Dissertationen zum Thema „Tfhe“

Avec la création et l'utilisation incessantes de données numériques, ces dernières années ont vu naître des inquiétudes au sujet des données sensibles et personnelles. De nouvelles lois, telle que le Règlement Général sur la Protection des Données, ont alors vu le jour pour assurer le respect de la confidentialité des données des individus. Cependant, l'externalisation grandissante du traitement des données notamment avec l'apparition du "machine learning as a service" soulève la question suivante: est-il possible de laisser un tiers traiter nos données tout en les gardant confidentielles ?Une solution à ce problème vient des chiffrements dits FHE, de l'anglais Fully Homomorphic Encryption. À l'aide de tels cryptosystèmes, des opérations peuvent être appliquées directement sur des messages chiffrés, sans jamais dévoiler ni le message d'origine, ni le message résultant des opérations. Ce corpus de techniques permet donc en théorie d'externaliser des calculs sans compromettre la confidentialité des données utilisées lors de ces calculs.Cela pourrait ouvrir la voie à de nombreuses applications telle que la possibilité d'ouvrir des services de diagnostic médicaux en ligne offrant une totale confidentialité des données médicales des patients.Malgré cette promesse alléchante, l'important coût computationnel des opérateurs FHE en limite la portée pratique. En effet, un calcul sur données chiffrées peut prendre plusieurs millions de fois plus de temps que son équivalent sur des données non chiffrées. Cela rend inenvisageable l'évaluation d'algorithme trop complexes sur des données chiffrées. Par ailleurs, le surcoût en mémoire apporté par les chiffrements FHE s'élève à un facteur multiplicatif de plusieurs milliers. Ce surcoût peut donc s'avérer rédhibitoire pour des applications sur des systèmes à basse mémoire tels que des systèmes embarqués.Dans cette thèse nous développons une nouvelle primitive pour le calcul sur données chiffrées basée sur l'opération de "bootstrapping fonctionnel" supportée par le cryptosystème TFHE. Cette primitive permet un gain en latence et en mémoire par rapport aux autres techniques comparables de l'état de l'art. Aussi, nous introduisons une seconde primitive permettant d'effectuer des calculs sous forme de circuit logique permettant un gain significatif de vitesse de calcul par rapport à l'état de l'art. Cette approche pourra notamment être intéressante auprès des concepteurs de compilateurs homomorphes comme alternative à l'utilisation de chiffrement binaire.Ces deux outils se veulent suffisamment généraux pour être applicables à un large panel de cas d'utilisation et ne sont donc pas limités aux cas d'usage présentés dans ce manuscrit.En guise d'illustration, nous appliquons nos opérateurs au calcul confidentiel de réseaux de neurones externalisés, montrant ainsi la possibilité d'évaluer des réseaux de neurones avec une relativement faible latence, même dans le cas de réseau de neurones de type récurrents.Enfin, nous appliquons nos opérateurs à une technique dite de transchiffrement permettant de s'affranchir des considérations de limitation en mémoire dûes à la grande taille des chiffrés FHE côté client
In recent years, concerns about sensitive and personal data arose due to the increasing creation and use of digital data. New laws, such as the General Data Protection Regulation, have been introduced to ensure that the confidentiality of individuals' data is respected. However, the growing outsourcing of data processing, particularly with the emergence of "machine learning as a service", raises the following question: is it possible to let a third party process our data while keeping it confidential?One solution to this problem comes in the form of Fully Homomorphic Encryption, or FHE for short. Using FHE cryptosystems, operations can be applied directly to encrypted messages, without ever revealing either the original message or the message resulting from the operations. In theory, this collection of techniques makes it possible to externalise calculations without compromising on the confidentiality of the data used during these calculations.This could pave the way for numerous applications, such as the possibility of offering online medical diagnostic services while ensuring the total confidentiality of the patients' medical data.Despite this promise, the high computational cost of FHE operators limits their practical scope. A calculation on encrypted data can take several million times longer than its equivalent on non-encrypted data. This makes it unthinkable to evaluate highly time consuming algorithms on encrypted data. In addition, the memory cost of FHE encryption is several thousand times greater than unencrypted data. This overhead may prove to be prohibitive for applications on low-memory systems such as embedded systems.In this thesis we develop a new primitive for computing on encrypted data based on the "functional bootstrapping" operation supported by the TFHE cryptosystem. This primitive allows a gain in latency and memory compared to other comparable techniques in the state of the art. We are also introducing a second primitive enabling calculations to be performed in the form of a logic circuit, providing a significant gain in calculation speed compared with the state of the art. This approach could be of particular interest to designers of homomorphic compilers as an alternative to the use of binary encryption.These two tools are intended to be sufficiently generic to be applicable to a wide range of use cases and are therefore not limited to the use cases presented in this manuscript.As an illustration, we apply our operators to the confidential computation of outsourced neural networks, thus demonstrating the possibility of evaluating neural networks with relatively low latency, even in the case of recurrent neural networks.Finally, we apply our operators to a technique known as transciphering, making it possible to overcome memory limitation on the client side coming with the large size of FHE ciphertexts

Dans notre vie de tous les jours, nous produisons une multitude de données à chaque fois que nous accédons à un service en ligne. Certaines sont partagées volontairement et d'autres à contrecœur. Ces données sont collectées et analysées en clair, ce qui menace la vie privée de l'utilisateur et empêche la collaboration entre entités travaillant sur des données sensibles. Le chiffrement complètement homomorphe (Fully Homomorphic Encryption) apporte une lueur d'espoir en permettant d'effectuer des calculs sur des données chiffrées ce qui permet de les analyser et de les exploiter sans jamais y accéder en clair. Cette thèse se focalise sur TFHE, un récent schéma complètement homomorphe capable de réaliser un bootstrapping en un temps record. Dans celle-ci, nous introduisons une méthode d'optimisation pour sélectionner les degrés de liberté inhérents aux calculs homomorphiques permettant aux profanes d'utiliser TFHE. Nous détaillons une multitude de nouveaux algorithmes homomorphes qui améliorent l'efficacité de TFHE et réduisent voire éliminent les restrictions d'algorithmes connus. Une implémentation efficace de ceux-ci est d'ores et déjà en accès libre
In our everyday life, we leave a trail of data whenever we access online services. Some are given voluntarily and others reluctantly. Those data are collected and analyzed in the clear which leads to major threats on the user's privacy and prevents collaborations between entities working on sensitive data. In this context, Fully Homomorphic Encryption brings a new hope by enabling computation over encrypted data, which removes the need to access data in the clear to analyze and exploit it. This thesis focuses on TFHE, a recent fully homomorphic encryption scheme able to compute a bootstrapping in record time. We introduce an optimization framework to set the degrees of freedom inherent to homomorphic computations which gives non-experts the ability to use it (more) easily. We describe a plethora of new FHE algorithms which improve significantly the state of the art and limit, (if not remove) existing restrictions. Efficient open source implementations are already accessible

La particularité de l'agence en droit de la concurrence, résultant du fait que l'agent est présent sur deux marchés à la fois - sur le marché sur lequel il conclut des contrats au nom et pour le compte de quelqu'un et sur le marché sur lequel il propose ses services en tant qu'agent - entraîne des difficultés dans la pratique et est intéressante du point de vue théorique. Le fait de qualifier de façon erronée le distributeur d'agent, est susceptible de conduire à des peines prononcées par les autorités de la concurrence. Les systèmes de distribution deviennent de plus en plus complexes et utilisent désormais différents schémas juridiques afin de satisfaire les besoins des producteurs. Toutefois, d'après l’interprétation de l'article 101 TFUE, étant donné que l'agent agit au nom et pour le compte de quelqu'un, le contrat susceptible d'être anticoncurrentiel, n'est finalement pas conclu entre les mêmes parties. C'est pourquoi, depuis 1962, la Commission Européenne, puis, la jurisprudence de la Cour de Justice des Communautés Européennes, ont introduit un critère d'application de l'article 101 TFUE. Toutefois, l'énigme n'était pas pour autant résolue puisque les solutions proposées étaient peu cohérentes. L'une se concentrait sur la doctrine de l'entité économique, considérant l'agent avec le donneur d'ordre comme une seule et même entité économique. La suivante constituait une théorie d'auxiliaire. La solution la plus récente se focalise sur les risques supportés par l'agent par rapport au contrat qu'il a négocié. La présente analyse démontre que d'autres éléments sont à prendre en compte, y compris parmi les effets de la conclusion du contrat
Distribution systems have become more and more complex and tend to use different legal figures to fulfill the producer's aims. One of them can be agency. From the perspective of competition law, agency holds a special status, resulting from the fact that the agent is present in two distinct relevant markets, in one representing this principal in the conclusion of a contract and in a second offering his services as an agent. This causes some practical difficulties and is interesting from theoritical point of view. The erroneous qualification of a distributor as an agent could result in fines imposed by competition authorities. However, as to the first market described above, according to an interpretation of article 101 TFEU, an anticompetitive agreement cannot be concluded between the same person which can lead to the agency being immune from the competition law requirements. It is imperative to recognize the difference, and therefore, the criteria of an application of article 101 TFEU to an agency have been indicated by the European Commission since 1962 and by the case law of the Court of Justice of the European Union. Nevertheless, the enigma is far from being resolved since the proposed solutions tend to be incoherent. One of them concentrates on the single economic entity doctrine. Other solutions refer to an auxiliary theory whereby. The most recent solution focuses on risks undertaken by an agent in relation to the contracts that he negotiated. The carried out analysis and solutions reached show that some other elements must be taken into account in assessing whether an agency relationship exists including an assessment of the effects of an agreement

Il est maintenant acquis que le système immunitaire occupe une place importante dans l’évolution les cancers (Hanahan et al., 2011). La compréhension actuelle de la réponse immunitaire adaptative en fait une cible de choix dans ce contexte. Il est apparu que les lymphocytes T CD4+, acteurs majeurs de la composante adaptative du système immunitaire, présentent des actions distinctes sur le contrôle de la croissance tumorale. Ainsi, les lymphocytes Th2 et Tfh, tous deux activateurs des lymphocytes B dans des conditions de lutte contre des infections pathogéniques, présentent des rôles ambivalents dans un contexte de cancer. En effet, de nombreuses études montrent que la présence de Th2 est corrélée à une progression de la maladie (notamment via l’action de l’IL-4 qu’ils sécrètent) (Koller et al., 2010 ; Roca et al., 2012) alors que les Tfh, seraient plutôt associés à un bon pronostic pour les patients (Gu-Trantien et al., 2013, 2017).Nos investigations actuelles nous ont permis de mettre en avant une caractéristique nouvelle de la biologie des lymphocytes Tfh. En effet, les Tfh expriment l’Hemathopoietic Prostaglandin D2 synthase (HPGDS). Cette enzyme de la voie de biosynthèse des eicosanoïdes est responsable de la production de Prostaglandine D2 (PGD2). Plusieurs travaux montrent que les cellules Th2 expriment le récepteur CRTH2, spécifique de la PGD2. Cette molécule agit sur ces cellules comme chemoattractant et permet également une augmentation de leur production cytokinique. Ainsi, nous posons l’hypothèse d’une communication potentielle entre lymphocytes Tfh et Th2 via la PGD2. Le projet présenté ici est alors axé sur la compréhension des mécanismes moléculaires et cellulaires sous-jacent à cette communication au sein des deux sous-types ainsi que sur son impact dans un contexte de cancer. Ce projet ayant également pour but de mettre en avant la PGD2 comme nouvelle cible thérapeutique dans le cancer
It is now accepted that the immune system plays a critical role in cancers evolution (Hanahan et al., 2011). In this context, current understanding of the adaptive immune response made it a prime target. T CD4 cells, the main players of the adaptive immune system component, are known to possess distinct roles in the control of tumour growth. Thereby, Th2 and Tfh cells, both known to activate B cells in pathogenic infections, present antagonistic roles in cancer. Indeed, numerous studies demonstrate that Th2 cells are correlated with disease progression (especially via IL-4 secretion) (Koller et al., 2010 ; Roca et al., 2012), whereas Tfh cells are associated with a good prognosis for the patients (Gu-Trantien et al., 2013, 2017) despite the actual limited amount of available data.Our current researches highlighted a new property of the biology of Tfh cells. We found that Tfh cells are able to express the Hemathopoietic Prostaglandin D2 synthase (HPGDS), an eicosanoid pathway enzyme involved in Prostaglandin D2 (PGD2) production. Moreover, different studies revealed that Th2 cells expressed CRTH2, the specific PGD2 receptor. PGD2 is known as a chemoattractant molecule for Th2 cells and lead to the increase of their cytokine secretion. We hypothesized that Tfh communicate with Th2 cells via PGD2 signalling. The present project is focused on the understanding of the underlying molecular and cellular mechanisms involved in this cross-talk and their impact in cancer. The last aim of this work is to favor the development of PGD2 as a new cancer therapeutic target

La GVH chronique (cGVHD) est une complication fréquente de l’allogreffe de cellules souches hématopoïétiques (CSH) dont la physiopathologie demeure partiellement comprise. Les données disponibles ont établi le rôle des lymphocytes T (LT) et B (LB) au cours de la cGVHD, mais la qualité de leur interaction et les sous-types de LT impliqués restent à définir. L’interaction entre les LT et les LB se fait au niveau du centre germinatif (CG) aboutissant à la production de LB mémoires et de cellules productrices d’anticorps de haute affinité grâce aux signaux d’aide reçus par les LT folliculaires helpers (TFH) finement contrôlés par une population régulatrice (TFR). La possibilité d’interroger les évènements se déroulant au niveau du CG par l’analyse de leur contingent circulant (c) nous a permis de mieux comprendre la physiopathologie de la cGVHD. En effet, la signature phénotype des cTFH suggère un gain de fonction au cours de la cGVHD, confirmée par étude fonctionnelle, et corrélant avec le phénotype des LB observé. De plus, les mécanismes de régulation apparaissent défectueux au cours de la cGVHD, puisque les cTFR présentent un défaut numérique expliqué par un défaut de résistance à l’apoptose et de prolifération. D’autre part, nous avons analysé une population de LT CD4+CD146+CCR5+, leur conférant une capacité de migration au travers des structures endothéliales et vers les sites inflammatoires. Cette population est significativement augmentée au cours de la cGVHD, et les modèles murins de cGVHD recevant des splénocytes de souris CD146-/- voient leur score clinique amélioré. L’expression de CD146 est associée à une polarisation Th17 justifiant un traitement par TMP778 (inhibiteur de RORγt) améliorant la cGVHD chez la souris. L’analyse de ces populations révèle des anomalies de la balance effecteurrégulateur et de potentielles cibles thérapeutiques à évaluer en clinique
Chronic GVHD (cGVHD) remains a major complication of allogeneic stem cell transplantation and its pathogenesis poorly understood. Previous reports established the role of T cells and B cells during cGVHD, but the quality of their interaction and T cell subsets involved remain to be defined. T cell – B cell crosstalk occurs in the germinal center generating memory B cells and high affinity antibody secreting cells consecutively to signals provided by T follicular helper cells (TFH) which are tightly controlled by a regulatory subset (TFR). The opportunity to interrogate events occurring in the germinal center through the analysis of their circulating contingent (c), allowed us to better understand cGVHD pathogenesis. cTFH phenotypic signature suggest an enhanced function during cGVHD, confirmed in functional studies, and correlating with observed B cell phenotype. In addition, regulatory mechanisms appeared defective during cGVHD, as cTFR showed a numerical deficiency, explained by a defect in resistance to apoptosis and low proliferative capacity. We also studied a T cell subset expressing CD4+CD146+CCR5+, giving the capacity to migrate through endothelial structures and toward inflammatory sites. This population is significantly increased during cGVHD, and cGVHD murine models receiving splenocytes from CD146-/- mice showed improved clinical score. CD146 expression is associated with a Th17 polarization justifying a treatment by TMP778 (RORγt inhibitor) improving cGVHD in mice. The analysis of these different populations revealed an abnormal effector-regulator balance and potential therapeutic targets to evaluate in clinic

Lysosomes are catabolic organelles devoted to the degradation of intracellular proteins and components. In addition, lysosomes are signalling hubs that orchestrate many intracellular responses. Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. ER-phagy receptors are ER membrane proteins or soluble proteins (such as CALCOCO1 and SQSTM1), that bind to cytosolic Atg8-family proteins via the LC3-Interacting Region (LIR) mediating the delivery of specific ER subdomains to lysosomes for degradation. Although the role of the ER-phagy in the regulation of ER size and cellular proteostasis has been well characterized, the upstream signalling pathway regulating ER-phagy in response to developmental and cellular needs is still largely unknown. Chondrocytes are highly secretory cells with an abundant ER, producing predominantly procollagen (PC) molecules in extracellular matrix during endochondral ossification. They reside in a poorly vascularized tissue, as the growth plate, with scarcity of nutrients, representing a good cellular model to study ER-phagy. We found that the nutrient responsive transcription factors TFEB and TFE3—master regulators of lysosomal biogenesis and autophagy—control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis is activated in chondrocytes by FGF signalling, a critical regulator of skeletal growth. We demonstrated that FGF18 induces lysosome biogenesis and ER-phagy in chondrocytes through the activation of FGFR3 and FGFR4 receptors, which in turn inhibit the PI3K/Akt-mTORC1 pathway and promote TFEB/TFE3 nuclear translocation and enhance Fam134b transcription. Notably, FAM134B is required for protein secretion in chondrocytes. In conclusion, this study identifies a new signalling pathway that allows ER-phagy to respond to developmental cues suggesting potential therapeutic approaches for the treatment of skeletal features in multiple human diseases.

