The metabolic state of an immune cell directly influences its ability to function and differentiate, ultimately affecting immunity, inflammation and tolerance. Different immune cell subsets have differing metabolic requirements. Macrophages, as the frontline, tissue-resident cells of the innate immune system, undergo profound metabolic reprogramming in response to environmental stimuli. To date, there has been little consideration how macrophage metabolism might be affected by humoral immunity. IgG antibodies are the soluble effector molecules of the adaptive humoral immune system. Fcγ receptors (FcγRs) mediate the cellular functions of IgG antibodies and are expressed on most immune cells including macrophages. FcγR cross-linking induced by IgG immune complexes (ICs) is important for defence against some infections but can also play a pathogenic role in autoimmunity. Here, I studied the metabolic reprogramming induced in macrophages by IgG IC ligation of FcγRs. I first investigated how FcγRs cross-linking might impact glucose metabolism. We show that macrophages undergo a switch to glycolysis in response to IgG IC stimulation. FcγR-associated glycolysis was dependent on the mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF)1α. Moreover, this glycolytic switch was required to generate a number of pro-inflammatory mediators and cytokines. Inhibition of glycolysis, or genetic depletion of HIF1α in macrophages resulted in the attenuation of IL1β and other inflammatory mediators produced in response to IgG IC in vitro. To determine the relevance of these observations to responses to IgG IC in vivo and, in particular, to IC-associated tissue inflammation in autoimmune diseases such as system lupus erythematosus (SLE), I developed three models to interrogate tissue macrophages. Following administration of IC to peritoneal macrophages, I observed IL1β-associated neutrophil recruitment that was abrogated by inhibiting glycolysis, or in the presence of HIF-1a deficiency. Similarly, following administration of intravenous IC, or nephrotoxic serum, kidney macrophage activation was abrogated by glycolysis inhibition or by myeloid HIF-1a deficiency. Together my data reveal the cellular molecular mechanisms required for FcγR-mediated metabolic reprogramming in macrophages and define a novel therapeutic strategy in autoantibody-induced inflammation. In the final part of the thesis I identified additional metabolic pathways that were altered by FcγR ligation, including cholesterol biosynthesis and fatty acid biosynthesis. This has important implications for protective immune responses and autoimmune susceptibility, since a number of intermediates in these pathways can directly regulate and contribute to immune responses. In summary, I have demonstrated the metabolic alterations triggered by FcγR ligation, reveal the cellular molecular mechanisms required for FcγR-mediated cellular respiration reprogramming in macrophages and define a potential therapeutic target in autoimmunity.

Glicocorticoides e outras drogas imunossupressoras são comumente utilizados para o tratamento de condições inflamatórias, como as Doenças Inflamatórias Intestinais (DIIs). Apesar dos avanços na terapia medicamentosa, a remissão da doença ainda é difícil de ser mantida. Devido às suas propriedades imunomodulatórias, as Células-Tronco Mesenquimais (MSCs – Mesenchymal Stem Cells) têm emergido como reguladoras da resposta imune, e sua viabilidade e propriedades migratórias são essenciais para o sucesso da terapia celular. Entretanto, pouco se conhece sobre os efeitos das drogas convencionalmente utilizadas no tratamento das DIIs no comportamento das MSCs. Portanto, o objetivo deste estudo foi avaliar a viabilidade, a morfometria nuclear, a polaridade celular, a distribuição da actina-F e da FAK (Focal Adhesion Kinase), e o comportamento migratório das MSCs na presença das drogas Azatioprina (AZA) e Dexametasona (DEXA). As células foram isoladas de membranas coriônicas humanas e caracterizadas pela diferenciação em adipócitos e osteócitos, bem como pela expressão de um painel de marcadores de superfície. As MSCs foram previamente tratadas com AZA ou DEXA por 24h ou 7d nas concentrações de 1μM ou 10μM, respectivamente. Ambas as drogas não afetaram a viabilidade celular analisada por MTT (3-(4,5-dimethyltiazol-2-yl)-2,5- diphenyltetrazolium bromide) e morfometria nuclear. Entretanto, a análise do índice de polaridade resultou em uma morfologia mais alongada após o tratamento com AZA, enquanto células mais arredondadas foram observadas na presença de DEXA. Os filamentos de actina foram marcados por Rodamina-Faloidina e sua análise mostrou que a AZA preservou parcialmente a formação de lamelipódios e aumentou a presença de fibras de estresse ventrais, enquanto que a DEXA inibiu a formação de lamelipódios, evidenciou uma maior presença de fibras de estresse ventrais e diminuiu a estabilidade das protrusões de membrana, observadas em vídeo. Através da análise de microscopia de série temporal, foi observado que as células sob o efeito da AZA por 7d migraram por maiores distâncias e tiveram um aumento em sua velocidade de migração (24,35%; P < 0,05; n = 4), ao passo que a DEXA diminuiu a velocidade migratória em 24h e 7d (-28,69% e -25,37%, respectivamente; P < 0.05; n = 4) e diminuiu a distância alcançada pelas células. Em conclusão, nossos dados sugerem que as drogas AZA e DEXA podem afetar diferentemente a morfologia e o comportamento migratório das MSCs, possivelmente afetando o resultado da terapia celular. O protocolo de migração celular utilizado neste estudo foi estabelecido por nosso grupo de pesquisa, sendo que um artigo científico contendo todas as etapas do protocolo foi escrito para que outros laboratórios possam utilizá-lo de maneira simples e eficaz. / Glucocorticoids and other immunosuppressive drugs are commonly used to treat inflammatory disorders, such as Inflammatory Bowel Disease (IBD) and, despite few improvements, the remission of IBD is still difficult to maintain. Due to its immunomodulatory properties, Mesenchymal Stem Cells (MSCs) have emerged as regulators of immune response, and its viability and activation of migratory properties are essential for a successful cell therapy. However, little is known about the effects of immunosuppressant drugs used on IBD treatment on MSCs behavior. In this way, the aim of this study was to evaluate MSCs viability, nuclear morphometry, cell polarity, F-actin and FAK (Focal Adhesion Kinase) distribution and cell migration properties in the presence of the immunosuppressive drugs Azathioprine (AZA) or Dexamethasone (DEX). MSCs were isolated from human chorionic membranes and characterized through adipogenic and osteogenic differentiations, as well as a panel of surface markers. Cells were previously treated with AZA or DEX for 24 hrs or 7 days at 1μM and 10μM, respectively. Both drugs had no effects on cell viability analyzed through MTT (3-(4,5- dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide) and nuclear morphometry. However, polarity index analysis showed that AZA treatment induced a more elongated cell shape while a greater presence of rounded cells was observed under DEX exposure. F-actin was stained by Rhodamine-Phalloidin and showed that AZA could partially preserve lamellipodia formation and increase the presence of ventral actin stress fibers, while DEX inhibited lamellipodia formation and increased the presence of ventral actin stress fibers while decreasing protrusion stability, observed in video. Through time-lapse microscopy, it was observed that after 7 days of treatment, AZA improved cell the spatial trajectory (ST) and increased migration speed (24.35%, P < 0.05, n = 4) while DEX impaired ST and migration speed after 24 hrs and 7 days treatment (- 28.69% and -25.37%, respectively; P < 0.05, n = 4). In conclusion our data suggests these immunosuppressive drugs can differently affect MSCs morphology and migration capacity, possibly impacting the success of cell therapy. The migration protocol used in this study was successfully established by our group, leading to the writing of a protocol paper to facilitate the usage of this technique by other laboratories in a simple and efficient manner.

