La co-culture d’amibes est utilisée afin d’isoler des microorganismes. Ainsi le premier virus géant,fut découvert. Cependant, les méthodes de culture sur protozoaires sont délicates et fastidieuses. De ce fait, le développement de ces cultures représente une difficulté pour les microbiologistes, limitant ainsi l’analyse d’un nombre important d’échantillons et la caractérisation de nouveaux virus. De nouvelles stratégies et des améliorations des modèles actuels sont donc nécessaires. Notre travail a été de développer de nouvelles stratégies permettant l’isolement de microorganismes associés aux amibes. Dans la 1ere partie nos travaux ont permis une amélioration des cultures avec le développement de nouveaux milieux de culture et l’utilisation ciblée d’antimicrobiens.La clé de ces stratégies est l’association des techniques rapides aux étapes améliorées de culture et leur application à un large panel de protozoaires pouvant abriter des microorganismes. Les résultats ont permis le développement d’un système d’isolement à haut débit très efficace. Nous avons notamment mis au point des techniques de tri de virus géants par cytométrie.Dans la seconde partie, nos travaux ont porté sur la description et la caractérisation des nouveaux isolats.Les résultats obtenus démontrent l’importance de poursuivre l’isolement et la caractérisation de ces microorganismes afin de mieux appréhender l’évolution de ces microorganismes, leur biotope et leur pathogénicité.De nouveaux outils sont nécessaires,notre manque d’imagination et l’absence de systèmes automatisés seront les limites aux nouvelles stratégies dans le monde de la microbiologie. / Amoebae are predators without distinction and they can also act as hosts to several different microorganisms that may coexist simultaneously. Some protozoa are sources of human pathogens where they act as reservoir of any human pathogens like Legionellae, Chlamydiaceae and others. In addition, the first giant virus, Acanthamoeba Polyphaga imivirus, was discovered using Amoeba as cell host. Since then, many other giant viruses have been isolated. For decades, amoebae were used as cell hosts in the culture- based process to isolate microorganisms, and allowed to recover new giant viruses and bacterial species from human and environmental samples. In contrast the co-culture system with protozoa is tedious and fastidious. Microbiologists are limited to routine culture methods, limiting by this the speed of screening potential samples and the efficiency of yielding new isolates. Much effort and improvement were needed. Our work consisted in the development of new strategies and techniques for the isolation of new microorganisms associated to protozoa. In the first part of this work, we described, all the improvements we brought to the protists culture system for the isolation of intracellular microorganisms especially giant viruses and Chlamydiaceae. Major improvements were based on modified culture enrichment steps, adapted culture media, and targeted use of specific drugs. The key of this new strategy was the implementation of high-throughput technologies to the ameliorated culture based systems, and the application of this later to a wide panel of protozoa used as potential host cells. These presented advances and strategies demonstrated significant time saving, and higher sensitivity than older techniques, they considerably increased the potential of collecting new environmental or clinical isolates and also new undiscovered microorganisms especially new giant virus familiesand particular Chlamydiaceae associated to amoebae. We continued to ameliorate the efficiency of the flow cytometric technology by reviewing its contributions to the virology field, then by applying it to the isolation system by sorting the new isolates as a new strategy for better genomic and proteomic analysis. In the second part of this work, we focused on the characterization of new isolates at the level of developmental cycle and genomic description. We used electron microscopy, and genome sequencing as main tools to describe our newly isolated giant viruses but also report new species of Chlamydiaceae and managed to decipher Chlamydiaceae species with a host dependent replication cycle, an issue that has not, thus far, been observed in protozoa-associated Chlamydiaceae. The strategies and results described herein show the importance of pursuing the isolation of new associatedamoebal microorganisms in order to give rise to new insights into the evolution of these microorganisms, their respective biotopes, and their potential or hidden pathogenicity. The more we need to search the more tools are needed, only our lack of imagination and appropriate automated systems will put limits on any needed strategy in the field of microbiology.