Même si les traitements immunosuppresseurs ont grandement contribué à améliorer la survie du greffon en transplantation, ils comportent des effets secondaires potentiellement graves (cancer, néphrotoxicité, infections…). Certaines équipes de recherche se sont donc intéressées spécifiquement à de très rares patients transplantés rénaux, appelés patients tolérants, qui conservaient une bonne fonction de leur greffon durant des années et ce, en l’absence d’immunosuppresseurs. Plusieurs travaux ont permis de mettre en évidence le rôle primordial des lymphocytes B (LB), avec notamment un défaut de différenciation in vitro des LB en plasmocytes rapporté chez ces patients tolérants. C’est à partir de ce résultat que nous nous sommes intéressés aux lymphocytes T folliculaires helper (TFH), connus comme étant des cellules indispensables à la différenciation des LB. Dans un premier temps, nous avons mis en évidence un défaut quantitatif et qualitatif des TFH chez les patients tolérants comparé à des patients transplantés ayant une fonction stable sous immunosuppresseurs. Aussi, nous avons suggéré une implication des TFH dans l’apparition des anticorps anti-donneur (DSA), ce qui pourrait expliquer la faible proportion d’immunisation anti-donneur reportée chez les patients tolérants. Dans un deuxième temps, nous avons exploré les mécanismes moléculaires mis en jeu en réalisant une analyse transcriptomique haut débit par RNA Sequencing à partir de TFH purifiés. A partir de cette analyse, nous avons pu mettre en évidence plusieurs gènes potentiellement intéressants dans la régulation des TFH et dans l’interaction avec les LB chez les patients tolérants
Immunosuppressive drugs largely contributed to a better graft survival over time in transplantation, but induced serious side effects (cancer, nephrotoxicity, infections…). In this context, some researchers focused on rare renal transplanted recipients, who maintain a good graft function without any immunosuppressive drugs during several years. These patients are named operationally tolerant patients and are of considerable interest to immunologists. Studies on these patients reveal a critical role of B cells, with particularly an in vitro B cell differentiation defect reported in tolerant patients. Based on this report, we focused on blood T follicular helper cells (TFH) which are known to be crucial for supporting B cell differentiation. At first, we reported a qualitative and quantitative TFH defect in tolerant patients compared to transplanted patients with stable graft function under immunosuppression. Moreover, we suggest a potential role of TFH in post graft immunization with donor-specific antibodies (DSA), which could explain the low incidence of post-graft DSA immunization reported in tolerant patients. Secondly, based on a transcriptomic analysis of purified TFH by RNA Sequencing, we have highlighted several TFH genes potentially interesting in tolerant patients, concerning the TFH regulation and the cooperation between TFH and B cells

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
O tetradecapeptídeo Eumenine mastoparano - AF (EMP-AF) extraído do veneno de vespa, em solução aquosa contendo trifluoretanol (TFE) mostra conformação helicoidal anfipática de acordo com os dados de Ressonância Magnética Nuclear (RMN) e Dicroísmo Circular (CD). A seqüência de aminoácidos tem o N-terminal amidado e três lisinas carregadas (5, 8 e 12). Nesse trabalho, investigamos a estrutura conformacional desse peptídeo em solução aquosa contendo TFE por simulação de dinâmica molecular usando o pacote GROMACS. As simulações foram feitas usando uma caixa cúbica que inclui o TFE (30%) e moléculas de água (70%). O método da troca de réplica foi usado para simular o sistema no intervalo de temperatura (280K - 350K) uniformemente distribuída em 14 processadores. Cada réplica numa dada temperatura T tem uma estrutura aleatória como conformação inicial. Em intervalos fixos de tempo, duas réplicas vizinhas tentam trocar conformações de acordo com a probabilidade de Boltzmann ( - onde = ( )( U), é 1/kBT e U a energia potencial). Apresentamos trajetórias que mostram claramente a formação de uma estrutura helicoidal (resíduos 3 e 12). Juntamente com a estrutura helicoidal, outras conformações tais como estruturas helicoidais parciais e folhas- também exibem estabilidade relativamente alta. A estrutura helicoidal está de acordo com as 20 estruturas disponíveis obtidas por RMN, com valores pequenos de RMSD. Também mostramos que a diversidade de estruturas obtidas por RMN está relacionada com flutuações globais da cadeia, como indicado pela análise de componentes principais. A projeção da trajetória de equilíbrio na primeira componente principal, de uma estrutura helicoidal obtida como conformação inicial, mostrou flutuações que aproximadamente reproduzem a diversidade de estruturas de RMN, que são devidas principalmente à flexibilidade do N- terminal do peptídeo.
Tetradecapeptide eumenine mastoparan-AF (EMP-AF) (14 residues) extracted from wasp venom, in solution with water and trifluoroethanol (TFE) show amphiphatic helical conformation, according with Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) data. The amino acid sequence has amidated N-terminus and three charged lysine (5, 8 and 12). In this work, we have investigated structural conformations of this peptide in TFE aqueous solution by molecular dynamics simulations using GROMACS package. The simulations have been done using a cubic box that included TFE (30%) and water molecules (70%). The replica-exchange method was used to simulate the system in the temperature range (280K - 350K) uniformly distributed in 14 processors. Each replica, at a given T has a coil structure as the initial conformation. At fixed time intervals, two neighboring replica try to exchange configurations with Boltzmann probability e-D, where D = (Db)(DU), b is 1/kBT and U is potential energy. We present trajectories, which clearly show the formation of the helix structure of the peptide (residues 3 to 12). Along with the helix structure, other conformations, such as partial helical structure and b-sheet, also show relatively high stability. The helical structure shows good agreement with the twenty available NMR structures, with relatively small values of RMSD. It is also shown that this diversity of the NMR structures is related to global fluctuations of the chain, as indicated by a principal component analysis. The projection of equilibrium trajectory, with the obtained helix as the initial conformation, on the first principal component, showed fluctuations that nearly reproduce the diversity of the NMR structures, which are due, mainly, to the flexibility of the N-terminus of the peptide.

Orientador: Jorge Chahine
Banca: Alexandre Suman de Araújo
Banca: José Roberto Ruggiero
Resumo: O tetradecapeptídeo Eumenine mastoparano - AF (EMP-AF) extraído do veneno de vespa, em solução aquosa contendo trifluoretanol (TFE) mostra conformação helicoidal anfipática de acordo com os dados de Ressonância Magnética Nuclear (RMN) e Dicroísmo Circular (CD). A seqüência de aminoácidos tem o N-terminal amidado e três lisinas carregadas (5, 8 e 12). Nesse trabalho, investigamos a estrutura conformacional desse peptídeo em solução aquosa contendo TFE por simulação de dinâmica molecular usando o pacote GROMACS. As simulações foram feitas usando uma caixa cúbica que inclui o TFE (30%) e moléculas de água (70%). O método da troca de réplica foi usado para simular o sistema no intervalo de temperatura (280K - 350K) uniformemente distribuída em 14 processadores. Cada réplica numa dada temperatura T tem uma estrutura aleatória como conformação inicial. Em intervalos fixos de tempo, duas réplicas vizinhas tentam trocar conformações de acordo com a probabilidade de Boltzmann ( - onde = ( )( U), é 1/kBT e U a energia potencial). Apresentamos trajetórias que mostram claramente a formação de uma estrutura helicoidal (resíduos 3 e 12). Juntamente com a estrutura helicoidal, outras conformações tais como estruturas helicoidais parciais e folhas- também exibem estabilidade relativamente alta. A estrutura helicoidal está de acordo com as 20 estruturas disponíveis obtidas por RMN, com valores pequenos de RMSD. Também mostramos que a diversidade de estruturas obtidas por RMN está relacionada com flutuações globais da cadeia, como indicado pela análise de componentes principais. A projeção da trajetória de equilíbrio na primeira componente principal, de uma estrutura helicoidal obtida como conformação inicial, mostrou flutuações que aproximadamente reproduzem a diversidade de estruturas de RMN, que são devidas principalmente à flexibilidade do N- terminal do peptídeo.
Abstract: Tetradecapeptide eumenine mastoparan-AF (EMP-AF) (14 residues) extracted from wasp venom, in solution with water and trifluoroethanol (TFE) show amphiphatic helical conformation, according with Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) data. The amino acid sequence has amidated N-terminus and three charged lysine (5, 8 and 12). In this work, we have investigated structural conformations of this peptide in TFE aqueous solution by molecular dynamics simulations using GROMACS package. The simulations have been done using a cubic box that included TFE (30%) and water molecules (70%). The replica-exchange method was used to simulate the system in the temperature range (280K - 350K) uniformly distributed in 14 processors. Each replica, at a given T has a coil structure as the initial conformation. At fixed time intervals, two neighboring replica try to exchange configurations with Boltzmann probability e-D, where D = (Db)(DU), b is 1/kBT and U is potential energy. We present trajectories, which clearly show the formation of the helix structure of the peptide (residues 3 to 12). Along with the helix structure, other conformations, such as partial helical structure and b-sheet, also show relatively high stability. The helical structure shows good agreement with the twenty available NMR structures, with relatively small values of RMSD. It is also shown that this diversity of the NMR structures is related to global fluctuations of the chain, as indicated by a principal component analysis. The projection of equilibrium trajectory, with the obtained helix as the initial conformation, on the first principal component, showed fluctuations that nearly reproduce the diversity of the NMR structures, which are due, mainly, to the flexibility of the N-terminus of the peptide.
Mestre

The ability of cells within the adaptive immune system to develop into specialized subsets allow for a robust and tailored immune response in the advent of an infection or injury. Here, CD4+ T-cells are a crucial component within this system, with subsets such as TH1, TH2, TH17, TFH and TREG cells playing vital roles in propagating cell-mediated immunity. For example, TH1 cells are essential in combating intracellular pathogens such as viruses, while TFH cells communicate with B-cells to optimize antibody responses against an invading pathogen. The development (and functionality) of these subsets is ultimately dictated by the appropriate integration of extracellular cues such as cytokines with cell intrinsic transcription factors, thereby promoting the necessary gene profile. Moreover, the observation that T-helper cells could exhibit a flexible nature (i.e having shared gene profiles and effector functions) not only demonstrate the efficiency of our immune system but also how such flexibility could have unintended consequences during adverse events such as autoimmunity. An important mediator of such flexibility is cytokines. However, the complete network of factors that come together to co-ordinate cytokine mediated plasticity remain unknown. Thus, the work in this dissertation hope to delineate the factors that collaborate to regulate cytokine induced T-helper cell flexibility. As such, we see that in the presence of IL-2, the Ikaros Zinc Finger (IkZF) transcription factor Eos is upregulated in TH1 cells, with this factor playing a significant role in promoting regulatory and effector functions of TH1 cells. Moreover, we show that Eos forms a novel protein complex with STAT5 and promotes STAT5 activity in TH1 cells. However, depleting IL-2 from the micro-environment leads to the upregulation of two other members within the IkZF family, Ikaros and Aiolos. Aiolos in turn collaborate with STAT3, induces Bcl-6 expression within these cells, thus promoting these cells to exhibit characteristic features of TFH cells. The work in this dissertation hopes to advance our understanding of the regulatory mechanisms involved in cytokine mediated T-cell flexibility thereby hoping to open new avenues for the development of novel therapeutic strategies in the event of autoimmunity.
Ph. D.
T-helper (TH) cells are an important component of the immune system, as these cells aid in the fight against pathogens by secreting factors that either accentuate the inflammatory response during infection or attenuate immune responses post infection. Such effects are made possible because T-helper cells can differentiate into a variety of subsets, with each subset being an important mediator in maintaining immune homeostasis. For example, the T-helper cell subset called TH1 plays a vital role in the fight against intracellular pathogens such as viruses and certain parasites, while T-follicular helper (TFH) cells aid in the production of antibodies specific to the invading pathogen. The development of such subsets occur when cell extrinsic signals, called cytokines, lead to the activation or induction of cell intrinsic proteins called transcription factors. Interestingly, research over the years have shown that T-helper cells are highly adaptable in nature, with one subset having the ability to attain certain characteristic features of other subsets. This malleable nature of T-helper cells relies on several factors, with cytokines within the micro-environment being an important one. Although this form of flexibility is efficient and beneficial at times, it can also be detrimental, as such flexibility is known to promote certain autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and type 1 diabetes. Such detrimental effects are thought to be due to cytokines within the environment. Therefore understanding how cytokines influence the flexible nature of T-helper cells is important; as controlling such flexibility (either by regulating cytokines or the transcription factors activated as a consequence) could prevent the propagation of undesired T-helper cell functions. As such, the work in this dissertation hopes to uncover how one such cytokine, termed Interleukin-2 (IL-2) mediates the flexibility between TH1 and TFH cells. The work highlighted in this dissertation broadens our understanding of how cytokines influence T-helper cell development and flexibility, and consequently allows the design of novel therapeutic strategies to combat autoimmune diseases.

Thesis: S.M., Massachusetts Institute of Technology, Department of Nuclear Science and Engineering, 2014.
Page 95 blank. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 67-70).
In this work we describe a preliminary design for a transportable fluoride salt cooled high temperature reactor (TFHR) intended for use as a variable output heat and electricity source for off-grid locations. The goals of the project were to design an economic reactor: a) Sized for the average load of a site but able to increase output to provide peaking power. b) With safety, security and safeguard requirements met by the choice of materials and form as opposed to relying on security forces and infrastructure. Powering remote sites such as mining stations, military bases, communities or even large ships could be a significant long term market for small nuclear reactors. However, the design basis is very different. The increased cost of transporting goods to the site and maintaining a large population of specialists means a reactor must be simpler to operate and able to defend itself against attackers and proliferators without a large security force. On the other hand, the increased cost of electricity in remote places means more can be spent to meet these goals. This report discusses these issues of operating at a remote site and a general strategy for meeting the resulting design criteria. The TFHR design puts these decisions into practice. The TFHR described is a 125MWth, thermal spectrum reactor using SiC-matrix coated particle fuel which can achieve single batch discharge burnups of up to 70MWd/HMkg over an 8 year cycle. Higher burnups are possible for larger cores. The neutronics properties of SiC-matrix coated particle fuel are explored in detail and various means by which they can be incorporated into a reactor are detailed. The TFHR uses a nuclear air Brayton combined cycle (NACC) for electricity generation, adapted from an off the shelf GE aero-derivative gas turbine. The NACC incorporates a combustible fuel injection port between the high and low pressure turbines which can be used to raise the temperature of the working fluid and boost the power extracted from the system by up to 50%. This increase of electric output occurs without changing the power drawn drawn from the reactor, avoiding any transients. The ability to peak the power output removes the need for a second power system or for the reactor to be sized for the maximum power demand, which is a significant cost saving. However, using an air Brayton cycle requires a high temperature reactor. A TFHR is a better match for this purpose than a gas cooled reactor as it operates at atmospheric pressure, making it easier to meet the security goals described above.
by Ruaridh R. Macdonald.
S.M.

The mechanistic Target Of Rapamycin Complex 1 (mTORC1) regulates cellular biosynthetic pathways in response to variations in nutrient availability. Activation of mTORC1 is mediated by Rag GTPases, that act as heterodimers and promote mTORC1 recruitment to the lysosome. Many studies have clarified the post-translational control of mTORC1, but little is known about its transcriptional regulation. Our study demonstrates that TFEB, TFE3 and MITF, members of the MiT/TFE family of transcription factors and master regulators of lysosomal and melanosomal biogenesis and autophagy, are nutrient-sensitive transcriptional activators of mTORC1 signaling. During starvation they induce the expression of the RagD gene and this enhances mTORC1 recruitment to the lysosome and its reactivation when nutrients become available. Thus, in periods of nutrient deprivation, this mechanism allows the cell to rapidly reactivate anabolic pathways and turn off catabolism when nutrient levels are restored. Furthermore this mechanism plays an important role in cancer growth. Up-regulation of the MiT/TFE genes in renal cell carcinoma and melanoma is associated to RagD-induced mTORC1 activation, causing cell hyperproliferation and cancer progression.

Le cellule follicolari T helper (TFH) sono una sottopopolazione separata di cellule CD4+ T helper specializzate nel fornire aiuto alle cellule B. Si sviluppano in maniera indipendente dalle altre sottopopolazioni di cellule T e sono fondamentali per l'immunità umorale, compresa la generazione di plasmacellule di lunga durata e ad alta affinità e di cellule B della memoria. Alterazione di geni specifici delle cellule TFH porta a difetti nella formazione dei centri germinativi (GC), con conseguenti disordini autoimmuni o immunodeficienze. I meccanismi molecolari alla base del differenziamento delle cellule TFH umane sono poco conosciuti. I microRNA (miRNA) sono piccole molecole di RNA non codificanti a singolo filamento e altamente conservati, che controllano l'espressione genica a livello post-trascrizionale legandosi alla regione 3’ non tradotta del mRNA bersaglio. Per studiare il ruolo dei miRNA nella biologia delle cellule TFH abbiamo eseguito RT-qPCR e un’analisi tramite deep-sequencing su cellule TFH e Naive CD4+ T isolate da adenoidi umane. Abbiamo identificato miRNA specifici delle cellule TFH umane e abbiamo convalidato in vitro i loro bersagli molecolari predetti. Abbiamo scoperto che miRNA specifici delle cellule TFH umane regolano trascritti specificamente espressi nella sottopopolazione di cellule TFH e noti per essere essenziali per la loro funzione. Abbiamo eseguito esperimenti di gain o loss-of function utilizzando vettori lentivirali volti a modulare l'espressione dei miRNA in cellule TFH umane. Attraverso questi esperimenti abbiamo valutato come questi miRNA specifici delle cellule TFH umane influenzano la capacità delle cellule TFH di fornire aiuto alle cellule B, e abbiamo chiarito il loro ruolo nella biologia delle cellule TFH.