Les GTPases Ral, RalA et RalB, sont des effecteurs proximaux de l’oncogène Ras.Malgré leur forte homologie, leurs activateurs communs (les RalGEFs) et des effecteurs communs (le complexe exocyste), ils apportent des contributions distinctes et parfois collaborent à diverses fonctions cellulaires.RalA est impliqué en prolifération en absence de substrat et l'exocytose polarisée.RalB est impliqué dans la migration cellulaire, l'autophagie et l'apoptose des cellules cancéreuses. Comment les GTPases Ral régulent ces différents fonctions n’est toujours pas connu.Une partie de ma thèse était consacrée à l'étude de la spécificité des fonctions de RalA et RalB, ainsi que la spécificité des RalGEFs et des éléments de l'interactome deRal, dans trois processus biologiques: la cytocinèse, la migration cellulaire et l'activation de la voie MAPK.Nous avons démontré que RalA et RalB ont des fonctions distinctes pendant la cytocinèse. RalA est nécessaire pour la correcte progression de la cytocinèse alors RalB est nécessaire pour l’abscission du pont intracellulaire. Nous avons montré également que RalA, mais pas RalB, régule l’activation de p38 et de Jnk à travers le complexe exocyste en réponse au stress osmotique. L'implication de RalB, mais pas RalA, dans la migration cellulaire a été établie antérieurement. Dans ces trois fonctions, nous avons montré que les GTPases Ral sont été régulées par des RalGEFs spécifiques.Nous avons effectué un crible par siARN de 91 gènes codant des protéines du réseau d’interactions protéine‐protéine autour de Ral (l'interactome de Ral), nous avons identifié 14 protéines impliquées dans la voie de RalA et 8 protéines impliquées dans la voie de RalB, en cytocinèse. Dans la migration cellulaire, nous avons identifié 22 protéines impliquées dans la voie de RalB. Nous avons identifié cinq protéines communes aux deux fonctions cellulaires.Parmi ces protéines, j'ai étudié la relation fonctionnelle entre RalA et Stk38, une kinase qui appartient à la voie Hippo, qui a un rôle suppresseur de tumeur. J'ai montré que RalA active Stk38 par une voie RalA/exocyste/Map4k4 en réponse au stress osmotique. J’ai démontré que cette voie est impliquée dans l’activation de la voie p38 et Jnk en réponse au stress osmotique. J'ai aussi montré que la régulation deStk38 par RalA est nécessaire pour l'apoptose induite par le TNFα.L'identification de nouveaux composants de la voie RalA ouvre de nouvelles perspectives dans la compréhension de la fonction des GTPases Ral dans les processus normaux et tumoraux. En outre, ce travail est le premier présentant RalA comme une protéine pro‐apoptotique, ce qui suggère que RalA pourrait posséder une fonction suppresseur de tumeurs. / The Ras‐like GTPases RalA and RalB are proximal effectors of oncogenic Ras.Despite their high homology, their common activators (the RalGEFs) and effectors(the exocyst complex), they make distinct and sometimes collaborative contributions to diverse cellular functions. RalA supports anchorage independent growth and regulates polarized exocytosis. RalB regulates cell migration and autophagy and inhibits apoptosis of cancer cells. How Ral GTPases achieve their differing functions is still elusive.One part of my thesis was dedicated to study the specificity of RalA and RalB functions, as well as the specificity of RalGEFs functions and of the components of the Ral interactome, in three biological processes: cytokinesis, cell migration and MAPK activation.We demonstrated that RalA and RalB have distinct functions during cytokinesis.RalA is necessary for correct progression of cytokinesis whereas RalB is necessary for abscission of the intracellular bridge. We showed also that RalA, but not RalB,regulates p38 and Jnk activation upon osmotic stress through the exocyst complex.The importance of RalB, but not RalA, in cell migration was established previously. In these three functions, we showed that the functions of Ral GTPases were triggered by specific RalGEFs.We carried out a siRNA screen of 91 genes encoding proteins participating to a protein‐protein interaction map rooted in Ral (the Ral interactome), we determined14 proteins as components of RalA pathway and 8 proteins as components of RalBpathway, required for cytokinesis completion. In cell migration, we determined 22 proteins as components of RalB pathway. We identified 5 proteins in common involved in both cellular functions.Among these proteins I have been studying the functional relationship betweenRalA and Stk38, a kinase that belongs to the tumour suppressor Hippo pathway. I showed that upon osmotic stress, RalA activates Stk38 by phosphorylation through aRalA/exocyst/Map4k4 pathway. I demonstrate that this pathway has the function to trigger p38 and Jnk activation upon osmotic stress. I showed that the regulation ofStk38 by RalA is required for apoptosis induced by TNFα.The identification of new components of Ral pathway opened new perspectives in understanding the Ral GTPases function in normal and tumour processes. Moreover,this is the first work presenting RalA as a pro‐apoptotic protein, suggesting that RalAmight have tumour‐suppressor like functions.