Récemment reconnus comme cancérogènes certains pour l'homme par l’IARC, la pollution atmosphérique et les particules fines (PM₂.₅) peuvent être inhalées et pourraient être retenues au niveau pulmonaire ou passer dans la circulation systémique. Ceci peut causer ou renforcer de nombreuses pathologies auxquelles les personnes âgées sont souvent plus sensibles. Cette thèse s’inscrit dans une démarche d’identification des processus impliqués dans la modulation du potentiel cancérogène des PM₂.₅, en lien avec l’âge ou le statut tabagique. Les particules ont été collectées à Dunkerque, agglomération présentant des influences maritimes mais également caractérisée par des activités industrielles et un trafic automobile importants. Pour évaluer l'influence de l'âge, des lymphocytes sanguins prélevés chez 90 patients issus de trois classes d'âge (25-30, 50-55 et 75-80 ans) ont été exposés ex vivo à des PM₂.₅ d’origine urbaine. Les lymphocytes isolés ont été exposés aux PM₂.₅ pendant 72 heures, avant de mesurer l'activité télomérase et la modulation d'expression de gènes tels que P16INK4A et MGMT. Les PM₂.₅ entraînent des variations de l'activité télomérase et de la longueur des télomères dans toutes les tranches d'âge indifféremment. L’expression du gène P16INK4A est significativement augmentée avec l'âge après exposition aux PM₂.₅. L'âge augmenterait l'expression du gène MGMT après exposition aux particules, en diminuant le niveau de méthylation de son promoteur uniquement dans le groupe des patients les plus âgés. Concernant le rôle du statut tabagique, 26 lavages broncho-alvéolaires ont été réalisés chez des patients fumeurs et non-fumeurs. Les macrophages issus de ces prélèvements ont été mis en culture avec des cellules épithéliales bronchiques BEAS-2B, avant exposition aux PM₂.₅ (3 et 15 µg/cm², 72 h). L’activité télomérase et la longueur des télomères varient après exposition aux PM2.5 et le statut tabagique modifie ces paramètres dans les cellules BEAS-2B et les macrophages alvéolaires. La méthylation des promoteurs et l’expression des gènes P16INK4A et MGMT ne sont pas modifiées dans les cellules BEAS-2B, alors que dans les macrophages alvéolaires les particules induisent l’expression de ces gènes par une diminution de la méthylation de leurs promoteurs. Le statut tabagique fumeur semble au contraire accroître la méthylation et limite l’expression de ces deux gènes. En conclusion, il apparaît que l’échantillon de PM₂.₅ étudié peut induire ex vivo plusieurs lésions décrites dans les étapes d’initiation et de promotion de la cancérogenèse broncho-pulmonaire. L’âge et le tabagisme sont susceptibles de moduler les effets toxiques des particules. Alors que les symptômes du cancer du poumon apparaissent seulement à une étape avancée de la maladie, nos résultats pourraient aider à la découverte de nouveaux marqueurs de diagnostic précoce permettant ainsi d’améliorer la survie. / Recently recognized as carcinogenic to human by IARC, air pollution and fine particulate matter (PM₂.₅) can be inhaled and could be retained into the lung or reach the systemic circulation. This can cause or worsen many diseases for which the elderly are often more sensitive. The PhD objective corresponds to the identification of the mechanisms of action involved in the modulation of carcinogenic potential of PM₂.₅, in connection with age or smoking status. PM₂.₅ were collected in Dunkerque, a French seaside city characterized by important industrial activities and heavy motor vehicle traffic. In order to estimate the influence of age, blood lymphocytes sampled from 90 patients from age classes (25-30, 50-55 and 75-80 years old) were ex vivo exposed to PM₂.₅ during 72 hours, before evaluation of telomerase activity and gene expression modulation of P16INK4A and MGMT. PM₂.₅ modulated telomerase activity and telomeres length in all age groups without any influence of age. P16INK4A gene expression increased significantly with age after exposure to PM₂.₅. Age could enhance MGMT gene expression after exposure to particles by decreasing the level of promoter methylation in the oldest group. Regarding the role of smoking status, 26 broncho-alveolar lavage were performed in smoker and non-smoker people. Macrophages were cultured with bronchial epithelial BEAS-2B cells before PM₂.₅ exposure (3 or 15µg/cm²; 72h). The telomerase activity and telomere length vary after exposure and the tobacco modify these parameters in BEAS-2B cells and alveolar macrophages. Methylation of P16INK4A and MGMT genes promoters and their expression are not modified in BEAS-2B cells. In alveolar macrophages, particles lead to a decrease of methylation of P16INK4A gene promoter. The smoking status seems also to increase methylation and to down-regulate expression of these two genes. In conclusion, it seems that the studied PM₂.₅ sample can induce ex vivo modifications described in the initiation and promotion of lung carcinogenesis. The age and smoking status may modulate the toxic effects of particles. Since lung cancer symptoms appear only at an advanced stage, our results could help in proposing new biomarkers of carcinogenesis allowing an early diagnosis to improve survival.

No description available.

