apoptotique Bcl-2 et de réguler positivement la perméabilisation de la membrane mitochondriale externe induite par le GzmB. L’activité non transcriptionelle de p53 au niveau des mitochondries joue donc un rôle clé dans le contrôle de l’apoptose induite par les CTL et les NK (Ben Safta et al. J Immunol 2015). Etant donné que le gène TP53 est muté dans plus de 50% des tumeurs humaines, nous avons également cherché à déterminer si la restauration d’une p53 sauvage dans des cellules tumorales portant une p53 non fonctionnelle pourrait potentialiser la réponse cytotoxique antitumorale. Nos résultats montrent qu'effectivement, la réactivation pharmacologique de l’activité sauvage de p53 dans une lignée d'adénocarcinome mammaire possédant une p53 mutée sensibilise ces cellules tumorales à la lyse induite par les cellules NK via l’activation d’un processus d’autophagie et d’une cascade d’événements moléculaires qui sont en cours d’identification. / Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate their tumor target cells through exocytosis and release of the cytotoxic granules (CG) content. These CG contain a pore-forming protein called perforin (PFN), and a family of cell death inducing serine-proteases, called granzymes (Gzms). Gzms enter the target cells in a PFN-dependent manner and activate various apoptotic signaling pathways leading to the death of the target cell. In this work, we studied the role of the tumor suppressor protein p53 in the molecular cascade leading to apoptosis induced by cytotoxic effectors via the PFN/Granzyme B (GzmB) pathway. We have shown that in response to GzmB or to cytotoxic effectors, wild-type p53 accumulates on target mitochondria in order to interact with the anti-apoptotic protein Bcl-2 and to positively regulate the GzmB-induced mitochondrial outer membrane permeabilization. Thus, the non-transcriptional activity of p53 in the mitochondria plays a key role in the control of apoptosis induced by CTL and NK (Ben Safta et al J Immunol 2015). Because the TP53 gene is mutated in more than 50% of human tumors, we also aimed to determine whether the restoration of a wild-type p53 fonction in tumor cells carrying a non-functional p53 could potentiate the cytotoxic antitumor response. Our results show that the pharmacological reactivation of a wild-type p53 activity in a mammary adenocarcinoma cell line harboring a mutated p53 sensitize these tumor cells to the NK cell lysis via the activation of autophagy and a cascade of molecular events that are being identified.