Les cellules sont organisées en tissus dont les propriétés physiques comme la rigidité et l'élasticité sont variables. La matrice extracellulaire (MEC) est produite et remodelée par les cellules qui s'adaptent en retour aux conditions physico-chimiques de cet environnement extracellulaire. Cela nécessite une communication bidirectionnelle entre la cellule et la matrice. Les intégrines sont des protéines transmembranaires impliquées dans l'adhérence, liant la MEC au cytosquelette d'actine via une plateforme protéique appelée adhérence focale, lieu d'une double signalisation (inside-out et outside-in). Des variations de tension intracellulaire imposées par l'environnement modifient la distribution et la taille de ces adhérences. Leur dynamique est aussi contrôlée par certaines protéines cytoplasmiques comme la protéine ICAP-1, partenaire de l'intégrine b1. En cherchant à comprendre le lien entre la tension interne et l'activation des intégrines, j'ai montré qu'ICAP-1 contrôle l'étalement, la contractilité interne et la migration cellulaire en présence comme en absence de l'intégrine b1, révélant un rôle ICAP-1 indépendant de son interaction avec l'intégrine b1. Ce contrôle semble passer par l'interaction ICAP-1/ROCK et a révélé un contrôle de l'intégrine b3 par l'intégrine b1. / The physical properties of cell tissues are variable and cells adapt their behaviour to the physical and chemical extracellular environment such as rigidity and composition of the extracellular matrix (ECM) which is produced and remodelled by cells. This implicates a bidirectionnal signalling between cells and the ECM. Integrins are transmembrane proteins involved in cell adhesion, linking the ECM to the actin cytoskeleton through adaptor proteins forming adhesion site called focal adhesion (FA) where take place an inside-out and an outside-in signallings. Intracellular tension can be controlled by extracellular cues, modifying the size and distribution of FA. FA dynamics is also regulated by cytoplasmic proteins such as ICAP-1 that interacts with b1 integrin. Looking for a better comprehension of the link between cell tension and integrin activation, I show that ICAP-1 controls cell spreading, cell contractility and cell migration both in presence or absence of b1 integrins meaning that ICAP-1 has an action without its interaction b1 integrin. This action seems to implicate the interaction between ICAP-1 and ROCK and revealed a control of b1 integrin on b3 integrin.

A cromoblastomicose é uma micose subcutânea, com alto índice de morbidade, sendo Fonsecaea pedrosoi (F. pedrosoi) considerado o maior agente etiológico dessa micose, caracterizando uma doença crônica, geralmente confinada na pele e tecidos subcutâneos. Raramente os indivíduos apresentam cura dessa doença, pois as terapias contemporâneas mostram-se deficientes e poucos trabalhos relatam a relação parasito-hospedeiro. As células dendríticas (DCs) são especializadas na apresentação de antígenos para linfócitos T naive induzindo respostas imunes primárias. Diante disso, propomos estudar a capacidade migratória de DCs após infecção com conídios de F. pedrosoi, uma vez que o processo de migração dessas células está intimamente ligado com a sua função sobre as células T, levando ao desenvolvimento de uma resposta imune adaptativa protetora. O fenótipo de DCs foi avaliado através de células obtidas dos linfonodos poplíteos, inguinais e patas de camundongos BALB/c após 12, 24 e 72 horas de infecção com conídios do fungo. Células obtidas foram marcadas com anticorpos específicos e analisadas por citometria de fluxo. Após 24 e 72 horas de infecção verificamos uma diminuição significativa na porcentagem de DCs nas patas, e um aumento significativo dessas células nos linfonodos após 72 horas. A expressão de marcadores de superfície como CCR7 e moléculas co-estimulatórias, mostraram-se diminuídas nas células obtidas das patas. Como no processo de imunofenotipagem podemos analisar DCs vindas de diversos locais, para melhor avaliar a capacidade migratória das DCs, células das patas foram marcadas in vivo injetando-se subcutaneamente o corante CFSE juntamente com conídios do fungo. Verificamos que após 12 e 72 horas, as DCs das patas dos animais infectados, migraram para os linfonodos regionais. Assim constatamos que o fungo F. pedrosoi é capaz de induzir a migração de macrófagos e neutrófilos para o sítio de infecção em poucas horas, leva ao aumento de células B nos linfonodos após 12 e 72 horas de infecção, e também leva ao aumento de linfócitos T CD4+ tanto no local de infecção quanto nos linfonodos. Os resultados também comprovam que o fungo F. pedrosoi foi capaz de ativar as DCs, induzindo sua migração para os linfonodos regionais. / The chromoblastomycosis is a subcutaneous mycosis with a high morbidity rate, Fonsecaea pedrosoi (F. Pedrosoi) being the largest etiologic agent of this mycosis, featuring a chronic disease, usually confined to the skin and subcutaneous tissues. Rarely do people have cure for this disease, because the therapies shown to be deficient contemporary and few studies report the host-parasite relationship. Dendritic cells (DCs) are specialized in presenting antigens to naïve T lymphocytes inducing primary immune responses. Therefore, we propose to study the migratory capacity of DCs after infection with conidia of F. pedrosoi, since the migration of these cells is intimately linked to its function on T cells, leading to development of a protective adaptive immune response. The phenotype of DCs was evaluated using cells obtained from popliteal lymph nodes, sub cutaneous tissue of BALB/c mice after 12, 24 and 72 hours of infection with conidia of the fungus. Cells were labeled with specific antibodies and analyzed by flow cytometry. After 24 and 72 hours of infection we found a significant decrease in the percentage of DCs in the sub cutaneous tissue, and a significant increase of these cells in the lymph nodes after 72 hours. The expression of surface markers such as CCR7 and costimulatory molecules, were reduced in cells obtained from the sub cutaneous tissue. Like the process of immunophenotyping we can analyze DCs coming from various locations, to better assess the migratory capacity of DCs, cells were stained paws in vivo by injecting dye subcutaneously with CFSE conidia of the fungus. We found that after 12 and 72 hours, DCs sub cutaneous tissue of infected animals migrated to regional lymph nodes. We found that the fungus F. pedrosoi is capable of inducing migration of macrophages and neutrophils to the site of infection within a few hours, leads to increased B-cells in lymph nodes after 12 and 72 hours of infection, and also leads to an increase of CD4 + T lymphocytes in both the site of infection and in the lymph nodes. The results also show that the fungus F. pedrosoi was able to activate the DCs, inducing their migration to regional lymph nodes.

