Made available in DSpace on 2015-05-14T13:00:02Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 7083796 bytes, checksum: 2aa6f174655d082d78cc059fc1eef2e0 (MD5) Previous issue date: 2014-03-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Mucormycoses are infections that have high rates of morbidity and mortality. They show high resistance to antifungal agents, and there is a limited therapeutic arsenal currently available, therefore, there is a great need to give priority to testing therapeutic agents for the treatment of mucormycosis. Along this line, the use of essential oils and phytoconstituents has been emphasized as a new therapeutic approach. The objective of this work was to investigate the antifungal activity of the essential oil (EO) of Thymus vulgaris, and its constituents thymol and p-cymene against Rhizopus oryzae and Rhizopus microsporus, through microbiological screening, determination of minimal inhibitory concentration (MICs) and minimal fungicidal concentration (MFCs), effects on mycelial growth and germination of sporangiospores, fungal morphology and interaction with ergosterol. Also was evaluated the preclinical acute toxicity in mice. In microbiological screening the T. vulgaris essential oil showed antifungal potential against resistant strains of R. oryzae.The MIC of EO and thymol varied 128 512 μg/mL, but the MFC of EO and thymol varied 512 1024 μg/mL and 128 1024 μg/mL, respectively. The results also showed that EO and thymol significantly inhibited mycelial development and germination of sporangiospores of both species of Rhizopus. Investigation of the mechanism of antifungal action showed that EO and thymol interact with ergosterol. These data indicate that EO of T. vulgaris and thymol possess strong antifungal activity, which can be related to their interaction with ergosterol, supporting the possible use of these products in the treatment of mucormycosis. In preclinical acute toxicology the doses of 125, 250, 500 and 1000 mg/kg intraperitoneally (i.p.) showed depressive activity on the central nervous system (CNS). In addition to these parameters was observed that the doses of 125 and 250 mg/kg did not change the body and organs weight of the animals, but it was observed change some of the hematological parameters of the mice. The EO showed DL50 of 250 mg/kg for male and 459.6 mg/kg for female; however the thymol showed DL50 of 222.3 mg/kg for male and 1551 mg/kg for female. These data indicate that EO of T. vulgaris and thymol possess strong antifungal activity, which can be related to their interaction with ergosterol. / As mucormicoses são infecções que possuem elevadas taxas de morbidade e mortalidade, limitado arsenal terapêutico, devido a resistência aos antifúngicos. Portanto, existe uma significativa necessidade de priorizar, testar e aplicar melhorias terapêuticas para o tratamento das mucormicoses. É nesse contexto, que os óleos essenciais e fitoconstituintes vem se destacando como uma nova abordagem terapêutica. O objetivo deste trabalho foi investigar a atividade antifúngica in vitro do óleo essencial (OE) de Thymus vulgaris L. e de seus componentes majoritários (timol e p-cimeno) contra Rhizopus oryzae e Rhizopus microsporus, através da triagem microbiológica, da determinação da concentração inibitória mínima (CIM) e fungicida mínima (CFM), avaliação dos efeitos dos fitoconstituintes no crescimento micelial, na germinação dos esporos fúngicos, na morfologia fúngica e interação com ergosterol. Também foi avaliada a toxicidade pré-clínica aguda em camundongos. Na triagem microbiológica o óleo essencial de T. vulgaris apresentou um dos melhores perfis antifúngicos contra cepas resistentes de R. oryzae. A CIM dos produtos variou entre 128-512 μg/mL, já as CFMs do óleo essencial e timol variaram entre 512-1024 μg/mL e 128-1024 μg/mL, respectivamente. Os resultados também mostraram que tanto o OE como o timol inibiram significativamente o desenvolvimento micelial e a germinação de esporos de ambas as espécies de Rhizopus. Em seguida foi mostrado que os produtos testados alteram a morfologia de R. oryzae e R. microsporus. Na investigação do mecanismo de ação antifúngica foi evidenciado que o OE e o timol interagem com o ergosterol, esterol presente na membrana dos fungos. No ensaio toxicológico pré-clínica agudo, as doses de 125, 250, 500 e 1000 mg/kg via intraperitoneal (i.p.) apresentaram atividade depressora do sistema nervoso central (SNC). Adicionalmente a estes parâmetros foi evidenciado que o OE e o timol nas doses de 125 e 250 mg/kg não promoveram alterações significativas na evolução ponderal e peso dos órgãos dos camundongos. Entretanto, ambas as doses das drogas-teste alteram alguns parâmetros hematológicos dos camundongos. Após 72 h de observação o OE apresentou DL50 estimada em 250 mg/kg para camundongos machos e 459,6 mg/kg para as fêmeas. Já o timol apresentou DL50 estimada em 222,3 mg/kg para os machos e 1551 mg/kg para as fêmeas. Estes dados indicam que o óleo essencial de T. vulgaris e timol, apresentam forte atividade antifúngica, que pode estar relacionada com a interação com ergosterol e consequentemente lise de membrana.