Ce manuscrit de thèse décrit la synthèse et la caractérisation d'ionomères conducteurs protoniques pour une application en tant que membrane pour pile à combustible. La stratégie adoptée pour la synthèse de ces polymères repose sur la polycondensation directe de monomères fonctionnels. Pour ce faire, et dans le cadre d'un travail important de chimie organique, la synthèse de trois monomères sulfonés ainsi que de trois monomères phosphonés a été réalisée. Différents polymères perfluorés (polyperfluorocyclobutane (PFCB) et polyaryléthers (PAE) (Polyétheréther cétone (PEEK) et Polysulfone (PS), ont été obtenus par polycondensation directe de ces monomères fonctionnalisés. Il a été possible de synthétiser des copolymères séquencés (à blocs) et comportant un ou deux types de fonctions conductrices protoniques. Afin d'établir d'éventuelles relations structure-propriété pour ces polymères et l'influence du solvant dans la morphologie de la membrane, une étude de la morphologie a été réalisée à partir de plusieurs séries de polymères PAE et leurs analogues « statistiques ». Les valeurs de conductivité des polymères à blocs sont, en général, supérieures à celles de leurs analogues « statistiques ». Un PAE obtenu est particulièrement intéressant, il possède à la fois une conductivité (216 mS.cm 1) nettement supérieure au Nafion® et un gonflement plus faible. Le polymère perfluoré (PFCB) obtenu est également très prometteur : la conductivité enregistrée pour ce ionomères est de 138 mS/cm. Ce travail de thèse est, à notre connaissance, le premier exemple de synthèse d'un PFCB sulfoné par polymérisation directe d'un monomère fonctionnel mais il constitue également le premier exemple de synthèse de poly(aryléther)s à blocs sulfonés / phosphonés par copolymérisation directe de deux types de monomères fonctionnels
This thesis describes the synthesis and characterization of proton conducting polymers for application as membrane for fuel cells (PEMFC). The approach for the synthesis of polymers consists in creating polymers from direct polycondensation of functional monomers, in order to better control their final IEC. Three sulphonated monomers and three phosphonated monomers have been first synthetized. Different types of polyarylethers (one of them is both sulphonated and phosphonated) and one perfluorinated polymer (PFCB) have been synthetised by direct polycondensation of functional monomers. In order to explain the influence of the solvent in the final morphology of the membrane, and the relation between its structure and properties, one morphological study has been realized to the obtained polymers but also to their analogues “statistical” polymers. In general, the blocks polymers obtained the highest values of conductivity. One polyarylether seems particularly interesting, because its conductivity value is much higher than Nafion®’s, and has a smaller swelling value. The perfluorinated polymer has also an interesting conductivity value (138 mS/cm). This thesis work is, to the best of our knowledge, the first example of the synthesis of a sulphonated PFCB obtained by direct polycondensation but also the first example of synthesis of a both sulfonated/phosphonated polyarylethers by direct copolymerisation of to types of functional monomers

Les lymphocytes T CD8+ spécifiques de l'antigène sont impliqués dans la réponse immunitaire adaptative et jouent un rôle essentiel dans la protection de l'hôte contre l'infection par des pathogènes intracellulaires. Cette protection de longue durée dépend de la génération de réponses lymphocytaires T CD8+ mémoires, hautement fonctionnelles en termes de fréquence et de fonctionnalité, après réinfection.Après présentation de l'antigène, une cellule T CD8 naïve subit une forte expansion clonale, générant une population hétérogène de cellules activées qui est dominée, au sommet de l'expansion, par des effecteurs CD8 de courte durée (SLEC). Cette expansion est suivie d'une phase de contraction massive par apoptose. Quelques cellules survivent à cette phase de contraction et finissent par se différencier en cellules mémoire hautement compétentes. Les processus par lesquels et le moment où se différencient les précurseurs de mémoire (MPECs) restent largement inconnus, tout comme les étapes ultérieures de leur maturation en cellules mémoire pleinement fonctionnelles. Les signaux d'aide provenant des cellules T CD4+ sont clairement requis tout au long du processus de maturation des MPEC.Notre équipe a montré que les lymphocytes T CD4+ régulateurs FoxP3+ (Tregs) favorisent la maturation des MPEC en limitant l'exposition à l'IL-2 et en fournissant des signaux inhibiteurs, mais ce n'est probablement qu'une facette de l'aide complexe et multiforme apportée par les cellules T CD4+ au MPEC. Les Tregs agissent sur des MPEC préexistants. Les réponses mémoire B et CD8+ partagent des caractéristiques communes, telles que l'expression du facteur de transcription Bcl-6. Les lymphocytes T CD4+ folliculaires (Tfh) sont les principaux producteurs de la cytokine IL-21. Bien que les mécanismes par lesquels les Tfh induisent l’expression de Bcl-6 dans les cellules B doivent être clarifiés, ils pourraient inclure l’IL-21 et l’interaction CD40-CD40L.Dans ce projet de thèse, nous avons étudié le rôle potentiel des Tfh dans l'initiation de la différenciation mémoire T CD8+, dans des modèles de souris transgéniques permettant une déplétion transitoire et sélective des Tfh, infectées par la bactérie recombinante Listeria monocytogenes-OVA.Nous avons montré que dès 2 jours après l'infection, les MPECs très précoces peuvent être identifiés par l’expression du récepteur de chimiokine CXCR5. Ces précurseurs précoces, qui ont un phénotype effecteur, se développent et migrent temporairement à la jonction des zones T et B, où ils interagissent avec les Tfh puis perdent leur expression CXCR5.Cette interaction avec les Tfh, considérés jusqu'à présent comme des auxiliaires exclusifs des cellules B, est nécessaire pour que les MPECs CD8+ deviennent des cellules mémoire compétentes sensibles à l'IL-21, capables de générer des réponses effectrices secondaires efficaces.Cette étude dévoile les premières étapes cruciales dans la génération de la mémoire CD8+, identifie CXCR5 comme le premier marqueur connu des MPECs CD8+, révèle l’implication fondamentale des Tfh dans le CD4 help et indique une coordination possible, via les Tfh, entre les voies de différentiation mémoire CD8+ et B. Ces résultats peuvent avoir des implications pour la conception du vaccin et de l'immunothérapie
Antigen-specific CD8 T cells are involved in the adaptive immune response and play a critical role in protecting the host from infection by intracellular pathogens. This long-lasting protection depends on the generation of memory CD8+ T cell responses, which are highly functional in terms of frequency and functionality, after secondary infection.Following antigen activation, a naive CD8 T cell undergoes strong clonal expansion, generating a heterogeneous population of activated cells that is dominated, at the peak of expansion, by short-lived CD8 effectors (SLECs). This expansion is followed by a phase of drastic contraction through massive apoptosis. A few cells survive this contraction phase and eventually become highly competent memory cells. Precisely when and how these memory precursors (MPECs) are generated is largely unknown, and so are the subsequent steps of their maturation into fully functional memory cells. Help signals from CD4+ T cells are clearly required throughout the MPEC maturation process.Our team has previously shown that FoxP3+ regulatory CD4+ T cells (Tregs) favor MPECs maturation by limiting exposure to IL-2 and by providing inhibitory signals, but this is probably only one facet of the complex and multifaceted help provided by CD4+ T cells to MPEC, and Tregs act on pre-existing MPECs.B-cell memory and CD8+ T cell memory share some common features, such as the expression of the transcription factor Bcl-6. Tfh are major producers of the cytokine IL-21. The mechanisms by which Tfh induces Bcl-6 in B-cells need to be clarified, they might include IL-21 and CD40-CD40L.In this PhD project, we have investigated the potential role of Tfh on the initiation of CD8 memory differentiation, in transgenic mice models, allowing transient and selective depletion of Tfh cells, infected by recombinant Listeria monocytogenes-OVA.We have shown that as early as 2 days after infection, very early memory precursors can be identified by their expression of the chemokine receptor CXCR5. These early precursors, which have an effector phenotype, expand and temporarily migrate to the junction of T-cell and B-cell zones, where they interact with follicular CD4 T cells (Tfh) then lose their CXCR5 expression.Remarkably, this interaction with Tfh, hitherto considered as exclusive B-cell helpers, is required for memory precursors to become competent memory cells responsive to IL-21 and capable of mounting efficient cytotoxic secondary effector responses.This study thus unveils critical early steps in the generation of CD8 memory, identifies CXCR5 as the earliest known marker of CD8 memory precursors, reveals a major helper role for Tfh, and points to possible coordination, through Tfh, between the pathways of CD8 and B-cell memory generation. These findings may have implications for vaccine and immunotherapy design

Organofluorine compounds find various applications ranging from pharmaceuticals to refrigerants, insecticides, high-value fluoropolymers and reagents in catalysis. However, the synthesis of organofluorine compounds depends on toxic chemicals such as hydrogen fluoride, chlorinated hydrocarbons, reactive F2 gas and environmentally persistent long-chain fluorosurfactants. Recently more sustainable, energy-efficient syntheses have been developed using base metal-catalyzed transformations of fluoroalkenes and the formation and functionalization of d6-8 fluorometallacycles. In this thesis, we use manganese complex precursors to prepare the first examples of d4 fluorometallacycles. Work in Chapter 2 describes the synthesis and one-electron reduction of manganese bis(diphosphine)- and tetrakis(phosphite) dibromide complexes, MnBr2(P-P)2 and MnBr2[P(O-i-Pr)3]4 and reactions of the corresponding reduced Mn(I)Br complexes with tetrafluoroethylene (TFE). Products proposed to be d4 perfluorometallacycles, MnBr[-CF2(CF2)2CF2-](P-P) proved to be unstable, reforming TFE upon application of vacuum. In Chapter 3 we show that photolysis of ligated manganese(I) carbonyl bromide complexes, MnBrLn(CO)5-n, in the presence of TFE, chlorotrifluoroethylene (CTFE) or perfluoro(methyl vinyl ether) (PMVE) in tetrahydrofuran affords the Mn-H insertion products, Mn(CF2CFXH)(L2)(CO)3 (X = F, Cl, OCF3) only for L2 = DPPE [1,2-bis(diphenylphosphino)ethane] as well as a solid by-product proposed to be MnBr2Ln. These reactions are accompanied by THF fluoroalkylation products, O[-(CH2)3CH(CF2CFHX)-]. By switching to methyl t-butyl ether solvent, we showed that exhaustive photolysis of MnBr(CO)5 + 3 equiv. of DPPE gave a new product proposed to be the first stable d4 fluorometallacycle, MnBr[-(CF2)4-](CO)(DPPE). Reactions of the fluoroalkenes with zerovalent Mn2(CO)10 also contributed to our understanding of potential reaction pathways to form these Mn-H-derived products. Previous work in the Baker group compared FeH2(dmpe)2 and [FeH(H2)(dmpe)2]+ as catalysts for the dehydrogenation of amine-boranes [dmpe = 1,2-bis(dimethylphosphino)ethane]. In Chapter 4 the catalytic reactivity and selectivity of MnH(H2)(dmpe)2 are compared with those observed using the Fe analogs and the catalyst resting state, Mn(2-BH4)(dmpe)2, is identified. Finally, in Chapter 5 we summarize the findings of this thesis and suggests future directions based on this work.

Implication de ROQUIN dans la physiopathologie du lymphome T angio-immunoblastique. Le T-LAI est un lymphome T périphérique qui de part sa rareté bénéficie de peu d'études contrairement aux lymphomes B. En France, le T-LAI est le PTCL le plus fréquemment rencontré. Malgré une évolution clinique variable, le T-LAI reste une tumeur agressive dont la survie globale est inférieure à 3 ans. Un des objectifs de notre équipe est donc de mieux comprendre la physiopathologie de ce lymphome et d'identifier des évènements oncogéniques conduisant à son développement. Dans le cadre de ce projet, notre étude s'est portée sur le gène ROQUIN qui code une E3 ubiquitine ligase de la famille RING et dont la mutation est associée à l'apparition d'un syndrome proche du T-LAI chez la souris sanroque.Bien que nous n'ayons détecté aucune mutation dans la séquence codante de ROQUIN nous avons identifié un nouveau transcrit alternatif appelé ROQUIN ØE17. Celui-ci code une protéine qui, comme la forme sauvage, se localise dans les granules de stress et les corps P et interagit avec certains ARNm et micro-ARN. Néanmoins il est le seul à inhiber l'expression de la molécule de costimulation ICOS. ROQUIN ØE17, qui est présent en concentrations variables dans les différentes populations lymphocytaires T n'est quasiment pas exprimé dans les T-LAI. De ce fait, la perte du transcrit ROQUIN ØE17 pourrait participer à la genèse et/ou développement de ce lymphome
Implication of ROQUIN in the physiopathology of angio-immunoblastic T cell lymphoma. AITL is a peripheral T cell lymphoma, poorly studied compared to B cell lymphomas due to its rarity. In France, AITL is the PTCL the most frequently encountered. Despite a variable clinical course, AITL is an aggressive tumor with an overall survival lower than 3 years. One of our goal is to better understand the physiopathology of this lymphoma and identify oncogenic events that lead to its development. In this project, our study was focused on ROQUIN gene that encodes a RING E3 ubiquitin ligase and whose mutation induces an AITL-like syndrom in sanroque mice.Although we did not detect any mutation in ROQUIN coding sequence, we identified a novel alternative transcript referred as ROQUIN ØE17. It encodes a protein that, like wild type protein, localizes to stress granules and P bodies and interacts with mRNAs and microRNAs. However, only ROQUIN ØE17 inhibits the expression of the costimulatory molecule ICOS. This transcript, whose expression varies between T cell populations, is hardly expressed in AITL. Consequently, the loss of ROQUIN ØE17 could be involved in the genesis and/or development of this lymphoma

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Protonectina e Protonectina 1-6 são peptídeos extraídos do veneno da vespa social Agelaia pallipes pallipes. O primeiro apresenta atividade hemolítica, de degranulação de mastócitos e quimiotática, enquanto o segundo apresenta somente a atividade quimiotática quando atuam isoladamente. A associação destes dois peptídeos, em proporção 1:1 produz um aumento nas atividades hemolíticas e de degranulação de mastócitos, enquanto a atividade quimiotática decresce acentuadamente, ou seja, em associação apresentam um comportamento mais acentuado de interação com membranas. Dados de dicroísmo circular mostram que, quando associados, há um pequeno acréscimo no percentual de aminoácidos em conformação de hélice-α. A proposta deste trabalho é analisar, usando dinâmica molecular, os aspectos conformacionais da Protonectina isolada e em associação com a Protonectina 1 – 6 em misturas de TFE-água, para entender se e como ocorre essa associação, procurando destacar quais fatores podem ser determinantes nesse processo. A Protonectina isolada foi estudada por meio de dinâmicas moleculares de equilíbrio e dinâmica associada ao método de troca de réplicas. Com esse método, obteve-se dezesseis trajetórias de 60 ns varrendo o intervalo de temperaturas de 287 a 342 K, usando uma conformação em hélice alfa ideal como estrutura inicial. Usando o software WHAM e parâmetros como o RMSD e a primeira das componentes de uma Análise de Componentes Principais, PCA, como coordenadas de reação, obteve-se a superfície de energia livre, que apresenta dois mínimos principais. O mínimo com energia mais baixa contém estruturas ordenadas, com parte dos resíduos em alfa-hélice, ou em hélice-5 ou, ainda, numa combinação de “turns” e B-bridges, enquanto conformações aleatórias são encontradas no segundo mínimo. Destes dados o tempo de transição...
Protonectin and Protonectin 1- 6 are peptides extracted from the venom of the social wasp Agelaia pallipes pallipes. While Protonectin presents hemolytic, mast cell degranulation and chimiotactic activities, protonectin 1-6 just presents the chimiotactic activity. When these peptides are mixed at 1:1 molar ratio the hemolytic and mast cell degranulation activities increase while the chemiotactic decreases significantly. Circular Dicroism data shows a small increase in the amount of residues in ordered structures, alfa helix, for example. The purpose of this work is to analyse the conformational features of these peptides isolated and in association in TFE-water mixtures using molecular dynamics simulation to understand if this association occurs or not and how. The Protonectin was studied using equilibrium MD simulations and associating Molecular dynamics and the replica exchange methods. With this method, sixteen trajectories of the 60 ns in the range 287 – 342 K was simulated, all the replicas starting from an ideal alfa helix. Using the WHAM software, RMSD parameters and the first Principal Components Analysis, PCA, we have the free energy surface, that presents two principals minimums. One corresponding to structures with some organization some residues in α-helix, 5-helix or turns associated with b-bridges. The other minimum presents structures in coil conformation. The “folding” time were stimated and the stability of the conformations are discussed. Starting from typical structures found in the equilibrium dynamic associated with the replica exchange method six 60 ns trajectories were simulated. These simulations comprove some stabilization of the structures found at the energy minimum. The association of Protonectin and Protonectin 1- 6 was studied using equilibrium molecular dynamics run during at ~300 ns in the ambient temperature. The results suggest that there... (Complete abstract click electronic access below)

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Os Mastoparanos são peptídeos curtos e catiônicos extraídos do veneno de vespas. Estes peptídeos dependem de sua seqüência primária, composição, hidrofobicidade, ângulo polar, anfipaticidade e amidação do C-terminal para apresentar diferentes atividades biológicas. A conformação em hélice anfipática parece ser fundamental para interação destes peptídeos com a bicamada membranar. Neste trabalho buscamos as correlações destas características com as atividades antimicrobianas e hemolíticas apresentadas por três mastoparanos conhecidos, usando simulações por dinâmica molecular em misturas de TFE-água. Misturas de TFE-água apresentam um caráter hidrofóbico e hidrofílico assim como o ambiente membranar. Um dos peptídeos estudados é o Eumenine Mastoparano-AF (EMP-AF), no qual constatamos que a desamidação do C-terminal acarretou na desestruturação da conformação helicoidal e na perda da anfipaticidade nesta região, devido à atração eletrostática entre a carga negativa do grupo carboxila e os grupos catiônicos das cadeias laterais de alguns resíduos. Por esse fato a atividade hemolítica é perdida. Outro peptídeo investigado é o Paulista-MPI, para o qual propomos como estrutura estável uma hélice-.. anfipática. Para este caso, também exploramos as razões da existência de atividade antimicrobiana e ausência da atividade hemolítica. O outro peptídeo estudado foi o Anoplin com e sem o grupo amida do C-terminal. O Anoplin natural, Anoplin-NH2 é um peptídeo antimicrobiano. Novamente esta atividade pode ser associada com sua estrutura helicoidal anfipática que se apresenta estável por fatores como o equilíbrio eletrostático entre os grupos carregados das cadeias laterais; pela predominância dos grupos apolares das moléculas de TFE na região dos átomos eletronegativos da cadeia principal; e pela solvatação da cadeia lateral...
Mastoparans is a class of peptides extracted from wasp venom, named after their first recognized biological action, the degranulation of mast cells. They are short and cationic peptides that depending on their primary sequence, degree of charge and amidation of Cterminal present different biological activities. Fundamental for their interactions with biological membranes seems to be an amphipatic helix conformation. In this work we search for correlations among these characteristics and the antimicrobial and hemolytic activities of three known mastoparans, using molecular dynamics simulation in TFEwater mixtures. TFE-water media shows both hydrophobic and hydrophilic character and in this way mimics a membrane-like environment. The peptides studied are the Eumenine Mastoparan (EMP-AF), for which the desamidation of the C-terminal leads to the lost of the helix conformation and amphipaticity at this region, due to the electrostatic attraction among the negative charge of carboxylate group and the cationic groups of the lateral chain of some residues. Consequently the hemolytic activity is lost. Another peptide studied is Paulista-MPI, for which an amphipatic ..-helix is proposed as a stable structure. We rationalize also the reasons for its antimicrobial activities and the absence of hemolytic activity. The last peptide studied is Anoplin, the C-terminal amidated wild type and its carboxylated analog. The former type, Anoplin-NH2, is an antimicrobial and nonhemolytic peptide. Again this can be associated to the amphipatic helical structure, that we show is maintained by factors as the electrostatic equilibrium between charged groups, predominance of apolar groups of TFE molecules around the electronegative atoms of the main chain and solvation of lateral side chains. Deamidation of C-terminal, rendering Anoplin-OH, abolishes its antimicrobial activity due to the unfolding of the helix terminal... (Complete abstract, click electronic address below