Dong Ming. / Thesis submitted in: December 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 118-152). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v / Abbreviation list --- p.xi / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- Preamble --- p.1 / Chapter 1.2 --- Neural Crest Cells (NCCs) --- p.2 / Chapter 1.3 --- Enteric Nervous System (ENS) and Vagal Neural Crest Cells (Vagal NCCs) --- p.4 / Chapter 1.4 --- Sacral Neural Crest Cells (Sacral NCCs) --- p.7 / Chapter 1.5 --- Signalling Mechanisms of Sacral Neural Crest Cells --- p.17 / Chapter 1.6 --- Hirschsprung's Disease (HSCR) --- p.20 / Chapter 1.7 --- Objective of the Study and Contents of the Following Chapters --- p.21 / Chapter Chapter 2 --- Migration from the dorsal neural tube to the pelvic mesenchyme / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.32 / Chapter 2.2.1 --- Animal --- p.32 / Chapter 2.2.2 --- Isolation of embryos from pregnant mice at E9.5 to --- p.32 / Chapter 2.2.3 --- Histological preparation of the caudal segments --- p.33 / Chapter 2.2.4 --- p75 immunohistochemical staining --- p.33 / Chapter 2.2.5 --- Preparation of rat serum --- p.34 / Chapter 2.2.6 --- Preparation of the culture medium --- p.34 / Chapter 2.2.7 --- Preparation of wheat germ agglutinin-gold conjugates (WGA-Au) --- p.35 / Chapter 2.2.8 --- Preparation of CMFDA --- p.35 / Chapter 2.2.9 --- Preparation of DiI --- p.36 / Chapter 2.2.10 --- "Microinjection of WGA-Au, DiI and CMFDA" --- p.36 / Chapter 2.2.11 --- Whole embryo culture --- p.37 / Chapter 2.2.12 --- Examination of cultured embryos --- p.37 / Chapter 2.2.13 --- Histological preparation of WGA-Au labelled embryos --- p.38 / Chapter 2.2.14 --- Silver enhancement staining of the sections of WGA-Au labelled embryos --- p.39 / Chapter 2.2.15 --- Cryosectioning of the embryos labelled with DiI --- p.39 / Chapter 2.2.16 --- p75 immunohistochemical staining of DiI-labelled cells --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Observations on embryos developed in vivo --- p.41 / Chapter 2.3.2 --- Closed yolk sac culture vs open yolk sac culture --- p.42 / Chapter 2.3.3 --- Neural crest cell labelling in the caudal part of embryos --- p.43 / Chapter 2.3.4 --- Neural crest cell labelling with DiI in the caudal part of the neural tube followed by in vitro culture from E9.5 to E11.0 --- p.45 / Chapter 2.3.5 --- Neural crest labelling with DiI in the caudal part of the neural tube followed by in vitro culture from E10.5 to E11.5 --- p.46 / Chapter 2.3.6 --- Focal labelling at the levels of the 26th and 29th somites followed by in vitro culture --- p.48 / Chapter 2.3.7 --- p75 immunohistochemical staining on the caudal part of the embryo at E10.5 --- p.49 / Chapter 2.3.8 --- p75 immunohistochemical staining on embryos labelled with DiI --- p.50 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Embryos at E9.5 cultured with an intact yolk sac membrane grew better than those with the yolk sac membrane cut open --- p.52 / Chapter 2.4.2 --- Migration at the levels of the 24th to 28th somite --- p.53 / Chapter 2.4.3 --- Migration at the levels of the 29th to 33th somite --- p.58 / Chapter 2.4.4 --- Sacral NCCs migrate along a straight dorsolateral pathway --- p.60 / Chapter 2.4.5 --- "Most of the DiI positive cells are p75 positive, but not all of the p75 positive cells are DiI positive" --- p.62 / Chapter Chapter 3 --- Migration from the pelvic mesenchyme to the hindgut / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Isolation of hindguts with or without adjacent tissues from embryos at E10.5 to E13.5 --- p.73 / Chapter 3.2.2 --- Microinjection of DiI into the pelvic mesenchymal tissue of the h indguts --- p.74 / Chapter 3.2.3 --- Preparation of the culture medium --- p.74 / Chapter 3.2.4 --- Preparation of the culture dish --- p.74 / Chapter 3.2.5 --- Gut culture --- p.75 / Chapter 3.2.6 --- Cryosections of the hindguts after in vitro culture --- p.75 / Chapter 3.3 --- Results --- p.76 / Chapter 3.3.1 --- "Hindguts isolated from embryos at E10.5, E11.5, E12.5 and E14.5" --- p.76 / Chapter 3.3.2 --- p75 immunohistochemical staining of the serial sections through the caudal part of the embryos --- p.78 / Chapter 3.3.3 --- Observations on hindgut without pelvic plexus cultured from E11.5 to E14.5 --- p.81 / Chapter 3.3.4 --- "Culture of hindguts with pelvic mesenchyme cultured from E11.5 to E14.25, E14.5 and E15.5" --- p.82 / Chapter 3.3.5 --- Culture of the whole length of the gut tube without pelvic mesenchyme from E11.5 to E15.5 --- p.84 / Chapter 3.3.6 --- Culture of the whole length of the gut tube with pelvic mesenchyme from E11.5 to E15.5 --- p.84 / Chapter 3.3.7 --- "Culture of hindguts with Dil labelling in the pelvic mesenchyme from E11.5 to E14.0, E14.2 5, E14.5 and" --- p.85 / Chapter 3.3.8 --- Culture of the whole length of the gut tube with DiI labelling in the pelvic mesenchyme from E11.5 to E14.5 --- p.86 / Chapter 3.4 --- Discussion --- p.88 / Chapter 3.4.1 --- Development of the hindgut and the urogenital system from E10.5 to E14.5 --- p.88 / Chapter 3.4.2 --- No p75 positive cells were found in the hindgut before E13.5 --- p.89 / Chapter 3.4.3 --- The sacral neural crest cells migrate into the hindgut at around E14.5 --- p.91 / Chapter 3.4.4 --- "The sacral neural crest cells migrated in the serosa, and entered the myenteric plexus prior to populating the submucosal plexus" --- p.95 / Chapter 3.4.5 --- Most of DiI labelled sacral neural crest cells in the hindgut also expressed p75 --- p.98 / Chapter Chapter 4 --- Migration from the neural tube to the hindgut / Chapter 4.1 --- Introduction --- p.101 / Chapter 4.2 --- Materials and Methods --- p.104 / Chapter 4.3 --- Results --- p.106 / Chapter 4.3.1 --- Morphology observations on the hindguts isolated from DiI-labelled embryos --- p.106 / Chapter 4.3.2 --- Distribution of the DiI-labelled cells and p75 positive cells before the culture of the hindgut explant --- p.106 / Chapter 4.3.3 --- Culture of hindgut explanted from Dil-labelled embyos for 3.5 days from E11.0 to E14.5 --- p.107 / Chapter 4.4 --- Discussion --- p.109 / Chapter Chapter 5 --- General discussion and conclusions --- p.113 / References --- p.118 / Figures and Legends --- p.153 / Tables and Graphs --- p.203 / Appendix --- p.209