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-17T13:33:41Z No. of bitstreams: 1 rebecamollima.pdf: 1811573 bytes, checksum: 85964eefdd3a90513fcac0f172c11491 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-18T13:41:24Z (GMT) No. of bitstreams: 1 rebecamollima.pdf: 1811573 bytes, checksum: 85964eefdd3a90513fcac0f172c11491 (MD5) / Made available in DSpace on 2017-04-18T13:41:24Z (GMT). No. of bitstreams: 1 rebecamollima.pdf: 1811573 bytes, checksum: 85964eefdd3a90513fcac0f172c11491 (MD5) Previous issue date: 2017-01-24 / A esporotricose é uma micose subcutânea de evolução subaguda a crônica capaz de afetar seres humanos e animais. Distribuída mundialmente, possui maior prevalência nas áreas de clima tropical e temperado, sendo a micose subcutânea mais comum na América do Sul. Seus agentes etiológicos são fungos do complexo Sporothrix schenckii, saprófitas e dimórficos, encontrados no ambiente. No Brasil, o aumento exponencial dos casos em humanos e animais vem sendo observado nas últimas décadas. Nos arredores do estado do Rio de Janeiro, já é observada uma epidemia de origem zoonótica. Aliado a isso, o tratamento desta micose vem sendo motivo de preocupação. O alto custo e toxicidade dos medicamentos, o tempo prolongado de tratamento, e o surgimento de linhagens resistentes aos fármacos de escolha justificam a demanda crescente pela descoberta de novos fármacos. Neste contexto, o presente estudo tem como objetivo identificar os componentes dos óleos essenciais de Myrtus communis L. e Thymus vulgaris L., investigar seus potenciais antifúngicos in vitro frente às principais linhagens causadoras de esporotricose no Brasil, e determinar a citotoxicidade preliminar dos óleos essenciais. Os óleos essenciais de mirta e tomilho foram obtidos comercialmente. O perfil cromatográfico obtido por CG para os dois óleos indicou elevados valores de monoterpernos, 87,1% para a mirta e 98,8% para o tomilho, tendo como componentes majoritários o 1-8 cineol e o timol, respectivamente. Para a avaliação da atividade antifúngica foram utilizadas as seguintes linhagens fúngicas: Sporothrix schenckii ATCC 1099-18, Sporothrix schenckii IPEC 15383, Sporothrix brasiliensis ATCC 5110 e Sporothrix brasiliensis IPEC 17943 e duas linhagens clínicas de Sporothrix schenckii, denominadas genericamente como A e B. O óleo essencial de mirta apresentou atividade antifúngica frente a todas as linhagens em concentrações que variaram de 31,25 a 62,5 µg/mL. Foi observada atividade fungicida frente a todas as linhagens em concentrações acima de 62,5 µg/mL. O óleo essencial de tomilho foi capaz de inibir todas as linhagens em concentrações que variaram de 125 a 250 µg/mL. Foi observada atividade fungicida frente a todas as linhagens em concentrações acima de 250 µg/mL. A anfotericina B e o itraconazol foram utilizados como fármacos de referência. Para estes foi observado que somente duas das linhagens estudadas apresentaram susceptibilidade à anfotericina B e ao itraconazol, todas as linhagens estudadas foram consideradas resistentes, com valores de CIM > 16 µg/mL. Estes resultados sugerem indícios de resistência fúngica das linhagens estudadas em relação aos fármacos de referência empregados no tratamento da esporotricose. As microscopias eletrônicas de varredura das linhagens fúngicas revelaram que, tanto nos fungos expostos aos tratamentos experimentais, quanto naqueles expostos aos fármacos de referência, deformidades na estrutura fúngica quando comparadas ao grupo não tratado foram observadas. Em relação aos ensaios de citotoxicidade realizados com queratinócitos humanos (HACAT) pelo método de redução do MTT, os óleos não apresentaram citotoxicidade nas concentrações equivalentes a seus valores de CIM. / Sporotrichosis is a subcutaneous mycosis with subacute to chronic evolution that can affect humans and