Ziel dieser Studie war die Etablierung einer Kokultur aus equinen endometrialen Epithel- und Stromazellen. Nach der erfolgreichen Umsetzung des Kokulturmodells sollte im weiteren Versuchsablauf durch die Zugabe von 17β-Östradiol (E2) und/oder Progesteron (P4) zum Nährmedium der Einfluss der Hormone auf die Zellen untersucht werden. Neben einer lichtmikroskopischen Auswertung der zytomorphologischen Charakteristika beider Zellarten sollte die Expression der Steroidhormonrezeptoren Östrogenrezeptor α und Progesteronre-zeptor sowie der uterinen Proteine Uteroglobin und CalbindinD9k immunzytologisch überprüft werden. Für die Etablierung der Kokultur wurden Endometriumproben von lebenden (n = 5) sowie frischtoten (n = 4) Stuten gewonnen. Eine jeweils parallel entnommene Gewebeprobe von jedem Tier wurde in Formalin fixiert und diente als Referenzmaterial (in situ). Auf die Zelliso-lierung (mechanisch und enzymatisch) folgte die Separation von Epithel- und Stromazellen (EZ/SZ) mittels Filtration, Dichtegradientenzentrifugation und Differenzialadhärenz. An-schließend wurden die EZ auf die Außenseite von Millicell®-Membraneinsätzen aufgebracht. Nach zwei Tagen erfolgte das Einsäen der bis zu diesem Zeitpunkt separat kultivierten SZ auf die Innenseite der Membranen. Als Nährmedium diente ein Gemisch aus DMEM und Ham’s F-12, wobei diesem 2,5 % fötales Kälberserum sowie verschiedene Additive zugesetzt wurden. Ab Kulturtag 4 wurden dem Medium definierte Konzentrationen und Kombinationen von E2 und P4 zugesetzt. Die Kultivierung erfolgte bei einem CO2-Partialdruck von 5 % in 37 °C warmer wasserdampfgesättigter Raumluft. Mit der polarisationsmikroskopisch er-fassbaren Ausbildung durchgehender Zellrasen („scheinbare Konfluenz“) wurden die Kokul-turen in Formalin fixiert und für die Lichtmikroskopie aufgearbeitet. Das Ausgangsgewebe zeigte mehrheitlich eine sekretorische Funktionsmorphologie (n = 6). Einzelne Endometrien befanden sich in einem Übergangsstadium von der Sekretions- zur Proliferationsphase (n = 1), bzw. vice versa (n = 1) oder wiesen eine irregulär proliferative Differenzierung (n = 1) auf. Im Rahmen der Kokultivierung bildeten die EZ innerhalb der Schnittebene vier und die SZ drei verschiedene morphologische Zelltypen aus. Dabei traten rundovale bis polygonale EZ (Typ 1) selten bis gelegentlich, spindelförmige EZ (Typ 2) gelegentlich bis häufig und iso-prismatische (Typ 3) sowie mehrschichtig wachsende EZ (Typ M) jeweils selten auf. Die SZ zeigten innerhalb der Schnittebene selten eine rundovale bis polygonale Zellform (Typ 1), sehr häufig eine spindelförmige Morphologie (Typ 2) und selten ein mehrschichtiges Wachstum (Typ M). Ein Zusammenhang zwischen der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung oder dem Hormonzusatz und der Häufigkeitsverteilung der Zell-typen sowie der Wachstumsgeschwindigkeit der kultivierten Zellen war nicht offensichtlich. Zytokeratin 19 wurde stets von EZ exprimiert, während es auf Seiten der SZ nur sporadisch in maximal 5 % der Zellen im Bereich mehrschichtig wachsender Zellrasen auftrat. Die Stero-idhormonrezeptoren konnten lediglich in einzelnen Kokulturen aus sekretorisch differenzier-tem Ausgangsgewebe detektiert werden. Uteroglobin wurde in vitro mit einer variablen Häufigkeit in den EZ-Typen exprimiert. Während ein übergreifender Zusammenhang zur hormonellen Supplementierung nicht abgeleitet werden konnte, wurde jedoch ersichtlich, dass im Bereich einschichtig wachsender EZ in Ansätzen aus sekretorisch differenzierten Endometrien unter niedrigen Hormondosen (Zusatz von entweder nur E2 oder nur P4) im Median häufiger Uteroglobin exprimiert wurde. Mit zunehmender Hormonkonzentration im Medium nahm der Anteil immunopositiver Zellen (Typen 1, 2 und 3) deutlich ab. Innerhalb der Stromazellpopulation wurde Uteroglobin selten und ausschließlich in Zellen aus sekretorisch differenziertem Ausgangsmaterial nachgewiesen. CalbindinD9k wurde in vitro vornehmlich intrazytoplasmatisch und sehr vereinzelt intranukleär exprimiert. Insgesamt konnte das Protein in vitro stets in wenigen Typ-1-EZ, sehr selten in Typ-2-EZ und in einer geringen bis mäßigen Anzahl von Typ-3- und Typ-M-EZ beobachtet werden. Innerhalb der Stromazellpo-pulation trat CalbindinD9k ausschließlich in einer geringen (Endometrien aus dem Östrus) bis mäßigen (Endometrien aus dem Interöstrus) Anzahl der Typ-2- und wenigen Typ-M-SZ auf. Insgesamt wurden keine deutlichen Einflüsse der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung und/oder der hormonellen Supplementierung in vitro auf die im-munzytologischen Charakteristika der kokultivierten Zellen ersichtlich. Abschließend betrachtet, konnte ein Kokultursystem equiner endometrialer Epithel- und Stromazellen erfolgreich etabliert und charakterisiert werden. Es bietet dabei, trotz der z. T. fehlenden Kongruenz zu den Gegebenheiten in situ, Ansätze für potenzielle Folgearbeiten, insbesondere hinsichtlich der Erfassung interzellulärer Wechselwirkungen sowie bezüglich der Vermittlung und Wirkung hormoneller Einflüsse auf zellulärer Ebene. / The