人類先天性巨結腸症（HSCR）主要表現為結腸末端的神經節缺失或稀少。 結腸末端的神經節來源於迷走源性神經脊細胞和骶源性神經脊細胞。迷走源性神經脊細胞是腸神經系統的主要來源，已被廣泛研究，而關於哺乳類包括人類的骶源性神經脊細胞的研究卻相當稀少。小鼠胚胎的骶源性神經脊細胞遷移途徑近期已被闡釋，其由神經管背側遷出並向腹側移動，於後腸附近聚集成旁神經節然後進入腸內。本研究運用一系列實驗鑒定了對小鼠骶源性神經脊細胞由旁神經節遷移至腸內過程有影響作用的因素。 / 研究發現骶源性神經脊細胞是沿著神經纖維並向口端方向遷移進腸內，同時由於迷走源性神經脊細胞於腸內向尾端遷移，它們在後腸末端相遇並相互作用，我們首先研究了這種相互作用以瞭解迷走源性神經脊細胞如何影響骶源性神經脊細胞的遷移。利用帶綠色螢光的小鼠骶源性神經脊細胞和可變螢光的小鼠迷走源性神經脊細胞（鐳射激發下由綠色變為紅色），以及鐳射共聚焦顯微鏡活細胞成像術，我們觀察了這兩種細胞在腸內相遇時的行為。當骶源性神經脊細胞和迷走源性神經脊細胞在神經纖維上相遇是，它們都停止了移動，不能前行。培養3天以後，骶源性神經脊細胞和迷走源性神經脊細胞在後腸中共同形成了神經細胞網路，其中骶源性神經脊細胞相對只占了小部分細胞。 / 由於骶源性神經脊細胞沿著由旁神經節發出的神經纖維遷移至腸內並且當在神經纖維上遇到迷走性源性神經脊細胞時便停止移動，我們進而研究了神經纖維在骶源性神經脊細胞遷移中的地位。活細胞成像術和免疫組化實驗表明旁神經節發出的神經纖維對骶源性神經脊細胞的遷移是非常重要的，它有助於細胞遷移同時，但當細胞到達纖維末端時它便也限制了細胞的遷移。儘管如此，體外實驗表明在一定培養條件下，骶源性神經脊細胞的遷移並不需要這些神經纖維。 / 由於有報導表明腸內微環境能影響腸神經脊細胞的遷移，我們利用2D電泳和質譜檢測了12.5天（骶源性神經脊細胞進入後腸之前）和13.5天（骶源性神經脊細胞進入後腸）胎鼠後腸中的蛋白表達情況。大多數鑒定到有差異表達的蛋白都與蛋白折疊、細胞生長和細胞骨架組織有關。我們選取了與細胞粘附和肌肉收縮有關的鈣離子依賴膜結合蛋白Anxa6，並結合腸內的平滑肌發育進行了進一步的研究。結果顯示在13.5天胎鼠中，旁神經節的口端方向有一段約600微米的腸的腹側的平滑肌還沒有發育，可能與腸神經脊細胞的遷移有關。但腸內平滑肌發育是否及如何影響腸神經脊細胞的遷移還需要進一步的研究。 / 綜上所述，骶源性神經脊細胞的遷移是一個複雜的過程，迷走源性神經脊細胞，神經纖維和腸內微環境都參與並能影響這個遷移過程。 / Hirschsprung’s disease (HSCR) in humans is characterized by the absence or reduction of enteric ganglia in the distal part of the colon. It is known that all enteric ganglia in the distal colon originate from neural crest cells (NCCs) at both vagal and sacral levels during embryonic development. Vagal NCCs have been well characterized as the main cellular source of the enteric nervous system (ENS), but however, information on the mammalian, including human, sacral NCCs is still scarce. Sacral NCCs in mouse embryos have been recently identified to be able to migrate from the dorsal neural tube to the mesenchyme, aggregate as pelvic ganglia adjacent to the hindgut and then enter the distal hindgut. In the present study, a series of experiments were performed to determine the factors that were involved in modulating their entry to the hindgut and their migration within the distal hindgut, using mouse embryos. / Having entered the hindgut, sacral NCCs migrated along nerve fibers in a caudal-to-rostral direction while vagal NCCs were colonizing the hindgut in a reverse, rostral-to-caudal direction. The migratory behaviors of the vagal and sacral NCCs were examined at the time when these two populations of NCCs met each other with live cell confocal imaging in the distal hindgut using GFP-expressing sacral NCCs (green fluorescent) and Ednrb-Kikume labeled vagal NCCs (red fluorescent) from transgenic mice. The rostral migration of sacral NCCs was observed to be temporarily affected when they met vagal NCCs on the nerve fiber. However, after 3 days in organotypic culture, sacral and vagal NCCs were found to intermingle with each other to form an interconnected cellular network in the hindgut with much greater cellular contribution from vagal NCCs than sacral NCCs. Hence, vagal NCCs were able to affect the migration and thus the final location of sacral NCCs within the hindgut. / Since sacral NCCs have been observed to enter the hindgut by migrating on the nerve fibers extending from pelvic ganglia, the role the nerve fibers in migration was then examined. Results obtained from time-lapse confocal live cell imaging and immunohistochemical localization indicated that nerve fibers extending from pelvic ganglia were very important for sacral NCCs migration. It was found that these nerve fibers could both assist in sacral NCC migration and also restrain their migration once the cells reached the distal tip of the fibers. However, under specific in vitro conditions, sacral NCCs were still able to migrate without the presence of nerve fibers. / The gut microenvironment surrounding the migrating NCCs has also been reported to affect NCCs migration. Therefore, protein molecules with differential expression levels prior to and after the entry of sacral NCCs to the distal hindgut between E12.5 to E13.5 were examined with 2-dimensional gel electrophoresis and mass spectrometry. The proteins identified with significant changes of expression (more than 1.5 folds) were grouped according to their predicted biological functions and involved in protein folding, cell growth and cytoskeletal organization. Among them, Anxa6, a calcium-dependent membrane binding protein related to cell adhesion and muscle contraction, was further examined for its relationship with the muscle development in the hindgut at E12.5 to E14.5. The results showed that a segment of the hindgut (about 600μm) rostral to pelvic ganglia exhibited an incomplete layer of smooth muscle at E13.5. Whether Anxa6 and the smooth muscle are involved in the sacral NCC migration is worth further investigations. / In summary, sacral NCCs migration is a complex process regulated by their interactions with nerve fibers, vagal neural crest cells and possibly molecules in the hindgut microenvironment through which they migrate. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Jielin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 201-214). / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.IV / Acknowledgements --- p.VI / Table of contents --- p.XII / Abbreviation --- p.XIII / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The enteric nervous system (ENS) --- p.1 / Chapter 1.1.1 --- Embryonic origin and development of the ENS --- p.2 / Chapter 1.1.2 --- Hirschsprung’s disease (HSCR) --- p.5 / Chapter 1.2 --- Enteric neural crest cells (ENCCs) --- p.7 / Chapter 1.2.1 --- Vagal neural crest cells (NCCs) --- p.8 / Chapter 1.2.2 --- Sacral neural crest cells (NCCs) and pelvic ganglia --- p.10 / Chapter 1.2.3 --- Interactions between neural crest cells (NCCs) --- p.14 / Chapter 1.3 --- Microenvironment within the gut --- p.16 / Chapter 1.3.1 --- Effect of molecules on ENCCs migration --- p.16 / Chapter 1.3.2 --- Effect of tissue age on ENCC migration --- p.19 / Chapter 1.3.3 --- Absence of ENCCs facilitates ENCC colonization --- p.20 / Chapter 1.4 --- Objectives of the present study --- p.22 / Chapter Figures and Legends --- p.25 / Chapter Chapter 2 --- Migratory behaviors of sacral and vagal neural crest cells in the distal hindgut --- p.32 / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.36 / Chapter 2.2.1 --- Mouse strains --- p.36 / Chapter 2.2.2 --- Isolation of gut tubes and pelvic ganglia --- p.36 / Chapter 2.2.3 --- Photo-conversion of Ednrb-kikume labeled neural crest cells within the gut --- p.37 / Chapter 2.2.4 --- Preparation of general culture medium and organ culture agarose gel --- p.38 / Chapter 2.2.5 --- Organotypic culture --- p.39 / Chapter 2.2.6 --- Time-lapse