animals. Distributed worldwide, it is more prevalent in tropical and temperate climates, being the most common subcutaneous mycosis in South America. Its etiological agents are fungi of the Sporothrix schenckii complex, saprophytic and dimorphic, found in the environment. In Brazil, the exponential increase in cases in humans and animals has been observed in recent decades. In the surroundings of the state of Rio de Janeiro, an epidemic of zoonotic origin is already observed. Allied to this, the treatment of this mycosis has been cause of concern. The high cost and toxicity of the drugs, the prolonged treatment time, and the emergence of resistant strains on drugs of choice justify the growing demand for the discovery of new drugs. In this context, the present study aims to identify the components of the essential oils of Myrtus communis L. and Thymus vulgaris L., to investigate their antifungal potentials in vitro against the main sporotrichosis strains in Brazil, and to determine the preliminary cytotoxicity of essential oils. The essential oils of myrtle and thyme were obtained commercially. The chromatographic profile obtained by GC for the two oils showed high values of monoterpere, 87.1% for myrtle and 98.8% for thyme, with 1-8 cineole and thymol, respectively. The following fungal strains were used: Sporothrix schenckii ATCC 1099-18, Sporothrix schenckii IPEC 15383, Sporothrix brasiliensis ATCC 5110 and Sporothrix brasiliensis IPEC 17943 and two clinical strains of Sporothrix schenckii, referred as A and B. Myrtle essential oil showed antifungal activity against all strains at concentrations ranging from 31.25 to 62.5 μg / mL. Fungicidal activity against all strains was observed at concentrations above 62.5 μg / mL. Thyme essential oil was able to inhibit all lineages at concentrations ranging from 125 to 250 μg / ml. Fungicidal activity was observed against all strains at concentrations above 250 μg / mL. Amphotericin B and itraconazole were used as standard drugs. For these, it was observed that only two of the studied strains showed susceptibility to amphotericin B and itraconazole, all strains studied were considered resistant, with MIC values > 16 μg / mL. These results suggest evidence of fungal resistance of the lines studied in relation to the reference drugs used in the treatment of sporotrichosis. Scanning electron microscopy of fungal lines revealed that, both in the fungi exposed to the experimental treatments and in those exposed to the reference drugs, deformities in the fungal structure when compared to the untreated group were observed. In relation to the cytotoxicity assays performed with human keratinocytes (HACAT) by the MTT reduction method, the oils did not present cytotoxicity at concentrations equivalent to their MIC values.

Nous avons évalué les différents aspects de la synergie entre des antibiotiques classiques et les huiles essentielles de certaines plantes, endémiques du Maroc, sur des bactéries résistantes impliquées dans les infections nosocomiales. Il s’agit de Thymus maroccanus, T. broussonetii, T. pallidus, T. riataraum et Rosmarinus officinalis. La combinaison de ces huiles essentielles avec des antibiotiques a révélé que parmi 80 combinaisons testées, 71% des combinaisons ont montré une synergie totale, 20% un effet synergique partiel et 9% des combinaisons n’ont pas d’effet synergique. De plus, les huiles essentielles de T. maroccanus et T. broussonetii ont pu augmenter considérablement la sensibilité de plusieurs isolats résistant au chloramphénicol. Elles peuvent restaurer la sensibilité au chloramphénicol en produisant une compétition vis-à-vis de son extrusion en bloquant les pompes impliquées dans son efflux. Par ailleurs, l’huile essentielle de T. maroccanus augmentait la perméabilité de la membrane des bactéries étudiées, et favorisait la libération des protéines intracellulaires dans le milieu extérieur. Elle perméabilise à la fois la membrane interne et la membrane externe des bactéries, mais sans causer une dégradation détectable des constituants cellulaires. Des altérations protéiques ont été associées à l'exposition prolongée aux composés naturels testés. Ces altérations se sont traduites par un efflux accru dont l'effet synergique est renforcé par la baisse du niveau de porines qui peuvent être impliquées dans la diffusion passive de ces antimicrobiens naturels, ce qui confère aux bactéries testées une tolérance accrue à ces composés. / We evaluated different aspects of the synergy between