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Made available in DSpace on 2016-04-04T12:35:27Z (GMT). No. of bitstreams: 2 rafaella_reis_ioc_mest_2014.pdf: 1575570 bytes, checksum: 5c1e218abcd29ee34857d52a4ba58b7a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A Síndrome de Wiskott-Aldrich (WAS) é uma imunodeficiência associada a infecções recorrentes, câncer e autoimunidade. Ela é causada por mutações no gene que codifica a molécula WASP (\201CWiskott-Aldrich syndrome protein\201D), envolvida na reorganização do citoesqueleto de actina em células de origem hematopoiética. Camundongos deficientes em Wasp (WKO) apresentam migração leucocitária e proliferação linfocitária deficientes, e desenvolvem colite. Esse processo inflamatório está associado a eventos de imunodesregulação importantes, como participação exacerbada de células Th2 e diminuição de células T regulatórias (Treg) no timo e baço desses animais. Assim, esse trabalho visa caracterizar as células Treg tímicas e avaliar os mecanismos potencialmente relacionados à diminuição dessas células no timo de camundongos WKO. Nas análises de CD25 e CD122, importantes para a diferenciação de células Treg, observamos uma diminuição na expressão de CD25 na população de células CD4+CD8-Foxp3+, além de uma diminuição no número percentual dessa população. As células Treg tímicas de animais WKO apresentaram níveis normais de CD122 e de moléculas de ativação, mas mostraram uma diminuição na expressão da molécula funcional CD39, responsável pela hidrólise tecidual de ATP Saliente-se ainda que estudos sobre a morte celular ex-vivo, a transmigração in vitro mediada por S1P e a localização intratímica de células Treg não revelaram diferenças significativas na comparação entre animais WKO e seus controles. De forma interessante, precursores tímicos de células Treg CD4+CD8-CD25+Foxp3- também se apresentaram diminuídos em WKO, assim como as células Treg Helios+. Além disso, observamos um aumento importante na expressão basal de pSTAT-5 nas células Treg nos camundongos WKO e também nos precursores dessas células. Nossos resultados indicam uma participação importante da proteína Wasp no desenvolvimento de células Treg tímicas, mas apontam para a necessidade de estudos mais detalhados sobre os mecanismos envolvidos na geração deficiente de células Treg em camundongos WKO / The Wiskott - Aldrich syndrome (WAS) is an immunodeficiency associated with recurrent infections, malignancies and autoimmunity. It is caused by mutations in the gene encoding WASP (Wiskott - Aldrich syndrome protein), a molecule involved in the actin cytoskel eton rearrangements in hematopoietic cells. Wasp deficient mice (WKO) show impaired leukocyte migration and lymphocyte proliferation, and also develop colitis. This inflammatory process is associated with important immunodysregulation events, such as exac erbated participation of Th2 cells and decreased numbers of regulatory T cells (Treg) in the thymus and spleen of these animals. Thus, this study aims to characterize the thymic Treg cells and evaluate the potential mechanisms involved in the decreased num ber of these cells in the thymus of WKO mice. In the analysis of CD25 and CD122, important molecules for the differentiation of Treg cells, we observed a decreased expression of CD25 in the CD4 + CD8 - Foxp3 + cells, and a decrease in the percentage number of t his population. WKO Treg cells showed normal levels of CD122 and activation molecules, but present a decreased expression of the functional marker CD39, molecule responsible for ATP hydrolysis in the tissues. Also, it is important to point out that studies about ex vivo cell death, S1P - mediated in vitro transmigration and intratymic localization of Treg cells revealed no significant differences between WKO animals and their controls. Interestingly, both CD4 + CD8 - CD25 + Foxp3 - Treg precursors and Helios + Treg c ells are reduced in WKO thymus. More over, we also observed an important increase in the basal expression of pSTAT - 5 in Treg cells of WKO mice and their precursors. Altogether, our results indicate an important role of Wasp in thymic Treg cell development, and point to the necessity of further studies on the mechanisms involved in the generation of Treg cells in WKO mice.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 3 uppsatser.