live cell confocal microscopic imaging --- p.39 / Chapter 2.2.7 --- Whole mount gut preparations for immunohistochemical staining --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Conversion of green fluorescent, Ednrb-kikume labeled vagal NCCs into red fluorescent --- p.42 / Chapter 2.3.2 --- Ednrb-kikume labeled cells were Sox10 immunorecative --- p.42 / Chapter 2.3.3 --- No discernible photo-toxicity after photo-conversion --- p.43 / Chapter 2.3.4 --- Sacral NCCs migration was hindered by vagal NCCs when they met on the nerve fiber --- p.43 / Chapter 2.3.5 --- Vagal NCCs migrated toward each other to form an interconnected network --- p.45 / Chapter 2.3.6 --- Sacral NCCs contributed much fewer cells than vagal NCCs in the terminal hindgut --- p.46 / Chapter 2.4 --- Discussion --- p.47 / Chapter 2.4.1 --- Ednrb-kikume mouse is a potentially ideal animal model for studies of NCCs migratory behaviors --- p.47 / Chapter 2.4.2 --- Migration of sacral NCCs in the hindgut was affected by vagal NCCs --- p.49 / Chapter 2.4.3 --- Migratory behaviors of vagal NCCs --- p.52 / Chapter 2.4.4 --- Vagal NCCs potentially preferred to move on nerve fibers --- p.53 / Chapter 2.4.5 --- Sacral NCCs contributed much less to the cellular network than vagal NCCs --- p.53 / Chapter 2.5 --- Summary --- p.55 / Chapter Table 2-1 --- Primary and secondary antibodies used in the experiments --- p.56 / Chapter Figures and Legends --- p.57 / Chapter Chapter 3 --- The relationship between nerve fiber extension and sacral neural crest cell migration in vitro --- p.81 / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials and methods --- p.86 / Chapter 3.2.1 --- Mouse strains --- p.86 / Chapter 3.2.2 --- Preparation of fibronectin (FN) coated coverslips and confocal dishes --- p.86 / Chapter 3.2.3 --- Preparation of media --- p.87 / Chapter 3.2.4 --- Isolation of pelvic ganglia --- p.87 / Chapter 3.2.5 --- In vitro culture of pelvic ganglia in 4-well plates or confocal dishes --- p.88 / Chapter 3.2.6 --- Live cell imaging using Nikon live cell imaging system --- p.88 / Chapter 3.2.7 --- WGA treatments on the pelvic ganglia culture --- p.89 / Chapter 3.2.8 --- Effect of embryonic cell proliferation medium and stem cell proliferation medium on pelvic ganglia growth in vitro --- p.90 / Chapter 3.2.9 --- Immunohistochemical staining --- p.90 / Chapter 3.3 --- Results --- p.92 / Chapter 3.3.1 --- Sacral NCCs and nerve fibers from the pelvic ganglia were in close association in vitro --- p.92 / Chapter 3.3.2 --- Migratory behaviors of sacral NCCs on the nerve fiber in vitro --- p.92 / Chapter 3.3.3 --- WGA treatments affected the growth of nerve fibers and sacral NCCs migration in vitro --- p.93 / Chapter 3.3.4 --- Sacral NCCs migrated without nerve fibers when cultured in proliferation media --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter 3.4.1 --- In vitro culture of pelvic ganglion --- p.97 / Chapter 3.4.2 --- Migratory behaviors of sacral NCCs in vitro --- p.98 / Chapter 3.4.3 --- Sacral NCCs migration was affected by the extension of nerve fibers from pelvic ganglia in vitro --- p.101 / Chapter 3.4.4 --- Nerve fibers from the pelvic ganglia were not necessary for sacral NCCs migration in vitro --- p.103 / Chapter 3.5 --- Summary --- p.106 / Chapter Figures and Legends --- p.107 / Chapter Chapter 4 --- Differentially expressed protein molecules in the distal hindgut before and after the entry of sacral neural crest cells --- p.128 / Chapter 4.1 --- Introduction --- p.128 / Chapter 4.2 --- Materials and methods --- p.132 / Chapter 4.2.1 --- Mouse strain --- p.132 / Chapter 4.2.2 --- Preparation of solutions for 2-dimensional (2D) gel electrophoresis --- p.132 / Chapter 4.2.3 --- Preparation of solutions for mass spectrometry --- p.133 / Chapter 4.2.4 --- Isolation of the distal hindgut and protein extraction --- p.133 / Chapter 4.2.5 --- Measurement of protein concentration --- p.134 / Chapter 4.2.6 --- 2D gel electrophoresis --- p.135 / Chapter 4.2.7 --- Mass spectrometry --- p.138 / Chapter 4.2.8 --- SDS-PAGE and Western blot --- p.139 / Chapter 4.2.9 --- Immunohistochemical staining of gut tubes and embryos --- p.140 / Chapter 4.2.10 --- Distal hindgut model reconstruction --- p.141 / Chapter 4.3 --- Results --- p.143 / Chapter 4.3.1 --- E13.5 was the critical stage at which sacral NCCs started to enter the hindgut --- p.143 / Chapter 4.3.2 --- Protein molecules identified by 2D electrophoresis and mass spectrometry --- p.144 / Chapter 4.3.3 --- Western blot analysis and immunostaining confirmed expression levels of Anxa6 --- p.145 / Chapter 4.3.4 --- Smooth muscle actin (SMA) and Anxa6 partially co-localized within the E13.5 hindgut --- p.146 / Chapter 4.3.5 --- Expression of SMA and Anxa6 before and after sacral NCC entry to the distal hindgut --- p.146 / Chapter 4.3.6 --- Reconstruction of distal hindgut images from serial sections with SMA and Anxa6 immunoreactivities --- p.147 / Chapter 4.4 --- Discussion --- p.149 / Chapter 4.4.1 --- Tissue age affected enteric NCC colonization --- p.149 / Chapter 4.4.2 --- Proteomics used in modern biological research --- p.150 / Chapter 4.4.3 --- Molecules differentially expressed in the distal hindgut at E12.5 and E13.5 --- p.151 / Chapter 4.4.4 --- Anxa6 and SMA expression in the distal hindgut --- p.153 / Chapter 4.4.5 --- The role of the smooth muscle development in sacral NCC entry into the hindgut --- p.155 / Chapter 4.5 --- Summary --- p.157 / Chapter Table 4-1 --- Identification of proteins by MALDI-TOF analysis and the MASCOT search program --- p.158 / Chapter Table 4-2 --- Differentially expressed proteins identified by 2-D electro-phoresis and MALDI-TOF/TOF and their predicted biological functions --- p.159 / Figures and Legends --- p.160 / Chapter Chapter 5 --- Conclusions and discussion --- p.182 / Chapter 5.1 --- Vagal NCCs hindered sacral NCC migration when they coalesced on the nerve fiber --- p.182 / Chapter 5.2 --- Nerve fibers from pelvic ganglia were important but not necessary for sacral NCCs migration in vitro --- p.186 / Chapter 5.3 --- Possible involvement of smooth muscle development in modulating sacral NCCs migration --- p.189 / Chapter 5.4 --- Future prospects --- p.191 / Chapter 5.4.1 --- Interactions of vagal and sacral NCCs within the hindgut of mouse embryos --- p.191 / Chapter 5.4.2 --- Role of nerve fibers for sacral NCCs migration ex vivo --- p.192 / Chapter 5.4.3 --- Role of Anxa6 in muscle development of the gut and NCCs migration --- p.193 / Chapter Appendix I --- Solutions used in 2-D electrophoresis --- p.195 / Chapter Appendix II --- Solutions for Colloidal Coomassie staining --- p.198 / Chapter Appendix III --- Procedures for embryo processing --- p.199 / Chapter Appendix IV --- Other solutions --- p.200 / References --- p.201