Resumo: Protonectina e Protonectina 1-6 são peptídeos extraídos do veneno da vespa social Agelaia pallipes pallipes. O primeiro apresenta atividade hemolítica, de degranulação de mastócitos e quimiotática, enquanto o segundo apresenta somente a atividade quimiotática quando atuam isoladamente. A associação destes dois peptídeos, em proporção 1:1 produz um aumento nas atividades hemolíticas e de degranulação de mastócitos, enquanto a atividade quimiotática decresce acentuadamente, ou seja, em associação apresentam um comportamento mais acentuado de interação com membranas. Dados de dicroísmo circular mostram que, quando associados, há um pequeno acréscimo no percentual de aminoácidos em conformação de hélice-α. A proposta deste trabalho é analisar, usando dinâmica molecular, os aspectos conformacionais da Protonectina isolada e em associação com a Protonectina 1 - 6 em misturas de TFE-água, para entender se e como ocorre essa associação, procurando destacar quais fatores podem ser determinantes nesse processo. A Protonectina isolada foi estudada por meio de dinâmicas moleculares de equilíbrio e dinâmica associada ao método de troca de réplicas. Com esse método, obteve-se dezesseis trajetórias de 60 ns varrendo o intervalo de temperaturas de 287 a 342 K, usando uma conformação em hélice alfa ideal como estrutura inicial. Usando o software WHAM e parâmetros como o RMSD e a primeira das componentes de uma Análise de Componentes Principais, PCA, como coordenadas de reação, obteve-se a superfície de energia livre, que apresenta dois mínimos principais. O mínimo com energia mais baixa contém estruturas ordenadas, com parte dos resíduos em alfa-hélice, ou em hélice-5 ou, ainda, numa combinação de "turns" e B-bridges, enquanto conformações aleatórias são encontradas no segundo mínimo. Destes dados o tempo de transição... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Protonectin and Protonectin 1- 6 are peptides extracted from the venom of the social wasp Agelaia pallipes pallipes. While Protonectin presents hemolytic, mast cell degranulation and chimiotactic activities, protonectin 1-6 just presents the chimiotactic activity. When these peptides are mixed at 1:1 molar ratio the hemolytic and mast cell degranulation activities increase while the chemiotactic decreases significantly. Circular Dicroism data shows a small increase in the amount of residues in ordered structures, alfa helix, for example. The purpose of this work is to analyse the conformational features of these peptides isolated and in association in TFE-water mixtures using molecular dynamics simulation to understand if this association occurs or not and how. The Protonectin was studied using equilibrium MD simulations and associating Molecular dynamics and the replica exchange methods. With this method, sixteen trajectories of the 60 ns in the range 287 - 342 K was simulated, all the replicas starting from an ideal alfa helix. Using the WHAM software, RMSD parameters and the first Principal Components Analysis, PCA, we have the free energy surface, that presents two principals minimums. One corresponding to structures with some organization some residues in α-helix, 5-helix or turns associated with b-bridges. The other minimum presents structures in coil conformation. The "folding" time were stimated and the stability of the conformations are discussed. Starting from typical structures found in the equilibrium dynamic associated with the replica exchange method six 60 ns trajectories were simulated. These simulations comprove some stabilization of the structures found at the energy minimum. The association of Protonectin and Protonectin 1- 6 was studied using equilibrium molecular dynamics run during at ~300 ns in the ambient temperature. The results suggest that there... (Complete abstract click electronic access below)
Orientador: José Roberto Ruggiero
Coorientador: Jorge Chahine
Banca: Pedro Geraldo Pascutti
Banca: Márcia Perez dos Santos Cabrera
Mestre

Orientador: José Roberto Ruggiero
Banca: Pedro Geraldo Pascutti
Banca: Luiz Carlos Gomide de Freitas
Banca: Mário Sérgio Palma
Banca: Valmir Fadel
Resumo: Os Mastoparanos são peptídeos curtos e catiônicos extraídos do veneno de vespas. Estes peptídeos dependem de sua seqüência primária, composição, hidrofobicidade, ângulo polar, anfipaticidade e amidação do C-terminal para apresentar diferentes atividades biológicas. A conformação em hélice anfipática parece ser fundamental para interação destes peptídeos com a bicamada membranar. Neste trabalho buscamos as correlações destas características com as atividades antimicrobianas e hemolíticas apresentadas por três mastoparanos conhecidos, usando simulações por dinâmica molecular em misturas de TFE-água. Misturas de TFE-água apresentam um caráter hidrofóbico e hidrofílico assim como o ambiente membranar. Um dos peptídeos estudados é o Eumenine Mastoparano-AF (EMP-AF), no qual constatamos que a desamidação do C-terminal acarretou na desestruturação da conformação helicoidal e na perda da anfipaticidade nesta região, devido à atração eletrostática entre a carga negativa do grupo carboxila e os grupos catiônicos das cadeias laterais de alguns resíduos. Por esse fato a atividade hemolítica é perdida. Outro peptídeo investigado é o Paulista-MPI, para o qual propomos como estrutura estável uma hélice-.. anfipática. Para este caso, também exploramos as razões da existência de atividade antimicrobiana e ausência da atividade hemolítica. O outro peptídeo estudado foi o Anoplin com e sem o grupo amida do C-terminal. O Anoplin natural, Anoplin-NH2 é um peptídeo antimicrobiano. Novamente esta atividade pode ser associada com sua estrutura helicoidal anfipática que se apresenta estável por fatores como o equilíbrio eletrostático entre os grupos carregados das cadeias laterais; pela predominância dos grupos apolares das moléculas de TFE na região dos átomos eletronegativos da cadeia principal; e pela solvatação da cadeia lateral... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Mastoparans is a class of peptides extracted from wasp venom, named after their first recognized biological action, the degranulation of mast cells. They are short and cationic peptides that depending on their primary sequence, degree of charge and amidation of Cterminal present different biological activities. Fundamental for their interactions with biological membranes seems to be an amphipatic helix conformation. In this work we search for correlations among these characteristics and the antimicrobial and hemolytic activities of three known mastoparans, using molecular dynamics simulation in TFEwater mixtures. TFE-water media shows both hydrophobic and hydrophilic character and in this way mimics a membrane-like environment. The peptides studied are the Eumenine Mastoparan (EMP-AF), for which the desamidation of the C-terminal leads to the lost of the helix conformation and amphipaticity at this region, due to the electrostatic attraction among the negative charge of carboxylate group and the cationic groups of the lateral chain of some residues. Consequently the hemolytic activity is lost. Another peptide studied is Paulista-MPI, for which an amphipatic ..-helix is proposed as a stable structure. We rationalize also the reasons for its antimicrobial activities and the absence of hemolytic activity. The last peptide studied is Anoplin, the C-terminal amidated wild type and its carboxylated analog. The former type, Anoplin-NH2, is an antimicrobial and nonhemolytic peptide. Again this can be associated to the amphipatic helical structure, that we show is maintained by factors as the electrostatic equilibrium between charged groups, predominance of apolar groups of TFE molecules around the electronegative atoms of the main chain and solvation of lateral side chains. Deamidation of C-terminal, rendering Anoplin-OH, abolishes its antimicrobial activity due to the unfolding of the helix terminal... (Complete abstract, click electronic address below
Doutor

Depuis sa découverte, le virus de l’immunodéficience humaine de type 1 (VIH-1) a causé la mort de 33 millions de personnes, et 36,7 millions sont actuellement infectées. Malgré l’existence des thérapies antirétrovirales, celles-ci ne conduisent pas à d’éradiquer le virus. En outre, il n’existe pas de vaccin. Les lymphocytes B, dont la fonction est de produire les anticorps sont dysfonctionnels au cours du VIH-1. Or la majorité des stratégies vaccinales se basent sur la production d’anticorps dépendante des cellules T CD4. Ainsi, la première partie de mon doctorat a été consacré à la compréhension de l’impact du VIH-1 sur les cellules T CD4 folliculaires auxiliaires (Tfh) essentielles à l’activation des lymphocytes B et à la production d’anticorps spécifiques, dans la rate l’organe majeur de la réponse des lymphocytes B. Dans la deuxième partie, j’ai analysé les cellules T CD4 mémoires, Tfh et les lymphocytes B dans les ganglions mésentériques: un site inducteur de la réponse immunitaire intestinale, qui alimente la lamina propria (site effecteur) de la muqueuse intestinale en cellules mémoires. Étant donné l’impossibilité d’étudier ces organes profonds en particulier en phase aiguë chez l’homme, j’ai utilisé le modèle du macaque rhésus infecté par le virus de l’immunodéficience simienne (VIS). Les résultats de ces études montrent que l’évolution vers le SIDA est associée à une déplétion précoce des cellules Tfh et T CD4 mémoires dans la rate et les ganglions mésentériques. Concomitant à cela, je rapporte une déplétion des lymphocytes B mémoires dans la rate et un faible titre d’IgG anti-VIS dans le sérum. En plus, les cellules Tfh commutent leur phénotype d'effecteur mémoire vers celui de centrale mémoire associé à l’expriment de CD127 (récepteur de l’IL-7) et de T-bet (marqueur de cellules Th1). De plus, je montre que la déstructuration des organes lymphoïdes secondaires, ainsi que les cytokines environnementales comme l’IL-7 ou l’IL-27 peuvent contribuer au dysfonctionnement des cellules Tfh puisque ces dernières induisant les facteurs de transcription inhibiteurs des cellules Tfh tels que T-bet, Foxo1, Stat5 et KLF2. En conclusion, mes résultats permettent de mieux comprendre que le dysfonctionnement des lymphocytes B et l’immunodéficience dans la muqueuse intestinale sont associés à la déplétion soudaine des lymphocytes T CD4 mémoires et Tfh dans la rate et les ganglions mésentériques. Par conséquent, prévenir la perte de ces cellules pourrait être une approche thérapeutique et vaccinale prometteuse pour la neutralisation du virus et pour une meilleure immunité intestinale, afin d’empêcher la translocation bactérienne.
Since its discovery, HIV-1 has caused the death of 35 million people, and 36.9 million are living infected. Although researches have led to the development of antiretroviral therapies, which not only improve life expectation but also life quality of infected individuals, these therapies are not capable of eradicating the virus, and unfortunately there is no vaccine. The pathogenesis of HIV-1 is linked to a dysfunction of CD4 T cells that favors progression to AIDS. Therefore, given that most vaccines are based on T cell-dependent antibody production, the first part of my PhD research is devoted to understanding the impact of HIV-1 on CD4 T Follicular helper (Tfh) cells, which are essential for B cell activation and the production of specific antibodies. These cells are particularly crucial in the spleen, which is the major organ for B cell response. In the second part, I have analyzed the dynamics of memory CD4 T, Tfh and of B cells in mesenteric lymph nodes: an inductive site of the immune response that provides memory cells to the lamina propria (effector site) of the intestinal mucosa. Given the difficulties to study these deep organs, particularly during the acute phase in humans, I have used rhesus macaques infected with the simian immunodeficiency virus (SIV) to study the dynamics of Tfh cells. My results show an early depletion of splenic Tfh cells during the acute phase; a depletion that persists during the chronic phase within macaques in which the infection rapidly progresses to AIDS. Concomitantly, we report a depletion of memory B cells and low titers of anti-SIV IgG titers in these macaques. Furthermore, I observed a massive depletion of memory CD4 T, Tfh and B cells in mesenteric lymph nodes, as well as a phenotypic change of Tfh cells that become central memory cells associated with the upregulation of the expression of CD127 (IL-7 receptor). My results also show that environmental cytokines such as IL-7 and IL-27 contribute to their dysfunction as support the expression of transcription factors that inhibit Tfh cells such as T-bet, Foxo1 and Stat5. In conclusion, my results provide a better understanding of B cell dysfunction related to the early loss of the Tfh cells during HIV/SIV infection. Moreover, I hypothesize that the loss of immunity in the intestinal mucosa is due to the sudden depletion of memory CD4 T, Tfh and B cells in the mesenteric lymph nodes. Therefore, maintaining Tfh and memory CD4 T cells during the early phase of infection could be a promising therapeutic and vaccine approach for neutralizing HIV/SIV, as well as preventing bacterial translocation.

Background: Age is an important risk factor for rheumatoid arthritis (RA), which often develops in middle age. However, how age-associated changes in immunity impact RA is poorly understood. Gut microbiota are known to be involved in the pathogenesis of RA, but the effects of microbiota in older subjects remain mostly unknown. Methods: We used segmented filamentous bacteria (SFB), a gut commensal species with immunomodulatory effects, and K/BxN mice, a T cell receptor (TCR) transgenic model, to study the effect of age and microbiota on autoimmune arthritis. Comparing young and middle-aged K/BxN T cells of the same TCR specificity allows us to study T cells with an age focus eliminating a key variable: TCR repertoire alteration with age. In addition to joints, we also studied pathological changes in the lung, an important extra-articular RA manifestation. We used flow cytometry to evaluate T follicular helper (Tfh) and T helper 17 (Th17) cells, as they both contribute to autoantibody production, a key disease index in both RA and K/BxN arthritis. Results: Middle-aged K/BxN mice had aggravated arthritis and pathological changes in the lung compared to young mice. Middle-aged mice displayed a strong accumulation of Tfh but not Th17 cells, and had defective Th17 differentiation and low expression of interleukin-23, a critical cytokine for Th17 maintenance. Although a soaring Tfh cell population accompanied by robust germinal center B cell responses were found in middle-aged mice, there was decreased cycling of Tfh cells, and SFB only induced the non-Tfh cells to upregulate Bcl-6, the Tfh master transcription factor, in the young but not the middle-aged group. Finally, the accumulated Tfh cells in middle-aged mice had an effector phenotype (CD62LloCD44hi). Conclusion: Age-dependent Tfh cell accumulation may play a crucial role in the increased autoimmune disease phenotype in middle-age. SFB, a potent stimulus for inducing Tfh differentiation, fails to promote Tfh differentiation in middle-aged K/BxN mice, suggesting that most of the middle-aged Tfh cells with an effector phenotype are Tfh effector memory cells induced at an earlier age. Our results also indicate that exposure to immunomodulatory commensals may allow the young host to develop an overactive immune system reminiscent of that found in the middle-aged host.

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ciência Política e Relações Internacionais na variante de Estudos Europeus
A atribuição ao Parlamento Europeu, no âmbito do Tratado da União Europeia, de uma prerrogativa que lhe permite solicitar à Comissão Europeia a introdução de propostas de lei, representou mais um desafio ao monopólio da iniciativa legislativa de que o executivo europeu formalmente dispõe. O nosso estudo foca-se em três dimensões desse fenómeno. Primeiro, na exposição da condição de agente da Comissão Europeia e no relacionar dessa qualidade com a atribuição do monopólio da iniciativa. Em segundo lugar, debruçamo-nos sobre as dinâmicas que contribuíram para a atribuição dessa prerrogativa ao Parlamento Europeu. Por fim, avaliamos o exercício dessa prerrogativa na actualidade e exploramos as suas implicações institucionais. Perante os dados recolhidos, testamos a possibilidade do Parlamento Europeu dispor de poder de facto para introduzir propostas de lei e tecemos algumas considerações relativamente ao futuro da prática da iniciativa legislativa na União Europeia.

The essential protein SecA plays a key role in post-translational protein secretion across the inner membrane of Escherichia coli. However, the extent of SecA-requirement in co-translational insertion of inner membrane proteins mediated by the signal recognition particle and its membrane-associated receptor FtsY is not clear. To inactivate SecA function a seven amino acid sequence that is specifically recognised by tobacco etch virus protease was introduced into the secA sequence. The resulting secA-constructs were integrated into the chromosome at the 1 attachment site via a specific lambda phage. Chromosomal wild-type secA was then eliminated by using the 1 Red recombinase system. Cleavage of nascent SecA 195 by a trigger factor-TEV protease hybrid protein resulted in effective inhibition of SecA activity. In addition, SecA830 under the control of the lacpromoter could be depleted in cells not supplemented with the inducer IPTG. Biotinylation assays showed that inactivation of SecA via site-specific proteolysis or depletion of SecA affected the proper secretion of the periplasmic protein alkaline phosphatase as well as the proper biogenesis of the inner membrane proteins FtsQ and MalF274. Similar experiments with Ffh- and FtsY-depletion strains confirmed the involvement of these proteins in inner membrane protein assembly. DNA macroarray technique was used to compare gene expression profiles of cells depleted either of Ffh, FtsY or SecA with the profiles obtained from undepleted cells. While depletion of Ffh and FtsY resulted in the induction of heat shock genes, depletion of SecA induced the expression of the phage-shock protein operon. The results strengthen the notion that SecA not only plays the central role in post-translational secretion of preproteins, but is also extensively involved in co-translational protein translocation. In addition, DNA array analysis indicated a possible involvement of cellular chaperones in targeting of inner membrane proteins.