conventional antibiotics and essential oils of some Moroccan endemic plants, on resistant bacteria involved in nosocomial infections. These plants were: Thymus maroccanus, T. broussonetii, T. pallidus, T. riataraum and Rosmarinus officinalis. Out of 80 combinations tested between these two essential oils and antibiotics, 71% showed total synergism, 20% had partial synergistic interaction and 9% showed no effect. Combination with carvacrol, the major constituent of T. maroccanus and T. broussonetii, showed also an interesting synergistic effect in combination with ciprofloxacin. Moreover, T. maroccanus and T. broussonetii essential oils increased chloramphenicol susceptibility of several resistant isolates. they could restore sensitivity to chloramphenicol producing a competition to its extrusion by blocking the efflux pumps expressed in the isolates and involved in its efflux. Furthermore, T. maroccanus essential oil increased permeability of studied bacteria membrane, and favoured intracellular proteins release into the external medium. Thymus maroccanus essential oil permeabilized both outer and inner membranes of tested bacteria, but without causing detectable degradation of cellular constituents. Protein alterations have been associated with prolonged exposure to natural compounds tested, an increased efflux associated with a lower level of porins that may be involved in the passive diffusion of these natural antimicrobials, conferring a native protection to bacteria towards these natural compounds.

Le virus HTLV-1 (Human T-cell Leukemia Virus Type 1) est l’agent étiologique de la Leucémie T de l’adulte (ATL) qui est caractérisée par la prolifération de cellules T CD4+ activées. L’absence de modèle animal fiable reproduisant la leucémogénèse associée à l’infection a ralenti la compréhension des étapes précoces du processus leucémogène et le développement de stratégies thérapeutiques efficaces. Récemment l’amélioration des modèles de souris humanisées a permis la reconstitution d’un système immunitaire humain dans des souris. L’injection de cellules souches hématopoïétiques purifiées à partir de sang de cordon humain dans des souris nouveau-nées de la lignée Rag2-/-γc-/- conduit à la formation de novo de cellules dendritiques, B et T humaines. Ces dernières étant la cible de l’infection par HTLV-1, nous avons infecté des souris humanisées avec des cellules productrices de HTLV-1. Cette inoculation conduit à l’infection stable des cellules humaines dans la souris humanisée et la formation de lymphome ou de leucémie à cellules T humaines activées. Cette infection altère le développement des cellules T dans le thymus conduisant à un phénotype plus mature des thymocytes. Ce modèle animal reproduisant l’infection et la pathogénèse associée nous a permis de suivre l’évolution de la clonalité du virus au sein des différents organes lymphoïdes. Basées sur ces observations, des tests préliminaires ont permis d’étudier une nouvelle approche thérapeutique potentiellement applicable en clinique humaine. Ce travail nous a également permis d’affiner le protocole conduisant à l’humanisation des souris afin d’obtenir une meilleure reconstitution humaine dans ce modèle. / Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-γc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The thymus is the major site for T-cell generation and thus is important for the adaptive immune system. Development of a properly selected, functional T-cell repertoire relies on interactions between developing T cells and a series of functionally distinct thymic stroma cell types including the cortical and medullary thymic epithelial cells (TECs). The thymus is one of the first organs to degenerate in normal healthy ageing. Related to this, there is strong interest in developing protocols for improving thymus function in patients by cell replacement or regenerative therapies. Thymic epithelial progenitor cells (TEPCs) represent a potential source of cells for thymus transplantation. However, the only source of these cells for transplantation is currently fetal thymus tissue. If TEPCs could be generated from pluripotent cells, this could provide an alternative source of cells for transplantation. The work described in this thesis therefore had two central aims (i) to test the stability of thymic epithelial progenitor cells in vivo and (ii) to investigate the possibility of generating TEPCs or TECs from mouse embryonic stem (ES) cells. The forkhead transcription factor, Foxn1, is