Câncer é a denominação de um conjunto de mais de cem doenças causadas pelo crescimento e multiplicação desordenados de células anormais capazes de invadir e se disseminar por diversos tecidos e órgãos. É considerado um problema de saúde mundial, sendo uma das maiores causas de morte. Dados da Organização Mundial da Saúde (OMS) indicam que 15% das mortes no mundo serão causadas por câncer em 2015. No Brasil, o Instituto Nacional do Câncer (INCA) estima 580 mil novos casos da doença para 2014. Apesar da vasta quimioterapia disponível, os tratamentos possuem alta toxicidade e estão sujeitos à resistência. Nesse contexto, a presente dissertação de mestrado tem como foco principal a identificação e caracterização de novos compostos com propriedades anticâncer. Os estudos foram realizados com base em dois alvos principais. O primeiro foi a proteína tubulina, um alvo anticâncer validado que é modulado por moléculas importantes como o taxol, a vimblastina e a colchicina. O segundo alvo foi a migração celular, característica relacionada ao processo de metástase, que é responsável por 90% das mortes por câncer. Uma série de acridinonas sintéticas foi avaliada in silico frente à proteína tubulina empregando métodos de modelagem molecular. Para tanto, fez-se uso de estruturas cristalográficas da proteína disponíveis no PDB (Protein Data Bank). Os resultados das análises de docagem molecular indicaram que as moléculas poderiam interagir com o sítio da colchicina e, dessa forma, atuariam como inibidoras da polimerização dos microtúbulos. Ensaios wound healing e em câmara de Boyden permitiram a identificação de quatro compostos com potente efeito de inibição da migração celular (valores de IC50 variando entre 0,294 e 1,7 &mu;M) em uma linhagem metastática (MDA-MB-231). Estes compostos foram submetidos a ensaios de citotoxicidade frente à mesma linhagem tumoral e também apresentaram boa potência, com valores de IC50 variando entre 0,110 e 3 &mu;M. Além disso, ensaios de citotoxicidade em células saudáveis mostraram que estes compostos inviabilizaram seletivamente células tumorais. Para a validação dos estudos in silico, ensaios de polimerização da tubulina foram conduzidos. Os resultados mostraram que as quatro moléculas ativas nos ensaios celulares atuam como inibidores de polimerização dos microtúbulos, com valores de IC50 variando entre 0,9 e 13 &mu;M. Estudos das relações entre a estrutura e atividade (SAR) revelaram alguns aspectos interessantes nesta série de acridinonas, como, por exemplo, a perda de atividade, que se deu tanto nos ensaios celulares quanto nos ensaios bioquímicos, causada pela substituição de um grupo nitro da posição meta- por para- em duas moléculas da série. A progressão do ciclo celular foi analisada por citometria de fluxo e os resultados mostraram que os compostos estudados são capazes de interromper o ciclo celular entre as fases G2 e M. A citometria fluxo também permitiu verificar a indução da apoptose celular devida à ação das moléculas. Em resumo, este trabalho possibilitou a identificação e caracterização de quatro compostos com propriedades antitumorais promissoras que serão utilizados como compostos líderes para posterior desenvolvimento como agentes anticâncer. / Cancer is a set of diseases with high diversity and aggressiveness. It is one of the major causes of death according to the World Health Organization (WHO), being responsible for 15% of worldwide deaths in 2015. In Brazil, the National Institute of Cancer (INCA) estimates 580,000 new cases of cancer for the year of 2014. Despite the vast availability of cancer chemotherapy, the treatments cause high toxic effects and are liable to resistance. In this context, the main goal of the present master\'s dissertation is the identification and the characterization of new compounds having anticancer properties. These studies were conducted based on two main targets. The first one was the protein tubulin, a validated anticancer target that is modulated by important molecules such as taxol, vinblastine and colchicine. The second target was the cellular migration, a feature related to the metastasis process, which causes 90% of the cancer deaths. A series of synthetic acridinones was studied in silico employing molecular modeling methods. For this, crystallographic structures of tubulin were collected from PDB (Protein Data Bank). The docking results indicated that the molecules could interact with the colchicine site and, thereby, could act as tubulin polymerization inhibitors. The series was assessed by the wound healing and Boyden chamber\'s assays using the metastatic cell line MDA-MB-231. In these assays, four compounds were identified as good inhibitors of cellular migration (IC50 values varying between 0.294 and 1.7 &mu;M). These compounds were evaluated in cytotoxicity assays using the same cell line and presented good potency, with IC50 values varying between 0.110 and 3 &mu;M. Furthermore, cytotoxicity assays using a healthy cell line showed that these compounds act selectively in tumor cells. For the validation of the in silico studies, tubulin polymerization assays were conducted. The results showed that the four active molecules in the cellular assays act as tubulin polymerization inhibitors, with IC50 values varying between 0.9 and 13 &mu;M. Structure-activity relationship (SAR) studies revealed interesting structural aspects in this series of acridinones, for instance, the exchange of the nitro group substituent from the meta- to the para- position in two molecules of the series led to a lack of activity in both cellular and biochemical assays. The cell cycle progression was evaluated by flow cytometry, and the results showed that the four compounds are capable of arrest cells in the G2/M phase. Moreover, the apoptosis induction was verified using flow cytometry. In summary, this work provided the identification and characterization of four new compounds with promising antitumor properties. These molecules will be used as lead compounds for further development as anticancer agents.