At the University of Pretoria's Fluoropolymer Laboratory, an important long-term project is the development of a waste polytetrafluoroethylene (PTFE) depolymerisation process where TFE can be produced, purified and polymerised to reproduce pure PTFE. At the start of this project, the process consisted of a batch depolymerisation system, a sub-zero distillation column, and a polymerisation reactor system. The batch depolymerisation system could not produce enough gas per session to operate the downstream processes efficiently. The main aim of this investigation was to adapt the batch depolymerisation system to enable continuous depolymerisation by designing, implementing and testing a continuous PTFE screw feeder. With the screw feeder in place, the operating limits, with regard to temperature, pressure, and Teflon® PTFE 807N feed rate, were determined. The effects of temperature and pressure on the tetrafluoroethylene (TFE), hexafluoropropylene (HFP) and octafluorocyclobutane (OFCB) fractional composition were examined and the optimum operating conditions to maximise these products were determined statistically. An investigative approach was used in designing the hopper system. The optimum hopper wall angle and Teflon® PTFE 807N feed mixture was determined experimentally by testing four hopper angles, pure Teflon® PTFE 807N, and two Teflon® PTFE 807N mixtures (Teflon® PTFE 807N mixed with larger, compressed Teflon® PTFE 807N, particles in a 70:30 wt % and 50:50 wt % ratio) and two motor speeds. At all of the hopper angles and Teflon mixture configurations, rat-hole formation prevented the feeder from producing a constant flow rate. A hopper wall angle of 20° (to the vertical) together with plain Teflon® PTFE 807N were selected, as these two variables together helped to delay the formation of rat-holes the most. A stirrer was inserted in the hopper to negate the rat-holing problem. The continuous feeder was successfully designed, manufactured, calibrated, and installed. The feeder consists of a wedge-shaped hopper with a constant pitch, a tapered shaft screw and is capable of providing a maximum Teflon® PTFE 807N flow rate of approximately 20 g·min-1 for up to 40 min. Experimental test runs of the continuous depolymerisation system indicated that the minimum operating reactor temperature was 650 °C due to heat transfer and or rate of reaction limitations. The maximum flow rate of Teflon® PTFE 807N was determined to be 11 g·min-1 for the current reactor system. The maximum operating temperature and pressure were limited to 750 °C and 40 kPa, respectively, to avoid operating conditions that could lead to the increased production of PFIB. A three-level full factorial experimental design was used to determine the temperature and pressure effects on the fractional distribution of TFE, HFP, and OFCB under steady operating conditions. For pressure control purposed no carrier gas was used. The PTFE flow rate and experimental run time were kept constant at 11 g·min-1 and 15 min, respectively. The pressure in the system was regulated manually by constricting the flow of product gas out of the system. A maximum TFE mole percentage of 97 % was achieved at operating conditions of 650 °C and 2 kPa. The maximum HFP mole percentage (31 %) was observed at operating conditions of 750 °C and 20 kPa. A maximum of 55 % was observed at 750 °C and 40 kPa for OFCB. Statistical analysis of the continuous depolymerisation results indicate that TFE formation is highly sensitive to changes in pressure, with higher TFE yield fractions achieved at low pressures. The production of OFCB is highly sensitive to pressure, whereas the formation of HFP is equally affected by pressure and temperature changes. However, changes in pressure have a larger effect on the HFP production than temperature when operating at pressures lower than approximately 20 kPa. At higher pressures the sensitivity has the inverse affect, with temperature having a larger effect. As opposed to TFE, an increase in temperature and pressure leads to an increase in the HFP and OFCB concentration. To achieve a TFE mole percentages of 95 % and higher, the operating temperature of the system has to be kept in the range of 650 °C 720 °C, together with a system pressure of 2 kPa or less. Within the operating range of 730 °C 750 °C and 35 kPa 40 kPa a mole percentage of 50 % and higher can be expected for OFCB. A mole percentage of 19 % and higher can be expected for HFP in the operating range of 744 °C 750 °C and 32 kPa 40 kPa. It was determined through a kinetic analysis of the system, that the residence time of the product gas in the reactor has a large effect on the production of HFP, with an increase in residence time leading to a sharp increase in the HFP concentration and a decrease in the OFCB and TFE concentrations. Analysis of the determined product specific kinetics indicate that the predominant HFP production pathway at low residence times (< 3 s) is via the reaction of TFE with difluorocarbenes. At higher residence times the dominant reaction pathway is the dissociation OFCB.
Dissertation (MEng)--University of Pretoria, 2016.
Chemical Engineering
MEng
Unrestricted

Durante le infezioni virali, le risposte adattative umorale e cellulare possono trovarsi in uno stato di coesistenza competitiva ed equilibrio per massimizzare l’eliminazione del virus. Tuttavia, in alcuni casi, questo delicato equilibrio può essere alterato, inducendo una risposta a prevalere sull’altra. Ad esempio, l'infezione da virus della stomatite vescicolare (VSV) induce risposte anticorpali neutralizzanti precoci e potenti, mentre l'infezione da virus della coriomeningite linfocitica (LCMV) induce forti risposte cellulari, ma deboli risposte neutralizzanti. Dati preliminari ottenuti nel nostro laboratorio hanno mostrato che lo squilibrio si osserva anche a livello delle risposte dei linfociti T CD4, con VSV che induce una forte polarizzazione verso T helper follicolari (TFH) che supportano le risposte neutralizzanti e LCMV al contrario che promuove la differenziazione verso T helper 1 (TH1). In questo progetto abbiamo analizzato i fattori determinanti la differenziazione delle cellule T CD4 che si sviluppano nel contesto delle infezioni virali. L'analisi delle nicchie di priming di VSV e LCMV ha portato all'identificazione della regolazione spaziotemporale dell'espressione di interferone (IFN) di tipo I come regolatore critico della polarizzazione delle cellule TFH. In particolare, nel contesto dell'infezione da VSV, la secrezione precoce di IFN di tipo I viene sentita dalle cellule dendritiche, che in risposta producono IL-6, inducendo la polarizzazione delle cellule TFH. Diversamente, l'esposizione tardiva all'IFN di tipo I nel contesto dell'infezione da LCMV determina una ridotta differenziazione delle cellule TFH. In aggiunta, abbiamo osservato una profonda eterogeneità tra le cellule TH1 che si sviluppano in risposta all'infezione da LCMV, che comprendono due sottopopolazioni: una che esprime TCF-1 e una che esprime GzmB. Abbiamo dimostrato che lo sviluppo di queste TH1 è indipendente da IL-12 e IFN di tipo I. Invece, abbiamo identificato l'IFN-γ come interruttore molecolare critico per la differenziazione delle cellule T CD4, spostando l'equilibrio verso la differenziazione TH1 a scapito dello sviluppo delle TFH e delle risposte delle cellule B del centro germinativo. Il meccanismo molecolare è ancora oggetto di studio, ma i dati preliminari suggeriscono un ruolo dell'IFN-γ nel sopprimere il differenziamento della popolazione TCF-1 verso TFH. I nostri risultati fanno luce sui nuovi meccanismi alla base della produzione inefficiente di anticorpi neutralizzanti in risposta a virus non citopatici come LCMV.
Although humoral and cellular immunity upon viral infections usually co-exist, sometimes one of the two responses emerges and is responsible for most of the antiviral activity. For example, vescicular stomatitis virus (VSV) infection induces early and potent neutralizing antibody (nAb) responses, whereas lymphocytic choriomeningitis virus (LCMV) infection induces strong cellular responses, but weak nAb responses. Preliminary data obtained in our laboratory showed that unbalance is observed also at the level of CD4 T cells responses, with VSV inducing strong TFH polarization that support nAb responses, and LCMV in contrast promoting TH1 differentiation. Here we dissected the determinants of CD4+ T cell differentiation upon viral infections. Analysis of the VSV and LCMV priming niches led to identification of the spatiotemporal regulation of type I IFN expression as a critical regulator of antiviral TFH cell polarization. In particular, in the context of VSV infection, early type I IFNs sensing by DCs induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN in the context of LCMV infection resulted in impaired TFH cell differentiation. Moreover, we unveiled a profound heterogeneity among the TH1 cells that arise in response to LCMV infection, that comprise a TCF-1+ subset and a GzmB+ subset. We proved that the development of these TH1 is independent of IL-12 and type I IFNs. Instead, we identified IFN-γ as a critical molecular switch of CD4+ T cell differentiation, tipping the balance towards TH1 differentiation at the expense of TFH development and GC-B cell responses. The molecular mechanism is still under investigation, but preliminary data suggest a role of IFN-γ in suppressing the commitment of the TCF-1+ population into TFH. Our results shed light on new mechanisms underlying the inefficient nAbs production in response to non-cytopathic viruses such as LCMV.

Humoral immunity provides protection against subsequent infections. Antigen-specific, high-affinity, class-switched antibodies are produced by B cells through rounds of proliferation, B cell receptor rearrangement and selection in the germinal centres (GC). T cells play an essential and indispensable role in this process and in the recent years the term T follicular helper cells (TFH) was coined to describe this cell subset. The aim of my thesis is to investigate whether there is more than one type of T cells within the TFH population and whether it has important functional consequences. Firstly, I use sheep red blood cell immunisation (SRBC) and Salmonella enterica infection to show phenotypical differences between TFH expressing high and low level of surface molecule PD-1. In order to investigate the relationship between different TFH populations gene profiling was carried out on the microarray platform. Detailed transcriptome analysis revealed the discrete nature of isolated TFH cell subsets and provided an overview of their genetic landscape. Secondly, I have investigated the dependence of TFH subsets on cognate interactions with B cell in SRBC model by generating BM chimeras. I have demonstrated that generation of PD-1HI TFH, but not of PD-1LO TFH, depends on antigen presentation by B cells. Furthermore, I have shown that provision of wild-type but not MHC II knock-out B cells rescues PD-1HI formation in BM chimeras after SRBC immunisation. Finally, I have explored plasticity within TFH subsets and showed that none of the populations is in a terminally differentiated state, as they can convert into one another. Thirdly, experiments with S. enterica model revealed that the absence of PD- 1HI TFH is independent of the splenic architecture disruption present within the first week of the response. Surprisingly, co-immunisation studies showed that PD-1HI population is not only present but even enhanced in the group which received both SRBC and S. enterica when compared to single immunisations. The work presented in the thesis documents that there is a significant and previously unappreciated heterogeneity within TFH subset. This knowledge is important for designing optimal vaccine strategies and treating autoimmune diseases, as in both processes the antibody production plays a crucial role and its manipulation (either enhancing or blocking antibody production, respectively) can significantly improve clinical interventions.

Mon travail, lors de ma thèse, avait pour but d’étudier la différentiation des lymphocytes Tfh, ainsi que leur fonction et leur régulation dans la pathogenèse de la DA. Pour cela, j’ai utilisé un modèle murin précédemment établi au sein de notre laboratoire consistant en l’application topique de MC903 (un analogue de la vitamine D3) induisant la production de TSLP par les kératinocytes et, par conséquence, la réponse immunitaire Th2 et la pathogenèse de la DA. mon travail doctoral s’est porté sur la différentiation des lymphocytes Tfh, leur production cytokinique ainsi que la formation des centres germinatifs dans le contexte d’un modèle murin de DA induite par le MC903. Mes études ont démontré un rôle critique joué par TSLP dans la réponse Tfh et ont exploré le rôle potentiellement joué par les cellules dendritiques langerine+ et la signalisation OX40L dans le développement des réponses Tfh et de type 2. Ceci nous a permis d’approfondir nos connaissances concernant les mécanismes sous-tendant la réponse immunitaire de type 2 dans la pathogenèse de la DA. Dans la deuxième partie de ma thèse, nous avons examiné le rôle de MC903 dans la régulation de l’inflammation due au psoriasis, en utilisant un modèle de psoriasis induit par l’Aldara. Nous avons montré que MC903 inhibe l’axe 23/IL-17/IL-22 chez les souris souffrant de psoriasis. De plus, cette inhibition semblait être dose-dépendante. Nous avons en outre exploré le rôle de TSLP et VDR dans la médiation de cet effet dû au MC903
My thesis aimed at studying the Tfh cell differentiation, function and regulation in AD pathogenesis. To this aim, I employed our previously established AD mouse model in which MC903 (a vitamin D analog) topical treatment on the skin induces TSLP production bykeratinocytes, promotes Th2 cell response and drives the pathogenesis of AD. my thesis work investigated Tfh cell differentiation, its cytokine expression and germinal center formation using MC903-induced AD mouse model. By exploring the role of TSLP,Langerin+ DCs and OX40L signaling in Tfh cell differentiation and regulation, my study provides novel insights into the mechanisms underlying the type 2 immune response in AD pathogenesis. In the second part of my study, we examined the role of MC903 in regulating the psoriatic inflammation using Aldara-induced psoriasis model. We showed that MC903 inhibited IL-23/IL-17/IL-22 axis in mouse psoriasis. Moreover, this inhibition exhibited a dose-dependent manner. We further explored the role of TSLP and VDR in mediating such effect of MC903

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Mastoparanos são peptídeos helicoidais, anfipáticos e catiônicos que apresentam diversas funções biológicas, entre elas temos a ação antimicrobiana, que está relacionada à sua afinidade por membranas aniônicas de bactérias e sua capacidade lítica. Recentes estudos têm mostrado que a formação de poros em membranas é facilitada pela agregação de peptídeos carregados. Esta situação favoreceria a hipótese de que a formação de poro é essencialmente similar a eletroporação molecular. Neste trabalho investigamos a estabilidade de um dímero do Eumenine Mastoparan-AF, um membro catiônico (+4) da família dos mastoparanos, em água e mistura TFE-água, mimetizando meio aquoso e meio membranar, respectivamente. Particular atenção foi colocada nas interações eletrostáticas de grupos carregados e polares, principalmente naqueles que participam de ligações de hidrogênio entre os dois peptídeos e na hidratação da cadeia principal e cadeias laterais apolares. Uma estrutura dimérica representativa foi inicialmente obtida por um método de docking rígido e submetida às simulações de dinâmica molecular usando o pacote GROMACS. Resultados de 50 ns de simulação em água mostram uma perda parcial do conteúdo helicoidal dos peptídeos e a estrutura dimérica se desestrutura devido às interações desfavoráveis dos resíduos hidrofóbicos com a água. Por outro lado, simulações em mistura TFE-água mostram que o dímero é estável durante o tempo observado, porque as moléculas de TFE se agrupam ao redor de resíduos hidrofóbicos criando um meio apropriado que protege as ligações de hidrogênio intra- e inter-peptídeos. Surpreendentemente, parece que a repulsão eletrostática não é a principal razão para a desagregação do dímero, o que reforça a importância da.
Mastoparans are helical, amphipathic and cationic peptides that display many biological functions, among which is the antimicrobial activity, which is related to its affinity for anionic membranes of bacteria and its lytic capacity. Recent studies have shown that pore formation on membranes is facilitated by the aggregation of charged peptides. This situation would favor the hypothesis that pore formation is essentially similar to the molecular electroporation. In this work, we investigate the stability of a dimer of the Eumenine Mastoparan-AF, a cationic (+4) member of the Mastoparan family, in water and TFE-water mixture, mimicking aqueous and membrane environments, respectively. Particular attention have been put on the electrostatic interactions of charged and polar groups, mainly those participating of hydrogen bonds between the two peptides and on the hydration of the backbone and apolar side chains. A representative dimer conformation was initially obtained by a rigid docking procedure and submitted to molecular dynamics simulations using the GROMACS package. Results of 50 ns of simulation in water show a partial loose of the helical content of the peptides and the dimer structure breaks down due to unfavorable interactions of hydrophobic residues with water. On the other hand, simulations in TFE-water mixture show the dimer is stable in the running time, because TFE molecules assemble around hydrophobic residues creating a suitable environment that protect the intra- and inter-peptides hydrogen bonds. Surprisingly, it seems that electrostatic repulsion is not the main reason for disaggregation of the dimer what reinforces both the importance of aggregation and the molecular electroporation mechanism for pore formation.

A principal função dos linfócitos T CD4+ é fornecer apoio a outras células no sentido de gerar uma resposta imunitária eficiente. As interações entre as células T e B são essenciais para a produção de respostas humorais, sendo que foi recentemente demonstrado que as células T foliculares auxiliares (Tfh) desempenham um papel crucial neste processo. Caracteristicamente, expressam o fator de transcrição Bcl-6, o recetor de quimiocinas CXCR5 e o marcador de superfície PD-1. A expressão destes marcadores é única e fundamental para que estas células possam aceder ao folículo de células B, onde orientam as reações no centro germinativo (GC), levando à consequente mudança de isótipo, maturação da afinidade, produção de anticorpos de alta afinidade e células B de memória. Neste projeto, foram testadas duas hipóteses opostas no sentido de caracterizar fenotipicamente as células Tfh. Propomos investigar se estas são especializadas no fornecimento de auxílio do tipo Th1 ou Th2, que designamos de células hipotéticas "Tfh1" e "Tfh2" (Hipótese 1) ou se são uma subpopulação genérica que responde igualmente na presença de diferentes antigénios, células Tfh (Hipótese 2). Deste modo, murganhos C57BL/6J e Balb/c foram imunizados na almofada plantar da pata traseira, utilizando proteína Ovalbumin (OVA) combinada com diferentes tipos de adjuvantes: CpG ODNs isoladamente e em combinação com TiterMax® Gold (TMX), Sigma Adjuvant System (SAS) e Montanide ISA 720 VG, testados como adjuvantes tipo 1, e por sua vez Incomplete Freund’s Adjuvant (IFA) e Alum experimentados como adjuvantes do tipo 2. A técnica de ELISA permitiu determinar no soro dos murganhos o tipo de resposta gerada, através da medição de imunoglobulinas específicas para OVA (IgG2a para Th1, IgG1 e IgE total para Th2). CpG ODNs e IFA foram considerados como os adjuvantes mais apropriados para induzir respostas Th1 e Th2, respetivamente. Células T que reconhecem especificamente OVA foram colhidas de murganhos OT-II Rag-/- e DO11.10 Rag-/- e transferidas para murganhos congénicos. De seguida, procedeu-se à imunização tal como descrito acima. Os nódulos linfáticos drenantes foram recolhidos no pico da reação do centro germinativo (11 dias após imunização), assim como as células Tfh específicas para OVA (CD4+CD44+ CXCR5+PD-1+ Thy1.2+Vβ5+Vα2+/DO11.10+) e as células T auxiliares ativadas específicas para OVA (CD4+CD44+CXCR5-PD-1- Thy1.2+Vβ5+Vα2+/DO11.10+). A caracterização molecular destas populações de células T está a ser analisada através da sequenciação dos seus transcritos pela técnica de RNA-sequencing. Além disso, a expressão de marcadores de Th1 e Th2 em células Tfh foi analisada através de citometria de fluxo e Reação em Cadeia da Polimerase quantitativa por Transcrição Reversa (RT-qPCR). Neste estudo, foi demonstrado que as células Tfh co-expressam Bcl-6 e T-bet e também produzem IFN-γ, quando sensibilizadas com OVA-CpG ODNs, características concordantes com os marcadores fenotípicos de uma célula Tfh e célula Th1. A expressão de Gata-3 (marcador Th2) só foi detetada sob estimulação IFA-OVA, embora em níveis mais baixos do que as determinadas para T-bet.
The major function of CD4+ T cells is to provide help to other lymphocytes to mount an efficient immune response. T and B cell interactions are essential for humoral responses and it was recently shown that T follicular helper (Tfh) cells play a crucial role in this process. They characteristically express the transcription factor Bcl-6, chemokine receptor CXCR5 and PD-1. These markers are unique as their expression is pivotal to acquire access to the B cell follicle and drive germinal centre (GC) reactions, leading to isotype switching, affinity maturation, and production of high affinity antibodies and memory B cells. In this project, two competing hypothesis investigating the phenotype of Tfh cells were tested. We propose to dissect whether Tfh cells are specialized in providing Th1 or Th2 help, which we call putative “Tfh1” and “Tfh2” cells (hypothesis 1), or if they are a more generic Th subset that responds equally in the presence of different antigens, which we designate as Tfh cells (hypothesis 2). Therefore, we immunized C57BL/6J and Balb/c mice in the footpad using Ovalbumin (OVA) protein combined with different adjuvant types: CpG ODNs only and combined with TiterMax® Gold (TMX), Sigma Adjuvant System (SAS) and Montanide ISA 720 VG, as type 1 adjuvant, and Incomplete Freund’s Adjuvant (IFA) and Alum as type 2 adjuvants. Using ELISA assays to determine the type of response generated by measuring serum immunoglobulins of distinct clones (OVA-specific IgG2a for Th1 and OVA-specific IgG1 and total IgE for Th2), we considered CpG ODNs and IFA as the most appropriate adjuvants to induce Th1 and Th2 responses, respectively. OVA-specific cells were transferred from OT-II Rag-/- and DO11.10 Rag-/- mice into congenic mice subsequent to immunization as described above. Draining LNs were collected at the peak of the GC reaction (day 11 post-immunization) and OVA-specific Tfh cells (CD4+ CD44+ CXCR5+PD-1+ Thy1.2+Vβ5+Vα2+/DO11.10+) and OVA-specific activated-Th cells (CD4+ CD44+ CXCR5-PD-1- Thy1.2+Vβ5+Vα2+/DO11.10+) were sorted. The molecular signature of these T cell populations is being analysed via RNA-Sequencing. Moreover, the expression of Th1 and Th2 markers on Tfh cells was investigated via flow cytometry and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). In this study, it could be shown that Tfh cells of mice immunized with OVA-CpG ODNs co-expressed Bcl-6 and T-bet and also produced IFN-γ, both concordant features with the phenotypic markers of a Tfh cell and of a Th1 cell. As for the expression of Gata-3, it has only been detected in mice under IFA-OVA stimulation, even though at levels lower than the ones determined for T-bet.