essential for the development of a functionally mature thymic epithelium, but is not necessary for formation of the thymic primordium or for medullary thymic epithelial sub-lineage specification. By reactivating Foxn1 expression postnatally in mice carrying a revertible hypomorphic allele of Foxn1, Foxn1R, I herein demonstrate that TEPCs that can express only low levels of Foxn1 mRNA can persist postnatally in the thymic rudiment in mice until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression. In parallel with this work, I confirmed the possibility of generating Foxn1-expressing cells from mouse ES cells by using a Foxn1 reporter cell line. As the thymic epithelium has a single origin in the third pharyngeal pouch (3pp) endoderm, I then tested whether or not TEPCs and /or TECs were generated during ES cell differentiation via existing protocols for generating anterior definitive endoderm differentiation cells from mouse ES cells. From this work, I showed that genes expressed in the 3pp and/or TEPC,-including Plet-1, Tbx1, Hoxa3 and Pax9, were induced by differentiation of ES cells using these protocols. I further showed that cells expressing both Plet-1, a marker of foregut endoderm and 3pp, and EpCAM, a marker of proliferating epithelial cells, were induced using a novel protocol (2i ADE) for generating ES cells from ADE. However, gene expression analysis and functional testing suggested that the majority of these cells were non-thymus lineage. I subsequently developed a novel protocol which combined this 2i ADE protocol with co-culturing of the differentiating ES cells with fetal thymic lobes, and demonstrated that this further induced 3pp and TEPC related genes. Finally, I modified the culture conditions in this protocol to conditions predicted to better support TEPC/TEC, and showed that in these conditions, the TEPC-specific markers Foxn1 and IL-7 were induced more strongly than in any other conditions tested. The data presented in this thesis therefore represent an advance towards an optimized protocol for successfully generating TEPCs from ES cells in vitro.

La tolérance immunologique assure le maintien de l’intégrité des organismes contre les pathogènes tout en respectant les constituants du soi. La dérégulation de ce mécanisme entraîne la survenue de maladies autoimmunes qui touchent de 5 à 10% de la population générale. Un mécanisme clé de la tolérance immunologique est la délétion clonale des lymphocytes T auto-réactifs après qu’ils ont reconnu leur antigène spécifique au cours de leur maturation dans le thymus. Il a été montré qu’un vaste répertoire d’autoantigènes est exprimé par les cellules épithéliales médullaires du thymus (mTECs) sous l’action de la protéine AIRE (AutoImmune Regulator).
Les études présentées dans ce travail participent à améliorer la compréhension du fonctionnement de AIRE. Au-delà de la fonction de transactivation de AIRE, nous avons montré, avec le reséquençage à haut débit (RNAseq) des mTECs, que les transcrits des autoantigènes induits par AIRE ont des extrémités 3’UTRs courtes associées à l’utilisation des sites de polyadénylation (pA) alternatifs. Nous avons identifié par analyse de données de CLIPseq une fixation préférentielle du complexe de terminaison de la transcription au niveau des pAs alternatifs des gènes sensibles à AIRE. Nous avons également mis en évidence l’interaction de AIRE au complexe de terminaison de la transcription. Parmi plusieurs partenaires de AIRE associés à ce complexe, nous avons montré par interférence d’ARN et RNAseq le rôle de CLP1 dans le choix des pAs alternatifs. De plus nous montrons que CLP1 est le seul membre du complexe de terminaison à être préférentiellement exprimé dans les mTECs matures. Fonctionnellement, nous avons mis en évidence une stabilité plus importante pour les transcrits des autoantigènes induits par AIRE en bloquant la transcription des mTECs ex-vivo par traitement à l’Actinomycine D. Nous montrons également l’existence d’un raccourcissement 3’UTR général dans les mTECs matures par rapport aux mTECs immatures et autres tissus de la souris, auquel se combine le raccourcissement spécifique des gènes dépendant de AIRE. Après avoir identifié par des analyses de Gene Ontology une activité cellulaire exacerbée dans les mTECs matures vs immatures, nous confirmons l’activité transcriptionnelle exacerbée des mTECs matures in-vivo grâce à l’incorporation de 5Ethynyl Uridine (EU) dans les ARN néosynthétisés après injection intrathymique. Le