Thesis (Th. M.)--Western Seminary, Portland, Or., 1999. / Abstract. Includes bibliographical references (leaves 115-119).

Made available in DSpace on 2016-05-02T13:07:00Z (GMT). No. of bitstreams: 2 bianca_franca_ioc_dout_2015.pdf: 11028145 bytes, checksum: e8f2a4ece5e213dd7f7e288656fd0f9d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A produção aumentada de eosinófilos pela medula óssea sustenta a inflamação crônica na asma, compensando a apoptose de eosinófilos maduros nos sítios inflamados. Outros trabalhos mostraram que a eosinopoiese poderia ser suprimida pela prostaglandina E2 (PgE2) por uma via iNOS-CD95L dependente. Sendo a PgE2 um ativador da produção de AMPc intracelular, testamos uma série de agentes capazes de mimetizar este aumento de AMPc. Foram realizadas culturas de medula óssea em meio líquido ou semi-sólido para a análise da eosinopoiese e neutropoiese a partir de camundongos mutantes e seus respectivos controles de tipo selvagem (WT), dos backgrounds BALB/c, C57BL/6. Todos os agentes testados, incluindo isoproterenol, mimetizaram os efeitos de PgE2, suprimindo a eosinopoiese pela mesma via utilizada para supressão pela PgE2. Outros agentes como a IL-17A, uma citocina pró-inflamatória, e a a- galactosilceramida (\03B1-GalCer), um glicolipídeo que ativa células iNKT de forma restrita por CD1d, também suprimiram a eosinopoiese pela via de iNOS-CD95L, mas cada agente utilizou um ponto de atuação e um mecanismo de ação próprio. Por outro lado, o ácido retinóico all-trans (ATRA) também suprimiu a eosinopoiese de forma dependente de CD95L, mas um papel para a iNOS só foi evidenciado, até o momento, para a supressão de formação de colônias pelo ATRA na presença de GM-CSF. Também evidenciamos uma interrelação entre a via da cicloxigenase (COX), a via da 5-lipoxigenase (5-LO) e a ação dos glicocorticóides (GC) na regulação da eosinopoiese Dados anteriores do laboratório mostram que: a) na ausência de 5-LO, a medula óssea estimulada por IL-5 não responde aos inibidores da COX, que potencializam a eosinopoiese em controles WT (Elsas et al., 2008)(Elsas et al., 2008)(Elsas et al., 2008)(Elsas et al., 2008)(Elsas et al., 2008)(Elsas et al., 2008)(Elsas et al., 2008)(Elsas et al., 2008); b) a ação supressiva da PgE2 é antagonizada pelos cisteinil-leucotrienos, produtos da 5-LO, e também pelos glicocorticóides (Gaspar-Elsas et al., 2009). Neste trabalho, mostramos que controles WT e mutantes WASP-KO (deficientes na proteína do Síndrome de Wiskott-Aldrich, que é um elemento importante na motilidade e função de defesa de várias células hematopoiéticas, por sua função no controle do citoesqueleto) respondem normalmente aos inibidores da COX na presença de 5-LO. Mutantes WASP-KO apresentaram menor celularidade da medula óssea recém-coletada, com um déficit significativo na população granulocítica, e não apresentaram resposta eosinopoiética a GC, nem in vivo nem in vitro, com respostas normais à PgE2. Os bloqueadores da COX reconstituíram a eosinopoiese in vitro na medula óssea mutante de forma dependente de 5-LO e leucotrienos. Portanto, WASP mostrou-se relevante tanto para a celularidade da medula óssea in vivo quanto para a sinalização da resposta ao GC. Essas observações reforçam o interesse de estudar a influência dos GC sobre a produção de granulócitos in vivo, e permitem distinguir com base em critério bioquímico (WASPdependência), a via de atuação dos GC daquela via utilizada pelos produtos da 5-LO para bloquear a eosinopoiese iNOS/CD95L-dependente / Increased eosinophil production by bone-marrow sustains chronic inflammation in asthma, by replacing mature eosinophils which undergo apoptosis in inflammatory sites. Other studies have shown that eosinopoiesis could be suppressed by prostaglandin E2 (PgE2) in vitro. Because PgE2 increases intracellular cAMP levels, we tested a panel of agents capable of induction or mimesis of intracellular cAMP rises. We established bone-marrow cultures, in liquid or semisolid media, to examine eosinopoiesis and neutropoiesis, from mutant mice and their respective wild-type (WT) controls of the BALB/c and C57BL/6 backgrounds. All these agents, including