As células T Natural Killer (NKT) são linfócitos que expressam o rearranjo V24V11 com TCR invariante e reconhecem glicolipídeos como o alfagalactosilceramida (-GalCer) apresentado no contexto da molécula MHC-não clássica chamada CD1d. As NKT são divididas em subgrupos distintos: os fenótipos CD4+ e CD4- em camundongos e humanos, e CD8+ e CD4-CD8- em humanos. Após estímulo, as NKT secretam citocinas Th1 e Th2. A frequência das NKT representa menos de 1% da população de linfócitos T no sangue periférico humano. O objetivo deste trabalho é primeiro, identificar a frequência, o fenótipo e a função da população de células NKT nos grupos de indivíduos saudáveis de procedências distintas. Segundo, avaliar a influência da idade, gênero e país de moradia na frequência das NKT, incluindo suas subpopulações, no sangue periférico; Terceiro, verificar se há variabilidade ou não na apresentação da molécula CD1d. Quarto, verificar se há alteração na função das NKT utilizando o estímulo -GalCer e por fim, verificar o perfil das NKT quanto ao seu estado de ativação e migração celular quando submetidas a expansão. As amostras são provenientes de São Paulo, Brasil; São Francisco, Estado Unidos da América e Estocolmo, Suécia. Os parâmetros imunológicos foram analisados por citometria de fluxo. Demonstramos que o grupo de São Paulo, não apresentou maiores valores de linfócitos T CD3+, no entanto, apresentou valores significantes de células NKT V24+/V11+. Não estabelecemos a expressão dos marcadores de ativação e migração nas NKT. Estabelecemos, no grupo de São Paulo, a capacidade funcional das NKT que expressaram IFN- e TNF- assim como de expansão, sob estímulo de -GalCer. Os resultados sugerem que vários fatores podem alterar a quantidade das células NKT na periferia. Mais estudos devem ser endereçados para esclarecer melhor estes fatos / Natural Killer T cells (NKT cells) are lymphocytes that express the invariant TCR rearrangement V24V11 and recognize glycolipid as alpha-galactosylceramide (-GalCer) presented in conjunction with a non-MHC pathway molecule named CD1d. The NKT cells are divided into distinct subsets: the CD4+ and CD4- phenotype in mice and humans and the CD8+ and CD4-CD8- phenotype in humans. After stimulation, the NKT secrete Th1 and Th2 cytokines. The frequencies of NKT cells represent <1% of the population of T lymphocytes in human peripheral blood. The objective of this study is to 1) identify the frequency, phenotype and function of NKT cell population in groups of healthy individuals from different racial/ethnic origins, 2) evaluate the influence of age, gender and country of residence on the frequency of NKT cells/subpopulations, 3) investigate the variability of presentation of the CD1d molecule, 4) investigate the functional variability of NKT cells using -GalCer stimulation, and 5) study the activation and migration profile of NKT cells after expansion. Immunologic parameters of clinical samples from Sao Paulo, Brazil; San Francisco, California and Stockholm, Sweden were analyzed by flow cytometry. We demonstrate that the São Paulo group does not have an elevated number of CD3+ T lymphocytes, but does have significantly more NKT V24+/V11+ cells. We have not yet determined expression of NKT markers for activation and migration. Thus, we have shown that among sample obtained in São Paulo, there is increased functional capacity of IFN- and TNF--expressing NKT cells along with NKT cells expansion under stimulation with the -GalCer. These results suggest that there are several factors that can modulate NKT cell number and function in the peripheral blood and additional studies are needed to further clarify these findings

Interactions between the Eph family of receptor tyrosine kinases, and their ligands, the ephrins, are required for the normal development and maintenance of many patterns of connectivity within the nervous system. Eph receptors and ephrins are expressed widely throughout both the developing and mature nervous system where they function as important regulators of cell migration and axon guidance. The studies presented in this thesis examine the role of one particular member of the Eph receptor family, EphA4, in regulating mechanisms that underlie the development and maintenance of certain neural connections within the nervous system. This thesis demonstrates that the EphA4 receptor is expressed within specific regions of the developing and mature nervous system, some of which are associated with the control of locomotor activity. Consistent with these observations are the locomotor defects exhibited by animals with a targeted disruption of the EphA4 gene. These animals exhibit abnormal bilateral limb movements and have severe disruptions of a number of major axonal pathways. One of these disrupted axonal pathways, the corticospinal tract (CST), is a particularly important mediator of locomotor activity. This thesis reveals that EphA4 is expressed on the axons that comprise the CST. It demonstrates that although EphA4 is not required for the initial development of the CST, repulsive interactions between EphA4-bearing CST axons and ephrinB3, a ligand for EphA4 that is expressed at the midline of the spinal cord, appear to prevent CST axons from aberrantly recrossing the spinal midline during development.