raccourcissement des transcrits des auto-antigènes associé à leur stabilité accrue suggère qu’ils échappent à la répression transcriptionnelle médiée par les microARNs. Ce travail a permis d’identifier les bases moléculaires de la régulation post-transcriptionnelle des autoantigènes dans le thymus. Dans l’étude faite en collaboration avec l’équipe de Jakub Abramson du laboratoire Weizmann, démontre que Sirt1, une désactylase ADN dépendante, est exprimé de façon abondante dans les mTEC AIRE+ et ce grâce à l’utilisation de profil d’expression génétique, de cytométrie en flux et d’analyses d’immunoblot de différents types cellulaires thymiques. De plus lorsque Sirt1 est inactivé, dans les lignées germinales et des lignées TEC, l’expression des gènes AIRE dépendants diminuent et donc avec elle la tolérance immune induite par AIRE. La capacité désacétylase de Sirt1 est nécessaire pour l’expression des gènes induits par AIRE dans les mTECs. Sirt1 cible surement d’autres molécules nucléaires, impliquées dans la voie de AIRE. Elle pourrait avoir un rôle plus étendu dans la régulation du système immunitaire et être présent à la périphérie. Cette étude a mis en évidence un rôle important de Sirt1 dans la tolérance centrale dans le thymus à travers la régulation des gènes induits par AIRE. (...) / No abstract

O tubarão-azul, Prionace glauca (Linnaeus, 1758) é uma espécie cosmopolita, de alto valor comercial, principalmente capturado por embarcações que operam em alto mar e vendido em mercados e feiras-livres. Poucos dados biológicos estão disponíveis sobre esta espécie, principalmente quanto à sua sanidade e, o conhecimento do desenvolvimento de seus órgãos linfoides pode trazer importantes informações neste contexto. Desta forma, o objetivo do trabalho é descrever o desenvolvimento embrionário dos órgãos linfoides em embriões de tubarão-azul: timo, órgão epigonal, baço e órgão de Leydig, quanto à estrutura e arquitetura macroscópica, microscópica e ultraestrutural, pelas técnicas de microscopia de luz e eletrônica de transmissão. Foram coletados cinco espécimes de cada fase representativa do desenvolvimento embrionário: I, II, III e IV e do animal adulto. O timo foi visível macroscopicamente nas fases III e IV e microscopicamente da fase I a IV. O órgão de Leydig está presente nas fases II, III e IV. O baço e o órgão epigonal estão presentes em todas as fases embrionárias e no adulto. O timo apresentou principalmente populações de timócitos em diversos estágios de maturação e melanomacrófagos, o baço apresentou melanomacrófagos linfócitos em diversos estágios de maturação, neutrófilos, trombócitos e grande quantidade de eritrócitos. O órgão epigonal apresentou um grande número de células imaturas, principalemente de linfócitos e células polimorfonucleares. A função do órgão de Leydig é perdida quando adulta, sendo substituída pelo órgão epigonal. Os resultados desse trabalho permitem sugerir que esses órgãos apresentam uma função hematopoiética desde o inicio da embriogênese até a fase adulta. / The blue shark (Prionace glauca) is a cosmopolitan species of high commercial value, easily caught by vessels operating on the high seas and sold in markets and street fairs. Few biological data are available on this species, mainly from their sanity. Studies on development of these lymphoid organs can provide important information in this regard. Thus, the aim of this study is to describe the gross, microscopic and ultraestrutural morphology of the embryonic development of lymphoid organs: thymus, epigonal organ, spleen and the Leydig organ by light microscopy and transmission electron techniques. Five specimens were collected from each representative stage of embryonic development: II, III and IV besides adult specimens. Thymus was visible macroscopically at phases III and IV and microscopically from phase I to IV. Leydig organ is presente in phases II, III and IV. Spleen and epigonal organ are present in all embrionic phases and adult. Thymus presented mainly thymocytes populations in several maturation stages and melanomacrophages, spleen presentes melanomacrophages and lymphocytes, neutrophyls, trombocytes and huge amount of erytrocytes. Epigonal organ presented many immature cells, mainly lymphocytes and polimorphonuclear cells. Leidigs organ function is lost in adulthood being replaced by the epigonal organ. The results of this work allow to suggest that these organs present a hematopoietic function since the early development until the adult phase.