isoproterenol, duplicated the effects of PgE2, suppressing eosinopoiesis by the same biochemical pathway previously characterized for PgE2. Other agents such as IL-17 A, a proinflammatory cytokine. and a-galactosilceramide (\03B1-GalCer), a CD1d-restricted glycolipid which activates iNKT cells, also supressed eosinopoiesis through the iNOS-CD95L pathway, but each agent entered the sequence at a distinctive point and used a distinctive mechanism of action. By contrast, all-trans Retinoic Acid (ATRA) also suppressed eosinopoiesis in a CD95L-dependent manner, but a role for iNOS was so far only demonstrated in suppression of colony-formation by ATRA in the presence of GM-CSF. We also demonstrated the three-way connectivity between the cyclooxygenase (COX) pathway, the 5-lipoxygenase (5-LO) pathway and glucocorticoid (GC) regulatory effects on eosinopoiesis. Our group previously reported that: a) in the absence of 5-LO, IL-5-stimulated bone-marrow did not respond to COX inhibitors, which enhance eosinopoiesis in WT bone-marrow; b) the supressive effects of PgE2 are antagonized by cysteinilleukotrienes, produced by 5-LO, and by GC (Elsas et al., 2008) Here we show that WT controls and WASP-KO mutants (lacking the Wiskott-Aldrich Syndrome Protein, a critical element in motility and immunological function of various hemopoietic cell types, due to its role in cytoskeleton regulation) respond normally to COX inhibitors in the presence of 5-LO. WASPKO mutants present lower cellularity in freshly harvested bone-marrow, with significantly decreased granulocyte numbers, and lack an eosinopoietic response to GC, in vitro and in vivo, but show normal responsivess to PgE2. COX inhibitors reconstituted eosinopoiesis in mutant bone-marrow in vitro in a 5-LO- and cysteinyl-leukotriene-dependent manner. Hence, WASP is relevant to bone-marrow cellularity in vivo, as well as to GC signaling. This highlights the interest of further studying the influence of GC on in vivo granulocyte production, and enables us to distinguish, based on a biochemical criterion (WASP-dependency), the targets of GC from the pathway used by cysteinyl-leukotrienes to prevent iNOS-CD95L-mediated suppression of eosinopoiesis / 2100-04-28

Made available in DSpace on 2016-05-16T12:55:49Z (GMT). No. of bitstreams: 2 larissa_fontes_ioc_mest_2016.pdf: 4617034 bytes, checksum: eee0cf93cb18ec86d4b978b114c27212 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A Síndrome de Wiskott-Aldrich (WAS \2013 \201CWiskott-Aldrich Syndrome\201D) é uma imunodeficiência primária associada a infecções recorrentes e ao desenvolvimento de doenças autoimunes e linfomas. Essa imunodeficiência se desenvolve em consequência de mutações no gene que codifica a proteína WASP (\201CWAS Protein\201D), expressa em células hematopoiéticas, e com funções reguladoras sobre os rearranjos do citoesqueleto de actina e na ativação de linfócitos T. Nesse sentido, camundongos deficientes em Wasp (Wasp \201Cknockout\201D \2013 WKO) apresentam desregulação de processos centrais para a resposta imunológica, resultando em migração leucocitária aberrante, proliferação linfocitária deficiente, e disfunção de células T reguladoras (Treg). De forma interessante, foi demonstrado que as células Treg, definidas pelo fenótipo CD4+CD25+Foxp3+ e com função imunossupressora, já se encontram em menor número no timo desses animais. Esse dado sugere que a deficiência em Wasp resulta em defeito na geração intratímica de células Treg. Nesse contexto, é importante salientar que a migração do timócito através do microambiente tímico é crucial para a formação das células T e, particularmente, a matriz extracelular exerce uma função determinante para os eventos de adesão e de-adesão da célula migrante. Assim, podemos postular que a matriz extracelular medeia interações cruciais para as células Treg tímicas (tTreg), fornecendo e facilitando o acesso aos diferentes sinais celulares durante o processo de maturação dessas células Nesse projeto, decidimos estudar a participação de ligantes e receptores da matriz extracelular na maturação de células tTreg e o possível envolvimento dessas moléculas na deficiência