Orientador: José Roberto Ruggiero
Banca: Mário Sérgio Palma
Banca: André Farias de Moura
Resumo: Mastoparanos são peptídeos helicoidais, anfipáticos e catiônicos que apresentam diversas funções biológicas, entre elas temos a ação antimicrobiana, que está relacionada à sua afinidade por membranas aniônicas de bactérias e sua capacidade lítica. Recentes estudos têm mostrado que a formação de poros em membranas é facilitada pela agregação de peptídeos carregados. Esta situação favoreceria a hipótese de que a formação de poro é essencialmente similar a eletroporação molecular. Neste trabalho investigamos a estabilidade de um dímero do Eumenine Mastoparan-AF, um membro catiônico (+4) da família dos mastoparanos, em água e mistura TFE-água, mimetizando meio aquoso e meio membranar, respectivamente. Particular atenção foi colocada nas interações eletrostáticas de grupos carregados e polares, principalmente naqueles que participam de ligações de hidrogênio entre os dois peptídeos e na hidratação da cadeia principal e cadeias laterais apolares. Uma estrutura dimérica representativa foi inicialmente obtida por um método de docking rígido e submetida às simulações de dinâmica molecular usando o pacote GROMACS. Resultados de 50 ns de simulação em água mostram uma perda parcial do conteúdo helicoidal dos peptídeos e a estrutura dimérica se desestrutura devido às interações desfavoráveis dos resíduos hidrofóbicos com a água. Por outro lado, simulações em mistura TFE-água mostram que o dímero é estável durante o tempo observado, porque as moléculas de TFE se agrupam ao redor de resíduos hidrofóbicos criando um meio apropriado que protege as ligações de hidrogênio intra- e inter-peptídeos. Surpreendentemente, parece que a repulsão eletrostática não é a principal razão para a desagregação do dímero, o que reforça a importância da.
Abstract: Mastoparans are helical, amphipathic and cationic peptides that display many biological functions, among which is the antimicrobial activity, which is related to its affinity for anionic membranes of bacteria and its lytic capacity. Recent studies have shown that pore formation on membranes is facilitated by the aggregation of charged peptides. This situation would favor the hypothesis that pore formation is essentially similar to the molecular electroporation. In this work, we investigate the stability of a dimer of the Eumenine Mastoparan-AF, a cationic (+4) member of the Mastoparan family, in water and TFE-water mixture, mimicking aqueous and membrane environments, respectively. Particular attention have been put on the electrostatic interactions of charged and polar groups, mainly those participating of hydrogen bonds between the two peptides and on the hydration of the backbone and apolar side chains. A representative dimer conformation was initially obtained by a rigid docking procedure and submitted to molecular dynamics simulations using the GROMACS package. Results of 50 ns of simulation in water show a partial loose of the helical content of the peptides and the dimer structure breaks down due to unfavorable interactions of hydrophobic residues with water. On the other hand, simulations in TFE-water mixture show the dimer is stable in the running time, because TFE molecules assemble around hydrophobic residues creating a suitable environment that protect the intra- and inter-peptides hydrogen bonds. Surprisingly, it seems that electrostatic repulsion is not the main reason for disaggregation of the dimer what reinforces both the importance of aggregation and the molecular electroporation mechanism for pore formation.
Mestre

A malária permanece um sério problema de saúde em países subdesenvolvidos. O estágio sanguíneo da infecção é responsável por todos os sintomas associados com a malária. Recentemente, tem sido mostrado que receptores imunes inatos são capazes de detectar sinais de dano, tais como a adenosina trifosfato ATP. O receptor P2X7 detecta altas concentrações de ATP extracelular. Ao avaliarmos a parasitemia e os parâmetros clínicos da doença em camundongos C57BL/6 e P2X7-/-, observamos uma semelhança em ambos os grupos até o dia 7 p.i., mas após este período os camundongos P2X7-/- tiveram dificuldade de controlar a parasitemia e restaurar os parâmetros clínicos. O ineficiente controle da parasitemia durante o período agudo e crônico em camundongos P2X7-/- foi associado com a baixa produção de IFNγ. Além disso, o receptor P2X7 aumenta a expressão de T-bet em células Th1 e controla o número de células Tfh. Este estudo mostra que o equilíbrio mediado pelo receptor P2X7 entre os fatores de transcrição Bcl-6 e T-bet ajusta a imunidade celular e humoral na malária.
Malaria remains a serious healthcare problem in developing countries. The blood stage of infection is responsible for all symptoms associated with malaria. Recently, it has been shown that innate immune receptors are able to detect signals as adenosine triphosphate (ATP). P2X7 receptor detects high levels of extracellular ATP. Evaluating the parasitemia and clinical parameters in C57BL/6 (B6) and P2X7-/- mice, we observed a similarity in both groups to day 7 p.i., but after this period the P2X7-/- mice had difficulty in controlling the parasitemia and restoring the clinical parameters. The inefficient parasite control in acutely and chronically infected P2X7-/- mice was associated with low production of IFNγ. Furthermore, P2X7 receptor increases the expression of T-bet in Th1 cells and controls the Tfh cell number. This study provides a new insight into immunology by showing that the balance between T-bet and Bcl-6 transcriptional factors tunes the cellular and humoral immunity in malaria.

TFH cells have been identified as a new T helper subset specialized to regulate the development of effector and memory B cells and long-lived plasma cells. The interaction between TFH and B cells leads to the activation of B cells and germinal centers (GC) formation. Considering the importance of TFH for B cell antibody response, novel vaccine adjuvants and intranasal vaccines to boost TFH number or function may be an attractive vaccination strategy to enhance vaccine efficacy in humans. Adenotonsillar tissues are major parts of nasopharyngeal associated lymphoid tissues (NALT) and they are important in response to upper respiratory tract pathogens and intranasal vaccination. This PhD project investigated the frequencies of TFH in human NALT and PBMC in children and adults. The effects of CpG-DNA and live attenuated influenza vaccine (LAIV) on TFH in human NALT and the TFH-mediated B cell immunity to influenza virus were studied. The importance of the cytokine IL-21 and plasmacytoid dendritic cells (pDC) in TFH cell-mediated B cell antibody production was also investigated. Adenotonsillar MNC and PBMC were isolated from adenotonsillar tissues and peripheral blood respectively. TFH (CD4+ CXCR5high ICOShigh) numbers and function were analysed by flowcytometry and intracellular cytokine staining. Purified TFH (CD4+ CXCR5hi) and non-TFH cells (CD4+ CXCR5-) were co-cultured with B cells in the presence of influenza virus antigen and CpG-DNA or of LAIV. Purified pDC were added to the TFH-B cell co-culture to study their importance in TFH -mediated B cell antibody production. Haemagglutinin (HA)-specific antibody production was analysed by ELISA and ELISpot assay. IL-21 receptor blocking by neutralization was used to study the importance of IL-21 in TFH-mediated B cell antibody production. A prominent number of TFH were found in human NALT which were considerably higher than in PBMC. There was an age-associated difference in TFH numbers in NALT and BPMC, i.e. the mean TFH number was higher in children than in adults. TFH in NALT were shown to express high levels of IL-4, IL-10 and IL-21 and that were important for B cell antibody production. A good correlation between the numbers of GC B cell and TFH in NALT was seen. Co-culture of purified TFH but not non-TFH with B cells promoted antibody production. Stimulation of adenotonsillar MNC by CpG-DNA significantly increased TFH number and that was correlated with HA-specific antibody production following influenza antigen stimulation. Co-incubation of TFH-B cell with pDC enhanced the CpG-DNA-mediated antibody production. We also found that stimulation with LAIV significantly increased TFH number and that was correlated with HA-specific antibody production. Blocking the IL-21R significantly reduced the number of TFH that was correlated with a significant reduction of HA-specific antibody production. Enhancing vaccine immunogenicity through modulation of TFH numbers or function in human NALT using immunological adjuvants such as CpG-DNA and through intranasal vaccination may be an effective vaccination strategy against respiratory pathogens.

La réponse immunitaire humorale désigne l’ensemble des processus menant à la production d’anticorps suite à une stimulation du système immunitaire. Les lymphocytes T folliculaires régulateurs (Tfr) forment une population essentielle dans le contrôle de l’immunité humorale. Dotés de fonctions régulatrices comme les lymphocytes T régulateurs conventionnels (Treg), les Tfr joueraient un rôle majeur dans les mécanismes de régulation de la production d’anticorps suite à une stimulation. Ils pourraient, par exemple, contrôler l’aide apportée par les lymphocytes T folliculaires helpers (Tfh) aux cellules B leur permettant de se différencier en plasmocytes producteurs d’anticorps dans une structure hautement spécialisée appelée centre germinatif (CG). Suite au constat de la non-réponse des Tfr à l’interleukine (IL)-2, nous avons observé l’absence d’expression de la sous-unité du récepteur à l’IL-2 (CD25) sur ces cellules et ainsi donné de nouvelles bases à la caractérisation phénotypique des Tfr. Cette redéfinition restrictive de la population cellulaire a permis une description plus approfondie de ces cellules et la découverte d’un axe de régulation du CG dépendant de l’IL-1ß. La dualité de régulations observée entre d’une part l’axe Treg - lymphocytes T effecteurs contrôlé par l’IL-2 en dehors du CG et d’autre part l’axe Tfr - Tfh contrôlé par l’IL-1ß dans le CG nous a amenés à nous poser la question de l’origine et de la spécificité des Tfr en partie élucidée par une analyse du répertoire de ces populations. Nous avons mis en évidence une similarité en matière de distribution et caractéristiques globales des répertoires des cellules folliculaires qu’elles soient régulatrices (Tfr) ou non (Tfh). Il est également apparu une proximité de spécificités entre le répertoire des Treg et des Tfr confortant l’hypothèse d’une origine commune de ces deux populations. Ce travail de thèse a permis la découverte d’éléments importants de la biologie des Tfr, population impliquée dans le contrôle des régulations humorales. Il ouvre des perspectives relatives au contrôle de la production d’anticorps tant sa limitation - dans le contexte de maladies auto-immunes - que son exploitation dans le développement de stratégies vaccinales
The immune humoral response offers the organism an efficient protection through the production of antibodies following an immune stimulation. Follicular regulatory T cell (Tfr) is an essential subset in the control of humoral immunity. These cells share with conventional regulatory T (Treg) cells regulatory functions and should play a major role in the control of antibody production following stimulation. As T follicular helper (Tfh) cells help is essential in the differentiation of B cell into antibody-producing plasma cells, one of the possible mechanisms of Tfr’s control could be the limitation of the Tfh cells’ help to the B cells. As a consequence of the non-response of Tfr cells to interleukin (IL)-2, we thoroughly revealed the CD25- phenotype of Tfr cells thus redefining the subset. This stringently-selected population allowed a fine-tuned characterization of Tfr cells and the discovery of an IL-1β axis regulating the germinal center responses. The dual regulation of T cells in secondary lymphoid organs, one between Treg and Teff cells regulated by IL-2 outside germinal centers and the other between Tfh and Tfr cells regulated by IL-1 inside GCs brought us to question the origin and specificity of Tfr cells. We partially answered this with a high-resolution analysis of these populations’ repertoires. We highlighted a similarity in the distributions and global characteristics of the follicular cells’ repertoires regardless their regulatory (Tfr) or not (Tfh) phenotype. We also brought out the major sharing between Treg and Tfr repertoires underpinning the hypothesis of a common origin for these populations. This work has disclosed important aspects of Tfr cells’ biology, a fundamental subset in the control of humoral immune responses. It opens many perspectives including the control of antibody production, negatively in the context of autoimmune diseases or its positive exploitation to enhance vaccine efficacy

L’aide fournie aux lymphocytes B par les lymphocytes T CD4+ est cruciale pour une réponse humorale de qualité. Les lymphocytes T helper folliculaires (Tfh) jouent un rôle majeur dans l’aide apportée aux lymphocytes B au sein des centres germinatifs et peu de choses sont connues sur la capacité d’aide des autres populations de cellules T CD4+. Le but de notre étude est d’évaluer la capacité d’aide aux lymphocytes B des lymphocytes T helper de type 2 (Th2), une population considérée, à l’origine, comme responsable de l’aide apportée aux lymphocytes B in vivo ainsi que d’analyser le rôle de l’axe IL6 / STAT3 dans la différenciation des lymphocytes Tfh et leur plasticité phénotypique. Nous montrons que les lymphocytes Th2 co-expriment des formes actives des facteurs de transcription STAT6 et STAT3 et que l’expression de STAT3 est requise pour la fonction d’aide aux lymphocytes B des lymphocytes Th2. Nous avons également pu montrer que la voie de signalisation IL-6 / STAT3 durant le développement des lymphocytes Tfh s’oppose à l’expression des gènes associés au programme de différenciation Th2.
Option Biologie moléculaire du Doctorat en Sciences
info:eu-repo/semantics/nonPublished

La tesi ha l’obiettivo di dimostrare l’opportunità di realizzare nelle Marche il Centro di Imprenditorialità Diffusa (CID) cioè un luogo dove stimolare e aiutare le imprese, specialmente le micro imprese (95% del tessuto produttivo regionale), ad affrontare sfide legate alla globalizzazione e alla trasformazione digitale dei modelli economici (e-transformation). Per fare questo, si ricorre all’analisi economica del diritto pubblico, metodo interdisciplinare per studiare i fondamenti logico-economici delle regole giuridiche e per valutare i loro effetti. In particolare, partendo dal piano nazionale Industria 4.0, lo studio punta a mettere le Marche davanti alla possibilità storica di inaugurare un nuovo modello di sviluppo della manifattura del XXI secolo. Ciò appare tanto più vero se si considerano le misure che riguardano la politica industriale europea, materia di coordinamento tra Stato e UE (art. 173 TFUE), alla quale lo studio guarda costantemente. Si vedrà come il livello normativo più vicino al contesto territoriale di riferimento debba attivarsi con forza per raggiungere determinati risultati. Con adeguate politiche pubbliche, quindi, il caso Marche (laboratorio ideale in quanto regione imprenditoriale, manifatturiera, esportatrice e con popolazione longeva) può diventare modello in Italia e in Europa: la tesi si sofferma sui dettagli di normativa e il tipo di organizzazione capaci di implementare un modello (CID) che tenga conto delle peculiarità del tessuto produttivo in un Paese seconda potenza manifatturiera in Europa. L’ente pubblico deve realizzare un lavoro sartoriale, considerando cinque categorie di imprese. Per affrontare questa complessità serve un luogo di contaminazione (CID) ed un ente pubblico pro attivo, catalizzatore, capace di agire su tre livelli normativi (regionale, nazionale, europeo), guidando un processo in cui, vista l’e-transformation, aggiungere una sola nuova regola: stimolare le imprese a guardare al mondo come il proprio mercato.
The thesis aims to demonstrate the opportunity to realize in le Marche the Centro di Imprenditorialità Diffusa (CID), i.e. a physical place to stimulate and help businesses, especially micro-enterprises (95% of the regional entrepreneurial system), in order to face challenges connected to globalization and the digital transformation of economic models (e-transformation). To do this, we consider the economic analysis of public law, an interdisciplinary method to study the logical-economic foundations of legal rules and to evaluate their effects. In particular, starting from the national plan Industria 4.0, my work wants to put le Marche in front of an historical possibility: proposing a new model to develop the manufacturing in the XXI century. Moreover, this is reinforced about measures concerning European industrial policy, a matter coordinated between the State and the EU (Article 173 TFEU) to which the study is constantly looking. We will see how local level it should be strongly activated to achieve certain results. With appropriate public policies, therefore, the case of le Marche (an ideal laboratory as the region is based on entrepreneurship, manufacturing, export, and with a long-lived population) can become a model in Italy and in Europe: my thesis focuses on the details of legal framework and on the type of organization are capable of implementing a model (CID) that takes into account the peculiarities of the entrepreneurial system in a country like Italy, that is the second manufacturing power in Europe. The public body must carry out a focused work, considering five categories of companies. To address this complexity, we need a place of contamination (CID) and a pro active public body, a catalyst, able to act on three regulatory levels (regional, national, European), leading a process in which, given the e-transformation, it will add a single new rule: stimulating companies to look at the world as their own market.