Cannabis sativa (maconha) é a droga ilícita mais conhecida e utilizada em todo o mundo. Recentemente, o uso recreativo da droga por jovens aumentou muito, especialmente entre as gestantes. Estudos indicam que seu uso na gestação causa efeitos fetotóxicos adversos, no entanto, poucos estudos avaliam esses efeitos durante este período crítico sobre o desenvolvimento do sistema imunológico. Neste estudo, avaliamos o impacto da exposição gestacional à fumaça de maconha resultante da queima de cigarros sobre o desenvolvimento do baço e do timo de proles de camundongos BalbC. Utilizamos um modelo que se aproxima das reais condições de uso humano (inalação) sob os aspectos de dose e média exposição. Camundongos fêmeas gestantes (n = 20) foram expostas ao fumo da maconha ou ao ar filtrado (grupo controle) do 5,5° ao 17,5° dia gestacional (DG) por 5 minutos diariamente. Para isso, foi utilizado um aparelho para inalação da fumaça desenvolvido em nosso laboratório, onde os cigarros de maconha (200mg de Cannabis sativa) foram queimados e a fumaça conduzida para a câmara de exposição dos animais (exposição somente inalatória). Os baços e timos das proles foram coletados no 18,5° dia gestacional (DG), 20° e 60° dias pós-natais (DPN) para a realização das análises histológica e histoquímica. Avaliações morfológicas e quantitativas dos órgãos foram realizadas utilizando-se métodos estereológicos e por meio da semi quantificação de fibras do sistema colágeno. Os resultados indicam um aumento no peso corporal dos animais com 60 DPN cujas mães foram expostas ao fumo de Cannabis sativa durante a gravidez. Os machos com 60 DPN expostos apresentaram redução no volume total do timo, assim como em seus principais compartimentos, medula e córtex, onde também foi observado um aumento de fibras do sistema colágeno, quando comparados aos animais controles. Além disso, o baço desses animais também apresentou redução significativa em seu volume, porém não houve redução significativa no volume total da polpa vermelha. Machos e fêmeas com 60 DPN do grupo exposto apresentaram redução no volume total da polpa branca e aumento de fibras do sistema colágeno na região das trabéculas do parênquima do baço. Em suma, a exposição gestacional à maconha causa efeitos fetotóxicos que podem ser observados por alteração no baço e no timo da prole (sendo os efeitos distintos para machos e fêmeas) e estas alterações se tornam mais marcantes com o avanço da idade do animal, com potencial comprometimento da resposta imune nesses indivíduos. / Cannabis sativa (marijuana) is one of the most well-known and used illicit drugs worldwide. Recently, recreational use of that drug among young people has increased greatly, especially among pregnant women. Studies indicate that its use in pregnancy causes adverse fetotoxic effects, however there are few studies evaluating the effects of marijuana use during this critical period on the developing immune system. In this study, we evaluated the impact of gestational exposure to smoke marijuana resulting from burning cigarettes on the spleen and thymus development of BalbC mice offspring. We have used a model that approximates the real conditions of human use (inhalation) under the dose and medium exposure aspects. Pregnant mice (n=20), were exposed to marijuana smoke or filtered air (control group) from 5.5° to 17.5° gestational day (GD) for 5 minutes daily. Thus, we used a smoke inhalation apparatus developed in our laboratory where the marijuana cigarettes (200mg of Cannabis sativa) were burned and conducted to exposure chamber reaching the animals (nose-only exposure). Spleen and thymus of the offspring were collected on the 18.5° gestational day (GD), 20° and 60° postnatal day (PND) for the histological and histochemical analysis. Morphological and quantitative evaluations of these organs were performed using stereological methods and semi quantification of collagen fibers. The results indicate an increase in the mice body weight at PND 60 whose mothers were exposed to Cannabis sativa smoke during pregnancy. The PND 60 males from exposed group showed lower thymus total volume, as well as its main compartments such as medulla and cortex, where collagen fibers was also observed, when compared with males from the control group. Also, the spleen of these animals also presented a significant reduction, however no significant reduction was showed in the total volume of the red pulp. In addition, males and females from the exposed group at PND 60 presented reduction in total volume in the white pulp and increased of collagen fibers in the trabeculae region of the spleen. In summary, gestational exposure to marijuana causes fetotoxic effects that can be observed due to alteration in the spleen and thymus of the offspring (the effects being different for males and females), and these changes being marked with the advancement of the age, with potential impairment of the immune response in these individuals.