numérica de células tTreg em camundongos WKO. Foi observada uma diminuição da rede de fibronectina na região medular do timo dos animais WKO em relação ao controle. Além disso, observamos uma redução de células tTreg expressando VLA-4 e VLA-5 nos animais WKO em relação ao controle. As análises de proliferação, ativação e morte demonstraram que as células Treg de animais WKO apresentam alterações em relação às células precursoras (CD4+CD8-CD25+Foxp3-). De forma interessante, as análises de adesão em fibronectina demonstraram que as células Treg dos animais WKO apresentam menos filopódios que as células Treg do controle, e que essa diminuição é proeminente comparando-se às células T CD4 SP. Esses resultados sugerem que a fibronectina tenha importância na diferenciação das células tTreg / Abstract: The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency associated with recurrent infection and development of autoimmune diseases and lymphomas. This immune deficiency develops as a result of mutations in the gene encoding the WASP protein ("WAS protein"), expressed in hematopoietic cells, and with regulatory functions on the actin cytoskeleton rearrangements and activation of T lymphocytes. Accordingly, mice deficient in Wasp (Wasp "knockout" - WKO) present disruption in central processes to the immune response, resulting in aberrant leukocyte migration, impaired lymphocyte proliferation and dysfunction of regulatory T cells (Treg). Interestingly, it was shown that Treg cells defined by the CD4+CD25+Foxp3+ phenotype and immunosuppressive function, already decreased in the thymus of the animals. This suggests that the Wasp deficiency results in defective intrathymic generation of Treg cells. In this context, it is important to note that the migration of the thymocyte through the thymic microenvironment is crucial for the formation of T cells and, particularly, the extracellular matrix has a decisive role in the events of adhesion and de-adhesion of migrant cell Thus, we postulated that the extracellular matrix mediates interactions crucial for thymic Treg cells (tTreg) by providing and facilitating access to different cellular signals during the maturation process of these cells. In this project, we decided to study the role of ligands and receptors of extracellular matrix in the maturation of tTreg cells and the possible involvement of these molecules in numerical deficiency tTreg WKO cells in mice. We observed a decrease in fibronectin network in the medulla of the thymus of WKO animals compared to the control. In addition, a decrease in cells expressing VLA-4 and VLA-5 in WKO animals compared to the control. The analysis of proliferation, activation, and death demonstrated that Treg cells animals in WKO showed changes from precursor cells (CD4+CD8-CD25+Foxp3-). Interestingly, adhesion on fibronectin showed that Treg cells from WKO animals present less filopodia compared to control Treg cells and that this is a marked decrease as compared to the CD4 SP cells. These results suggest an important role for fibronectin in the differentiation of Treg cells

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, autoimmunity, and predisposition to malignancy. Mutations in the WAS gene lead to dysfunction of the Wiskott-Aldrich syndrome protein (WASP), a multifunctional regulator implicated in various hematopoietic and immune processes. While some disease phenotypes have been linked to classical WASP's actin nucleation function, recent advances have unveiled additional nuclear functions, such as involvement in R-loop formation, transcriptional regulation during T helper 1 cell differentiation, and homology-directed repair. However, a comprehensive understanding of WASP's multifaceted functions remains elusive. We employ induced pluripotent stem cells (iPSCs) and Clustered Regularly Interspaced Palindromic Repeats (CRISPR) technology, specifically the CRISPR-Cas9 system, as powerful tools to investigate the mechanisms underlying WASP's functions. We first explore the consequences of WASP loss on RNA splicing. We reveal its critical role in RNA splicing. WASP-deficiency causes widespread alterations RNA splicing