Le lupus érythémateux disséminé est une maladie auto-immune systémique très invalidante dont les atteintes sont multiples, les plus fréquentes étant cutanées, articulaires et rénales. Dans ce type de maladie, le système immunitaire, hyperactif, ne se limite pas à lutter contre des agents extérieurs mais s'attaque à ses propres cellules, entre autres par le biais d'auto-anticorps. Ces anticorps délétères sont produits par des plasmocytes, cellules issus de la différenciation des lymphocytes B. Ce processus se déroule principalement au sein des centres germinatifs (GC) dans les organes lymphoïdes secondaires, et fait intervenir de nombreux acteurs moléculaires et cellulaires. Mon projet de thèse a porté sur l'étude de la contribution du GC et de ses constituants, tels que les cellules auxiliaires folliculaires (Tfh) et l'IL-21, au cours du lupus. Au cours de ce travail, nous avons mis en évidence une altération à la fois quantitative et qualitative des cellules Tfh chez des patients lupiques et dans un modèle murin, altération entre autres responsable de taux anormalement élevés d'IL-21. Nous avons également observé une sensibilité accrue des cellules B de souris lupiques à cette cytokine, dont la cause est une surexpression de molécules clés telles que STAT3, et dont la conséquence est un surcroit de différenciation plasmocytaire. Tous les éléments sont donc présents pour favoriser l'interaction "Tfh-B" et la réaction du GC, et amplifier la réponse autoimmune. Enfin, la découverte de l'existence de GC ectopiques fonctionnels dans les reins de souris lupiques permet d'envisager l'existence de réponses locales au sein même des organes cibles. Les données obtenues, fondamentales, sont prometteuses et laissent entrevoir de nouvelles perspectives de biothérapies, plus ciblées, pour le traitement de la maladie lupique
Systemic lupus erythematosus is a disabling chronic autoimmune disease characterized by B cell hyperactivity leading to the production of autoantibodies, some of which exerting pathogenic effects. Autoantibodies are produced by plasma cells, which originate from the differentiation of B cells through a process that mainly takes place in germinal centers (GC) in secondary lymphoïd organs and involves many molecular and cellular parameters. The aim of my PhD project was to analyze the individual contribution of GC components, such as follicular helper T cells (Tfh) and IL-21, to lupus development. During this work, we have shown both a quantitative and qualitative impairment of Tfh cells in lupus patients and in a mouse model, leading, among other things, to high IL-21 levels. We also observed that B cells from lupus mice display a specific intrinsic sensitivity to this cytokine, due to over-expression of key molecules such as STAT3, which results in increased plasma cell differentiation. Thus, all elements are gathered that favor "Tfh-B" cell interactions and the GC reaction, and therefore the autoimmune response. Finally, the discovery of functional ectopic GC in the kidneys of lupus mice allows envisaging that local responses occur within the target organs and likely participate to kidney injury. The fundamental data we obtained are promising and anticipate new and better targeted biotherapies for lupus treatment

Le lupus érythémateux disséminé (LED) est une maladie auto-immune systémique caractérisée par une inflammation provoquant des lésions dans de nombreux organes tels que les reins, les poumons ou la peau. Dans cette pathologie, une activation excessive du système immunitaire conduit à la production d’auto-anticorps dirigés, le plus souvent, contre du matériel nucléaire. La différenciation des lymphocytes B (LB) en cellules productrices d’anticorps requiert une communication entre les LT et les LB. Ce dialogue est régulé par de nombreux acteurs cellulaires et moléculaires afin de permettre la mise en place d’une réponse humorale efficace en cas d’infections, mais aussi de prévenir le développement de maladies auto-immunes. Mon projet de thèse a consisté à étudier l’implication de deux de ces acteurs, l’un favorisant la différenciation des LB en plasmocytes, à savoir, les cellules T folliculaires auxiliaires (TFH) et le second régulant négativement l’activation lymphocytaire, le récepteur co-inhibiteur BTLA (pour B and T Lymphocyte Attenuator) dans le LED chez l’Homme. Au cours de cette étude, nous avons d’une part amélioré les connaissances concernant les sous-populations de TFH circulantes humaines, en décrivant que parmi les cellules TFH CXCR3-CCR6- sont retrouvées des cellules aux propriétés suppressives. De plus, nous avons suggéré que la contraction des TFH1 (CXCR3+CCR6-) au profit des TFH2 (CXCR3-CCR6-), observées chez les patients lupiques, pourrait être le reflet d’une migration des TFH1 vers les organes inflammés. D’autre part, nous avons mis en évidence un défaut fonctionnel de BTLA dans les LT CD4+ de patients lupiques. Ce défaut, restauré en normalisant le métabolisme lipidique des LT CD4+, semble associé à la sévérité de la pathologie. En parallèle de ces observations, nous avons démontré un défaut d’expression de BTLA sur les LB et les LT CD4+ régulateurs de patients lupiques. L’ensemble de nos données sont prometteuses et ouvrent de nouvelles perspectives thérapeutiques pour le traitement du LED
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by lesions in several organs such as kidneys, lungs and skin for instance. In this pathology, an excessive activation of the immune system leads to the production of autoantibodies targeting mainly nuclear antigens. B cell differentiation into antibody-secreting cells requires a close collaboration between T and B cells. This cross-talk is regulated by various cellular and molecular factors in order to mount an efficient humoral response in case of infection, but also to prevent autoimmune disease development. The aim of my thesis was to study two regulating factors of the B cell response, one promoting the B cell differentiation into plasma cells, i.e the follicular helper T cells (TFH) and the other one inhibiting lymphocyte activation, i.e a co-inhibitory receptor called BTLA (« B and T Lymphocyte Attenuator ») in human SLE. In this study, we first improved our knowledge concerning human circulating TFH cells, by describing among the CXCR3-CCR6- TFH cell subset, a population with suppressive capacities. Moreover, we suggested that the decreased frequency of TFH1 in lupus patients’ blood could be explained by the migration of these cells into inflamed tissues. We also highlighted a BTLA functional deficiency in lupus CD4+ T cells. This deficiency, which can be restored by normalizing the lipid metabolism, seems to be associated to disease severity. Furthermore, we described an altered expression of BTLA in lupus B cells and regulatory T cells. Altogether, our data show promising results and suggest new potential therapeutic strategies for lupus treatment

This thesis deals with the question as to whether the public and the private enforcements of European competition law, as currently shaped, are able to generate a sufficient and effective deterrence level. Proposals aimed at addressing certain possible gaps and shortages in this respect, in particular the criminalization of cartel conducts, are eventually put forward. More in details, sections I and II carry out a preliminary assessment of the real level of deterrence stemming from public and private enforcement tools with respect to violations of European competition law. Section III poses the question as to whether a possible criminalization of certain anticompetitive conducts in breach of European competition law, in particular cartels, along the line of what already occurred in many jurisdictions over the last decades, might contribute to enhance the current level of deterrence in this context. Section IV analyzes specific legal issues connected to a possible criminalization of cartel conducts in breach of art. 101 TFUE through directive, such as the identification of an appropriate legal basis pursuant to the Treaties of the European Union, the definition of the criminal offence, the penalties that could be provided, the interplay between criminal sanctions and leniency programs, the coordination between competition authorities and criminal judges, and certain further issues.

L'alternance de deux groupes de polarités très différentes, CH2 et CF2, permet au poly fluorure de vinylidène (PVDF) d’être un polymère industriellement très intéressant. Cependant, cette spécificité mène aussi à d’importantes inversions du monomère lors de la polymérisation vinylique. Pendant la polymérisation, en complément de la propagation tête-queue, CH2CF2CH2CF2, les monomères inversés conduisent à l’addition en queue-queue, CF2CH2CH2CF2, et tête-tête, CH2CF2CF2CH2. Le taux de transformation de polymère se trouve expérimentalement entre 3 et 7%. Ce pourcentage élevé entraine sans aucun doute la modification de propriétés macroscopiques. En utilisant la dynamique moléculaire, cette thèse a pour but de montrer l'effet de ces défauts sur la température de transition vitreuse (Tg), la dynamique locale et sur la température de fusion (Tm) du PVDF. En phase amorphe, le PVDF avec différents pourcentages de régio-défauts a été étudié : 3.6, 4.1, 9.3 et 23%. Cette étude permet de prédire le comportement de polymères qui ne sont pas synthétisés. Étant donné que les Tg simulées et expérimentales concordent avec précision, les motifs moléculaires qui donnent lieu à l'effet plastifiant de l'inversion de monomères peuvent être envisagés. En plus d'accentuer leur effet de plastifiant, la conclusion significative est que la relaxation de la chaîne peut être révélée en abordant explicitement des mouvements locaux. Car cette procédure ne peut pas être déduite de la connaissance du Tg, nous avons basé notre analyse sur le fait cela : 1) Nous avons démontré que des relations linéaires directes entre Tg et l'énergie d'activation conformationnelle de transition (Ea) extraite à partir d'un graphe d'Arrhenius, existent. Ce diagramme correspond au logarithme naturel des taux de transition entre les états rotameriques contre l'inverse de la température. La pente de cette courbe rapporte directement à cet Ea efficace. Un tel lien a été seulement spéculé dans la littérature. 2) Nous avons calculé des relations d'Arrhenius pour différents genres de torsions le long de la chaîne d'épine dorsale. En conséquence, une barrière d'énergie potentielle, ea, est associée à la rotation d'un lien dans un environnement spécifique. L'addition de ces énergies pesées par le pourcentage de chaque lien le long de l'épine dorsale, donne un ea moyen qui est équivalent à l'ea efficace. À l'aide de cette procédure, nous avons maintenant accès au mouvement local de la chaîne entière. 3) Nous avons vérifié cette procédure pour calculer une valeur pour le Tg du copolymère alternatif du l'éthylène-tétrafluoroéthylène (E-TFE), qui possède les segments qui sont présents le PVDF changé. L'ambiguïté concernant la valeur de la Tg du copolymère E_TFE peut être résolue grâce à cette approche, puisque le PVDF avec 50% de défauts régio-isomériques conduit à l'E_TFE. D'ailleurs, nous avons étudié les temps de relaxation pour la fonction d'autocorrélation de torsion au-dessus d'un large éventail de température. La dynamique locale est alors spécifiquement étudiée. L'équation Vogel-Fulcher-Tammann (VFT) est utilisée pour décrire le processus de relaxation associée aux mouvements coopératifs des segments le long de la chaîne. Nous avons également étudié le possibilité d'utiliser le Kohlrausch-Williams-Watts (KWW), fonction exponentielle étirée, afin de décrire la dépendance temporelle du processus de relaxation, ce travail a été effectué à différentes températures. Les résultats concordent bien avec les données expérimentales. L'objectif principal de cette section est d'étudier conjointement la fréquence des transitions conformationnelles et le temps de relaxation obtenu par la fonction d’autocorrelation de torsion, sur une plage importante de température, afin d’établir un entre les fréquences des transitions conformationnelles et le comportement de type VFT. Nous montrons pour la première fois qu’une relation linéaire peut être établie entre la barrière de transition conformationnelle et l’énergie d’activation effective. Nous montrons pour la première fois qu'une relation linéaire peut être établie entre la barrière de transition conformationnelle, Ea et l'énergie d'activation effective, B, responsables de la dynamique locale. Parmi les cinq phases cristallines que présente le PVDF, les cristaux α et ß présentent des propriétés particulières intéressantes et ont fait l'objet d'une attention significative. Ces deux structures cristallines sont celles que l’on rencontre le plus souvent, la phase α est la plus thermodynamiquement stable le cristal β possède des propriétés ferroélectriques. Toutefois, le comportement lors de la fusion de ces deux phases cristallines n’est pas encore totalement compris. Certains chercheurs pensent que la température de fusion de la phase β est supérieure à la phase alpha . D'autres affirment que le pic endothermique vu sur le thermogramme obtenue par calorimétrie différentielle à balayage (DSC) a été attribué par erreur à la phase β, cela à cause d’une confusion dans les références . À cet égard, le comportement de la Tm des cristaux α et β par rapport à leur épaisseur est obtenu par la dynamique moléculaire. Différents types de nanocristaux composés de chaînes de PVDF, sans ou avec 10% de régio-défauts, ayant des longueurs différentes ont ainsi été simulées dans les phases α et β. On applique l'équation de Gibbs-Thomson (G-T) afin de déterminer l'énergie de surface et l’enthalpie de fusion des nanocristaux. Les valeurs déterminées sont en accord avec les données expérimentales. Nous avons montré que le PVDF en phase β pur a une température de fusion inférieure à celle du PVDF en phase α pur. Cependant, en insérant des défauts à l'intérieur du cristal, la phase α modifiée présente une température de fusion inférieure à celle de la phase β modifiée.
Abstract : Alternating two groups, CH2 and CF2, of very different polarities along the backbone chain of polyvinylidene fluoride (PVDF) leads to very interesting properties, such as ferroelectricity. However, these properties are affected by the presence of regioisomerism defects (monomer inversion) that appear during the synthesis. During the polymerization, in addition to the Head-to-Tail (HT) sequences, CH2CF2CH2CF2, the reversed monomer units lead to formation of Tail-to-Tail (TT), CF2CH2CH2CF2, and Head-to-Head (HH), CH2CF2CF2CH2, links. The rate of this chain alteration experimentally lies between 3 and 7 %. This percentage undoubtedly brings changes in macroscopic properties. The aim of this thesis is to reveal the impact of these defects on the glass transition temperature (Tg), local dynamics and melting temperature (Tm) of PVDF by using Molecular Dynamics (MD) simulation. In amorphous phase, PVDF chains with different percentages of regiodefects were investigated: 0, 3.6, 4.1, 9.3, and 23 %. This study makes it possible to predict the experimental behavior of polymers which have not yet been synthesized. Once Tg is acquired, the relaxation of the chain can be investigated through the calculation of the activation energy (Ea) of the conformational transition. The significant conclusion is that the relaxation of the chain can be revealed by addressing the local motions. More specifically: a) We demonstrate a direct linear relationship between Tg and Ea extracted from an Arrhenius plot. This diagram corresponds to the natural logarithm of transition rates between rotameric states versus the inverse of the temperature. The slope of this curve yields directly Ea. Such a link was only speculated in the literature. b) A significant finding of this work is that the mobility of the chain can be associated with different types of bonds in PVDF with regiodefects. c) Based on the analysis of Ea for the different bond contributions, we proposed a value for the Tg of ethylene-tetrafluoroethylene (E-TFE), an isomeric polymer of PVDF with 50% regiodefects. Experimentally, the available data for the Tg of E-TFE are limited and highly variable. For example, it has been reported as varying from -108 °C to 145 °C. The ambiguity of Tg for this copolymer can be resolved with this approach. Furthermore, we studied the relaxation time associated with the torsional autocorrelation function (TACF) over a wide temperature range. The Vogel-Fulcher-Tammann (VFT) equation was used to describe the temperature dependence of the relaxation time. The Kohlrausch Williams Watts (KWW) stretched exponential function is then applied to fit the time dependence of the relaxation process at various temperatures. The results obtained from this work were found to be in good agreement with the experimental data. A particular interest in this study is the question of how the non-Arrhenius VFT of relaxation process is related to the Arrhenius behavior of conformational jump rates near the glass transition. In both cases, the energies (the conformational transition energy (Ea) and the effective activation energy (B) in VFT equation), were very close to the value of a single torsional barrier. However, in contrast to the relaxation time associated with TACF, the rates of conformational jumps show the activation energy higher than the single barrier value. We have shown that a linear relationship can be established between the conformational transition energy and the effective activation energy. In crystalline PVDF, among the five typical phases, the α and β crystals are of particular interest. The α phase is the most thermodynamically stable form and the β crystal possesses ferroelectric properties. The melting behaviour of these two crystal phases is not so clear. Some researchers believe that the melting temperature of the β phase is higher than that of the α phase. Others have claimed that the higher melting temperature of the peak in Differential Scanning Calorimetry (DSC) has been mistakenly attributed to β phase melting, due to confusion in the referencing of literature sources. In this regard, the melting temperatures of α and β crystals (with and without regiodefects) with respect to their thickness are captured by MD simulation. We then applied the Gibbs-Thomson (G-T) equation to determine the melting temperature, as well as the surface energy and enthalpy of fusion, for α and β nanocrystals. We have shown that pure β phase PVDF has a lower melting temperature than pure α phase PVDF. However, by inserting regiodefects randomly inside the crystal, the α phase with regiodefects shows a lower melting temperature than that of the β phase with regiodefects. We attributed this behaviour to the different structures of the two phases.