patterns and epigenetic activation of splicing factor gene promoters. Additionally, we uncover its involvement in liquid-liquid phase separation, forming phase-separated condensates to dynamically regulate the splicing machinery. In the second part of this thesis, our investigation uncovers the presence of WASP within the nucleolus and its interactions with key nucleolar proteins. Intriguingly, depletion of WASP leads to significant reduction in nucleolar size, disrupted nucleolar morphology, and decreased ribosomal RNA transcription, unveiling its critical role in nucleolus structure and function. Furthermore, we successfully recapitulated nucleolus changes and ribosomal RNA profile in patient samples. Lastly, we investigate immunosenescence, a crucial aspect of aging-related immune dysregulation, in the context of WAS. Through the use of WASP-deficient macrophage cells, our study revealed several distinctive features associated with immunosenescence in WASP-KO-iMPs. These include increased senescent cell proportions, heightened expression of senescence-associated secretory phenotype genes, nuclear deformation, loss of heterochromatin, and enhanced susceptibility to DNA damage. These preliminary findings offer valuable insights into our understanding of immunosenescence within the framework of WASP-deficient macrophages and its association with conditions related to WAS. In conclusion, the mechanistic study of WASP has unveiled its novel roles in regulating RNA splicing, nucleolus structure and function, as well as its potential involvement in immunosenescence.

Plusieurs populations de cellules immunitaires possèdent une activité cytotoxique permettant l'élimination de cellules altérées. Cette fonction cellulaire est ainsi déterminante dans le contrôle des infections, des processus tumoraux, ou encore des maladies inflammatoires chroniques. Mon projet de thèse se concentre sur des aspects fondamentaux de l'activité lytique de deux populations de lymphocytes T cytotoxiques : les lymphocytes T CD8+ et les lymphocytes T CD4+ régulateurs de type 1. Pour cela, l'exploration des mécanismes de cette activité a été conduite au travers de deux modèles, pathologique et thérapeutique, à différentes échelles biologiques : au niveau de la population ou de la cellule individuelle, mais aussi différentes échelles d'organisations moléculaires : cellulaire et nanoscopique. Nous avons pu démontrer que l'activité de lyse de lymphocytes T CD8+ cytotoxiques face à un excès de cellules cibles est efficace sur des temps prolongés, reposant sur une capacité individuelle fortement hétérogène à effectuer une lyse multiple. L'importance de cette activité de lyse soutenue a été renforcée par l'identification d'un défaut lytique particulièrement prononcé sur le long- terme chez des lymphocytes T CD8+ issus de patients atteints du syndrome de Wiskott-Aldrich. Ce défaut est lié à une activation réduite de l'intégrine LFA-1 et un délai dans la délivrance du coup létal. De plus, la protéine WASP permet de restreindre LFA-1 de haute-affinité en nanoclusters denses ainsi que de permettre l'organisation en un ring de LFA-1 et la localisation des granules lytiques à l'intérieur de celui-ci. Par ailleurs, les lymphocytes T CD4+ régulateurs de type 1 développés dans le cadre d'une thérapie cellulaire (Ovasave(r)) démontrent une capacité de lyse envers les cellules myéloïdes, en complément d'une activité immunosuppressive sur les lymphocytes T conventionnels. Cette activité est mise en place sur du long-terme, jusqu'à atteindre une efficacité optimale, lié à un délai dans la délivrance du coup létal. De manière surprenante, malgré une spécificité pour l'ovalbumine, l'activité cytotoxique semble être indépendante de l'activation du TCR. En outre, la lyse est granzyme-dépendante mais perforine-indépendante. Ainsi ces lymphocytes T thérapeutiques manifestent une activité cytotoxique alternative. Pour conclure, mon projet de thèse a permis de caractériser une activité de lyse soutenue basée sur une capacité individuelle hétérogène. Cette habilité à soutenir une lyse sur du long-terme implique une stabilité de la synapse, où WASP joue notamment