Les Contrôleurs du VIH sont de rares individus capables de contrôler spontanément la réplication virale en l’absence de traitement. De nombreuses études montrent que les Contrôleurs développent des réponses T antivirales remarquablement efficaces. Les cellules T CD4+ spécifiques de Gag pourraient jouer un rôle particulier car cette population est préservée en comparaison aux patients traités et corrèle négativement avec la charge virale. Afin d’étudier cette population, nous avons réalisé une analyse transcriptionnelle et protéique multiplexée sur cellule unique, à partir de cellules T CD4+ détectées ex vivo par marquage tétramère de CMH-II contre le peptide Gag293 (Tet+). Nous avons comparé l’expression de 44 gènes et 6 protéines membranaires chez 9 patients Contrôleurs et 9 patients traités. Nous avons d’une part validé la forte fréquence de cellules T CD4+ Tet+ chez les Contrôleurs en comparaison aux patients traités et, d’autre part, montré que les cellules T CD4+ Tet+ des Contrôleurs, étaient activées et engagées dans une différenciation Th1 avancée et présentant un profil cytotoxique. De plus, les cellules T CD4+ Tet+ de Contrôleurs ont montré un état d’épuisement limité, reflété par une expression faible de PD-1, qui pourrait être l’une des raisons du maintien de leur fréquence et de leurs fonctions. Dans une deuxième étude, nous avons étudié les cellules T folliculaires « helper » (Tfh) dans la population T CD4+ spécifique de Gag chez les Contrôleurs du VIH. Les Tfh jouent un rôle essentiel dans la maturation d’affinité des anticorps en aidant les cellules B. Afin de déterminer si ce sous-type cellulaire joue un rôle dans le contrôle de l’infection à VIH, nous avons analysé le phénotype et la fonction des Tfh circulantes (cTfh) : cellules T CD4+ CD45RA- CXCR5+). Nous avons utilisé un marquage tétramère de CMH-II contre le peptide Gag293, pour détecter les cTfh spécifiques du VIH (cTfh Tet+), et nous avons montré que cette population est préférentiellement maintenue chez les Contrôleurs du VIH. L’analyse phénotypique de la population cTfh Tet+ a montré une intensité d’expression (MFI) de PD-1 plus importante dans le groupe de patients traités, suggérant une activation immune anormale chez ces patients. La fonction des cTfh, analysée pour leur capacité à induire la sécrétion d’IgG en coculture avec des cellules B mémoires autologues, n’a pas montré de différences majeures entre les groupes en terme de production d’IgG totales. Cependant, la production d’IgG spécifiques anti-VIH est significativement plus efficace chez les Contrôleurs, en particulier pour la réponse anti-Env qui est plus de 30 fois supérieure à celle des patients traités. Enfin, la fréquence des cTfh Tet+ a corrélé positivement avec la production d’IgG spécifiques, supportant l'idée d'un rôle important de la fonction Tfh dans la réponse humorale anti-VIH. L’ensemble de ces résultats indique que la population T CD4+ spécifique de Gag supporte chez les Contrôleurs les deux bras de la réponse immunitaire antivirale : d’une part, une réponse de type cellulaire Th1 montrant un profil cytotoxique et, d’autre part, une réponse de type humorale, reflétée par des interactions cTfh/B préservées, résultant en une réponse B mémoire vigoureuse. Le maintien de la fonction et de la fréquence de ces cellules spécifiques de Gag pourrait donc jouer un rôle important dans le contrôle du VIH
HIV Controllers are rare individuals able to spontaneously control viral replication in the absence of treatment. Several studies showed that controllers develop effective anti-viral T cell responses. Gag-specific CD4+ T cells could play a particular role in HIV control, because this population is preserved in comparison with the treated patients and correlates negatively with the viral load. In order to study this population, we performed a multiplexed single cell transcriptional and protein analysis from CD4+ T cells detected ex vivo by MHC-II tetramer labeling against the Gag293 peptide (Tet+). We compared the expression of 44 genes and 6 surface proteins in 9 Controllers patients and 9 treated patients. Firstly, we validated the high frequency of Tet+ CD4+ T cells in controllers compared to the treated patients, then we showed that Tet+ CD4+ T cells from controllers were activated and engaged in advanced Th1 differentiation with a cytotoxic profile. In addition, Tet+ CD4+ T cells from controllers showed a limited state of exhaustion, reflected by a lower expression of PD-1, which could be one of the reasons for maintaining their frequency and functions. In a second study, we studied follicular helper T cells (Tfh) among the Gag-specific CD4+ T cell population of HIV controllers. Tfh plays an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4+ T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh: T cells CD4+ CD45RA- CXCR5+). We performed a MHC-II tetramer labeling against Gag293 peptide to detect HIV-specific cTfh (cTfh Tet +), and showed that this population is preferentially maintained in HIV controllers. Phenotypic analysis of Tet+ cTfh population showed a higher intensity of PD-1 expression (MFI) in the treated group suggesting abnormal immune activation in these patients. The function of cTfh, analyzed by the capacity to promote IgG secretion in cocultures with autologous memory B cells, did not show major differences between groups in terms of total IgG production. However, the production of HIV-specific IgG is significantly more efficient in the controller group, especially for the anti-Env response that is more than 30-fold greater than those of the treated patients. Finally, the frequency of Tet+ cTfh correlated positively with the production of specific IgG, supporting the idea of an important role of Tfh function in the humoral antiHIV response. Taken together, these results indicate that Gag-specific CD4+ T cell population supports the two arms of the antiviral immune response in HIV controllers: the cell-mediated response through a preferential differentiation toward Th1 cell type showing a cytotoxic profile, and the humoral response, reflected by preserved cTfh / B interactions, resulting in a vigorous memory response. Maintaining the function and frequency of these Gag-specific CD4+ T cells could therefore play an important role in HIV control

O sistema de secreção tipo IV (T4SS) da família de bactérias Xanthomonadaceae transfere efetores (X-Tfes) com a capacidade de matar outras bactérias, conferindo uma vantagem em comunidades bacterianas mistas para colonizar diferentes nichos como o solo ou as superfícies das plantas. Os X-Tfes possuem diferentes domínios putativos com atividades hidrolíticas contra componentes do envelope celular bacteriano do tipo: glicohidrolases, transglicosilases, amidases e lipases. Os X-Tfes por sua atividade biológica inata podem ocasionar dano intracelular para a bactéria que os produz. Para se proteger contra estas atividades, também são produzidas lipoproteínas com função inibitoria (X-Tfis) localizadas no periplasma. Os genes que codificam os X-Tfes e os X-Tfis estão organizados em operons, o que permite gerar os pares efetor/inibidor simultaneamente. Entre os potenciais X-Tfes do fitopatógeno Xanthomonas citri estão Xac1918 e Xac0574. Xac1918 é uma proteína com um domínio da superfamília da lisozima e um domínio conhecido como RTX (Repeats in Toxin) de ligação ao cálcio, enquanto Xac0574 tem um domínio da superfamília da lipase 3. Os seus possíveis inibidores, Xac1917 e Xac0573 respectivamente, apresentam um peptídeo sinal no N-terminal contendo o lipobox representativo das lipoproteínas. As proteínas Xac0574 e Xac0573 são monômeros em solução que formam um complexo estável 1:1, favorecido termodinamicamente (ΔG°= -12 Kcal/mol) com uma constante de dissociação de 2,45 nM, garantindo que a bactéria fique protegida contra os efeitos nocivos de Xac0574 quando é produzida intracelularmente. Xac0574 é uma fosfolipase A1, sem atividade lisofosfolipase, com a capacidade de hidrolisar os três fosfolipídios majoritários que compõem a membrana celular bacteriana, fosfatidilglicerol (PG), cardiolipina e fosfatidiletanolamina (PE), mostrando uma aparente preferência pelo último. A atividade enzimática de Xac0574 explica a forte inibição do crescimento celular em E. coli após da sua indução heteróloga, já que gera uma diminuição de quase 10 vezes da população celular comparada com a cultura não induzida com a mesma construção. Poroutro lado, Xac0573 inibe efetivamente a atividade enzimática de Xac0574 ao formar o complexo, além de não ter atividade fosfolipase nem lisofosfolipase. Foram produzidos cristais da Xac1918 e Xac0573 que difrataram com uma resolução de 3,0 e 2,5 Å, respectivamente. Porém, só foi gerado um modelo de Xac0573. Xac0573 está composta por duas folhas β antiparalelas com uma topologia característica de β sanduíche Com uma pequena hélice e duas voltas. Um alinhamento de homólogos de Xac0573 identificou nas extremidades da proteína as regiões conservadas, constituindo duas possíveis interfaces de interação que podem ser as responsáveis por bloquear o acesso dos fosfolipídios ao sítio catalítico ou impedir os rearranjos estruturais de Xac0574 que são necessários para a sua atividade enzimática. Adicionalmente, a topologia da Xac0573 é semelhante do domínio C2, conhecido em eucariotos como domínio de ligação ao lipídio e ao cálcio, e está envolvido em processos de sinalização de segundos mensageiros lipídicos, proteínas de trafego de membranas e mecanismos de fusão de membranas. Nossos resultados apontam para uma nova função biológica do domínio C2 como um inibidor enzimático intracelular em bactérias.
The type IV secretion system (T4SS) of the bacteria family Xanthomonadaceae transfers effectors (X-Tfes) with that can kill other bacterial cells, conferring an advantage to the bacterial community during colonization of different niches in the soil or on the plant surface. The X-Tfes possess different putative domains with hydrolytic activity against components of the bacterial cellular envelope, including glycohydrolase, transglycolase, amidase and lipase domain. The innate biological activity of X-Tfes can cause intracellular damage. Therefore, the bacteria that produce them also produce lipoproteins with inhibitor function (X-Tfis) located in the periplasm for their protection. The genes that code for X-Tfes and X-Tfis are organized in operons that allow for their simultaneous expression. Among the X-Tfes of the phytopathogen Xanthomonas citri are Xac1918 and Xac0574. Xac1918 is carries a lysozyme superfamily domain, as well as a domain known as RTX (Repeats in Toxic) predict to bind calcium, while, Xac0574 has a domain belonging to the lipase 3 superfamily. Their possible inhibitors, Xac1917 e Xac0573 respectively, carry an N-terminal signal peptide containing a lipobox found in bacterial lipoproteins. The Xac0574 and Xac0573 proteins are both monomers in solution, They can form a stable 1:1 complex, that is thermodynamically favored (ΔG°= -12 Kcal/mol) with a dissociation constant of 2,45 nM. This affinity ensure that the bacterium is protected against the harmful effects of Xac0574 when it is produced intracellularly. We show that Xac0574 is a phospholipase A1, without lisophospholipase activity, and is able to hydrolyze the three most common phospholipids found in the membranes of Gram negative bacteria, namely phosphatidylglycerol (PG), cardiolipin and phosphatidylethanolamine (PE), presenting an apparent preference for PE. The enzymatic activity of Xac0574 explains the strong inhibition of growth of E. coli cells after its heterologous induction: a nearly 10-fold decrease in the cell population is observed when compared to the non-induced culture with the same construct. On the other hand, Xac0573 effectively inhibits the enzymatic activity of Xac0574. Furthermore, Xac0573 does not possess when forming the complex, besides not having phospholipase nor lysophospholipase activity.Crystals of Xac1918 and Xac0573 were produced which diffracted with to resolution of 3.0 and 2.5 Å, respectively. However, we were able to resolve the structure of only Xac0573. Xac0573 is composed of two anti-parallel sheet that form a β-sandwich with three small helices. An alignment to Xac0573 homologs identified conserved regions at the ends of the protein that constitute two possible interfaces of interaction that may be responsible for blocking the access of the phospholipids to the catalytic site or impede the structural rearrangements of Xac0574 that are necessary for its enzymatic activity. Additionally, the topology of Xac0573 is similar to that to C2 domains, known in eukaryotes to bind lipids and calcium and to be involved in signaling processes mediated by lipid second messengers, membrane trafficking and membrane fusion mechanisms. Our results point to a new biological function of the C2 domain as an intracellular enzyme inhibitor in bacteria.

L'infection par le VIH est une pathologie caractérisée par la dissémination du virus dans l'ensemble de l'organisme et une destruction progressive des lymphocytes T CD4+ (LT CD4+) résultant en une immunité altérée. L'épuisement et l'apoptose des cellules T mémoires induites par le virus amènent à l'apparition d'un SIDA. Il a été montré que les centres germinatifs (GCs), structures anatomiques spécialisées au sein des follicules B des organes lymphoïdes secondaires, représentent des lieux privilégiés de la persistance virale. Parmi les organes lymphoïdes, la rate et les ganglions mésentériques seraient des sanctuaires viraux du fait de leur rôle dans la réponse immunitaire. La rate est le principal organe de la réponse B, et les ganglions mésentériques sont essentiels dans l'établissement de la réponse immune mucosale. Les GCs sont principalement constitués de lymphocytes B mais également de lymphocytes T folliculaires (TFH), sous-population de LT CD4+ effecteurs mémoires jouant un rôle essentiel dans le développement de la réponse B, en particulier dans la maturation des anticorps de haute affinité. Des études dont celles du laboratoire ont montré que l'altération de la réponse B observée au cours de l'infection VIH serait due à une perte et à un défaut de différentiation des lymphocytes TFH. Plus récemment, il a été observé que les GCs sont également constitués de lymphocytes T mémoires CD8+ folliculaires (LT CD8+ folliculaires), exprimant le récepteur de chimiokine CXCR5. De récents travaux suggèrent un rôle de ces cellules dans le contrôle de l'infection par le VIH. Le but de ma thèse a été d'étudier la dynamique des lymphocytes TFH au sein de ces deux organes que sont la rate et les ganglions mésentériques, et de déterminer dans quelle mesure l'administration d'un inhibiteur d'apoptose permet de restaurer des fonctions effectrices telle que la production d'anticorps. Ces travaux ont été réalisés chez le macaque rhésus infecté par la souche SIVmac251. Mes résultats montrent que l'infection persistante des ganglions mésentériques est associée à une perte et une altération des follicules B ainsi qu'à une déplétion et une altération des lymphocytes TFH. Ils montrent également une altération de l'expression de la chimiokine CXCL13, ligand de CXCR5, qui serait à même de créer un environnement défavorable à la différenciation des cellules B. Ils montrent aussi que l'administration d'un inhibiteur d'apoptose au cours de la phase de primo-infection chez les singes infectés induit une diminution de l'apoptose des LT CD4+ comme attendu, une diminution des cytokines inflammatoires, et une réponse B systémique spécifique plus importante chez les singes traités. Ceci pourrait contribuer à un meilleur contrôle de la réplication virale. Enfin, mes travaux démontrent qu'au niveau de la rate, on observe une augmentation du nombre de LT CD8+ qui infiltre les follicules B et que ceux-ci n'expriment pas majoritairement la protéine CXCR5 chez les singes infectés en particulier progresseurs. De même, on observe une augmentation de la chimiokine MIP1b, susceptible de jouer un rôle dans le recrutement de LT CD8+ conventionnels chez les singes infectés, et une augmentation de l'expression des marqueurs de cytotoxicité chez les singes non-progresseurs comparativement aux singes progresseurs. Enfin, ces travaux semblent indiquer que les LT CD8+ folliculaires des individus progresseurs ne contrôlent pas l'infection par le VIS. En conclusion, l'infection par le VIS induirait la formation d'un réservoir viral au niveau des follicules B et des GCs induisant une réponse B altérée, associée à une altération des follicules B, à un défaut des lymphocytes TFH, ainsi qu'à un enrichissement en LT CD8+ folliculaires potentiellement défectifs. Ensemble, mes travaux portant sur l'étude des lymphocytes T folliculaires ont mis en exergue et confirmé l'importance de ces cellules T dans les relations hôtes-pathogènes
HIV infection is characterized by a viral spread in the host body and a progressive destruction of CD4+ T cells, leading to a defect in the immune system. The memory T cells apoptosis and exhaustion induced by the virus leads the outcome of an AIDS. It was showed that germinal centers (GCs), specialized anatomical structures present in the B follicles of the secondary lymphoid organs, represent privileged viral reservoir. Among secondary lymphoid organs, spleen and mesenteric lymph nodes would be viral sanctuaries because of their role in the generation of the immune response. Spleen is the main organ of the B cell response, and mesenteric lymph nodes are essentials in the establishment of the mucosal immune response. GCs are mainly composed by B cells but also by TFH cells, a sub-population of effector memory CD4+ T cells, crucial for the generation of the B cell response, especially for the maturation of the highly efficient antibodies. Previous works from our laboratory showed that the defect of the B cell response during HIV infection is due to a loss and a defect of the TFH cells. Recently, it was showed that GCs are also composed of follicular memory CD8+ T cells, expressing the chemokine receptor CXCR5. Recent works suggested that those cells are implicated in the control of HIV infection. Thus, the aim of my thesis was to study the dynamic of follicular T cells in these two organs, spleen and mesenteric lymph nodes, and to set how well the administration of a pan-caspase inhibitor during acute phase of SIV infection help to restore effective functions as the antibody production. Those works were done in rhesus monkeys infected with the strain SIVmav251. My results show that the persistent infection of mesenteric lymph node is associated with a loss and a defect of B follicles and with a defect of TFH cells. They also show a defect in the expression of the CXCL13 chemokine, ligand of the CXCR5 receptor, potentially capable of creating a bad environment for B cell differentiation. My results show also that the administration of a pan-caspase inhibitor during the acute phase of infection leads to a decrease in the CD4+ T cells apoptosis as expected, a decrease in the inflammatory cytokines expression and in a better specific systemic B cell response in treated monkeys. These could help in the control of the viral replication. Finally, my results show that in the spleen, there's an increase of the follicular CD8+ T cells filtering the B follicles, which mainly doesn't express the CXCR5 receptor in infected monkeys, especially the progressor ones. They also show that there's an increase in the expression of the MIP1b chemokine, possibly responsible of the recruitment of conventional CD8+ T cells in the infected monkeys, and an increase in the expression of cytotoxic markers in the non-progressors monkeys compared to the progressors ones. Thus, these work seems to show that follicular CD8+ T cells of progressor monkeys fail to control SIV infection. Thus, SIV infection induce an altered B cell response, associated to a defect of B follicles, a defect of TFH cells, and an increase in follicular CD8+ T cells potentially inefficient. Altogether, my works focused on the study of follicular T cells show and confirm the strength the importance of these T cells in the host-pathogen relationship

Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the acute and persistent GC B cell reaction following respiratory IAV infection is lacking, as is the characterization of IAV-induced T follicular helper (TFH) cells that support GCs. Additionally, it remains unclear as to whether IAV-induced GC B cells are subject to control by regulatory T cells (Tregs). To address this, GC B cell and TFH cell responses were analyzed in mice following pulmonary challenge with IAV. Studies demonstrated that marked GC reactions were induced in lung-draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude, kinetics, and isotype switching patterns of the response was site-specific, and largely depended on the magnitude of IAV-induced TFH cell populations. TFH cell magnitude peaked prior to that of GC B cells in all tissues, and TFH cells purified from dLNs generated IL-21 and IFN-gamma upon activation, although CD4+CXCR5- T effector cells produced higher levels of all cytokines. IgA+ GC B cells were infrequent in most sites, but composed a significant subset of the switched GC population in NALT. Further, splenectomized mice withstood a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection. Additionally, GC B cell populations were analyzed at distal time points to assess the understudied, persistent GC B cell response after IAV infection. Our analysis demonstrated that persistent GC B cell populations in mouse lungs directly correlated with infectious dose, pathogenicity of the virus, as well as the presence of long-term CD4+ T cell help. Finally, experiments showed that Tregs contribute to the control of GCs induced in the spleen by IAV challenge. This was demonstrated by a marked increase in the number of total and switched GC B cell numbers when Tregs were either depleted or disrupted in vivo proximal to IAV exposure.