un rôle clé pour l'activation et l'organisation de LFA-1. Les lymphocytes T régulateurs thérapeutiques démontrent aussi une activité de lyse soutenue, cependant les acteurs moléculaires sont non conventionnels. De manière générale, une activité de lyse soutenue permettrait de calibrer une réponse cytotoxique prolongée en rapport à la taille de la population cible, ainsi que le partage avec d'autres fonctions cellulaires comme la sécrétion de cytokines. / During different pathological conditions such as infections, tumoral processes or chronic inflammation diseases, altered cells are eliminated through a cytotoxic activity mediated by several immune cell populations. This cellular function is therefore crucial for carrying out the action of the immune system. My thesis project focuses on fundamental aspects of the lytic activity of two cytotoxic lymphocyte populations: CD8+ T cells and type-1 CD4+ regulatory T cells. To explore the mechanisms of this activity, this study has been driven on two cases, pathological and therapeutic models, at the population and single-cell levels and also at the cellular and nanoscopic scales of the molecular organisation. We have been able to demonstrate that the CD8+ T cell lysis activity against an excess of target cells is effective over prolonged periods, relying on a highly heterogeneous individual capacity to perform multiple lysis. The importance of this sustained cytotoxic activity was reinforced by the identification of a lytic defect, particularly pronounced on a long time period, of CD8+ T cells from Wiskott-Aldrich syndrome patients. This defect is related to a reduced activation of the LFA-1 integrin and delay in the lethal hit delivery. In addition, the WASP protein allows to restrict high affinity LFA-1 to dense nanoclusters as well as the assembly of LFA- 1 ring and the localization of the lytic granules inside this ring. Moreover, type-1 CD4+ regulatory T cells from a cellular therapy (Ovasave(r)) demonstrated a cytotoxic activity toward myeloid cells, additionally to an immunosuppressive activity on conventional T cells. This activity is implemented over long time periods, until reaching optimal efficiency, and is related to a delay in the lethal hit delivery. Surprisingly, despite a specificity for ovalbumin, the cytotoxic activity measured in absence of the antigen suggests a TCR independence. In addition, lysis is not mediated by perforin but is exclusively granzyme-dependent. Thus, these therapeutic T cells exhibit an alternative cytotoxic activity. To conclude, my thesis project permits to characterize a sustained lysis activity relying on a heterogeneous individual capacity. This ability to sustain a lytic activity involves stability of the synapse, where WASP plays a key role towards the activation and organization of LFA-1. The therapeutic regulatory T lymphocytes also demonstrated a sustained cytotoxic activity, however the molecular actors are unconventional. On the whole, sustained lytic activity would be key to the calibration of cytotoxic responses in relation to the size of the target population, as well as sharing with other cellular functions such as cytokine secretion.

This study reveals how the Pacific war changed at Coral Sea and Midway due to a little known but important cadre of sailors on USS Yorktown (CV5). Those US victories resulted from not only clever code breakers and courageous airmen but equally from the determined Damage Control (DC) crews aboard Yorktown. DC crews were the ship’s first responders. They fought fires, kept power and propulsion operable, controlled the ship’s stability, and patched her flight deck to keep aircraft flying. DC teams saved Yorktown multiple times, and their story is memorable for their contributions at Coral Sea and Midway. Without DC efforts, CV5 would not have participated in the battle of Midway. Without Yorktown, the commitment of only two American carriers (with one being virtually inexperienced) against four Japanese carriers with their skilled airmen would have yielded disaster for the United States at Midway instead of victory.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.