aim of the present study was the establishment of a coculture system of equine endome-trial epithelial and stromal cells. Subsequent to the successful development of the coculture model the culture medium should be supplemented with 17β-estradiol (E2) and/or progester-one (P4) in order to study the influence of the hormones on the cellular level. In addition to the examination of cytomorphological characteristics of both cell types via light microscopy, the expression of the steroid hormone receptors (estrogen receptor α and progesterone receptor) as well as of the uterine proteins Uteroglobin and CalbindinD9k was investigated. For the establishment of the coculture system endometrial samples were obtained from living (n = 5) as well as freshly deceased mares (n = 4). A simultaneously taken tissue specimen of each animal was fixed in formalin and served as in situ reference material. After an initial mechanical and enzymatical isolation the epithelial and stromal cells (EC/SC) were separat-ed via filtration, density gradient centrifugation and differential adhesion. Subsequently, the EC were applied to the outer surface of Millicell® inserts. The SC were cultivated separately for 2 days before they were seeded onto the inner surface of the same insert. The culture medium used was comprised of a DMEM and Ham‘s F-12 basis as well as 2.5 % foetal calf serum and different additives. Starting on day 4 of cultivation the standardised medium was supplemented with different concentrations and combinations of E2 and P4. Throughout the study the cultures were kept in a humidified atmosphere of 37°C and a 5 % partial pressure of carbon dioxide. Once the cocultures formed continuous cell layers, as determined via a polarisation microscope (“apparent confluency”), the membranes were fixed in formalin and routinely processed for light microscopical evaluation. The initial tissue samples predominantly showed a secretory functional morphology (n = 6), while single specimens were obtained during the transition from the secretory to the prolifera-tive phase (n = 1) or vice versa (n = 1). One endometrial sample exhibited an irregular proli-ferative differentiation. In the course of cocultivation the EC formed 4 and the SC 3 different cellular morphologies within the section plane. EC with a round-oval to polygonal cell form (type 1) were rarely to occasionally encountered, while spindle-shaped EC (type 2) were occasionally to frequently seen and EC with a cuboidal morphology (type 3) as well as such cells growing in stratified layers (type M) were only infrequently detected. The SC only rarely showed a round-oval to polygonal cell form (type 1) or areas of a stratified cell growth (type M), whereas spindle-shaped SC (type 2) were observed very often. A correlation of the endometrial functional morphology at the time of cell isolation or the hormonal supplementation and the frequency distribution of the cell types as well as the growth rate of the cultivated cells was not evident. The EC always expressed Cytokeratin 19, while on the side of the SC only up to 5 % of the cells in areas of stratified cell growth exhibited this filament. Solely in individual cocultures from secretory differentiated endometrial tissue the steroid hormone receptors could be de-tected. Uteroglobin was expressed in vitro in EC with a variable frequency. An overall corre-lation of the hormonal supplementation and the Uteroglobin expression could not be derived. However, under low hormone doses (only E2 or only P4 supplement) Uteroglobin was detect-ed in EC in areas of single-layered cell growth more often (median value). With an increase in hormone concentration the amount of immunopositive cells (types 1, 2 and 3) diminished noticeably. In SC the protein could only rarely be seen and exclusively in cells from endome-tria with a secretory functional morphology. In vitro CalbindinD9k was predominantly detected intracytoplasmatically, while single cells showed an additional intranuclear expression. Alto-gether, CalbindinD9k could always be observed in a few type-1-EC, rarely in type-2-EC and with a variable frequency in small to moderate numbers of type-3- and type-M-EC. In SC the protein was exclusively expressed in a small (endometrial samples form the oestrous phase) to moderate (endometrial tissue from the interoestrous phase) number of type-2-SC and a few type-M-SC. Generally, no distinct influence of the endometrial functional morphology at the time of tissue sampling and/or of the hormonal supplementation in vitro on the immuno-cytochemical characteristics of the cocultured cells could be observed. In summary, a coculture system of primary equine endometrial epithelial and stromal cells was successfully established and characterised. Despite of the partly absent congruence to the in situ conditions/prerequisites, the present study offers a basic approach and scaffold for further investigations, particularly regarding the ascertainment of intercellular dependencies or the mediation and effectiveness of hormonal influences on the cellular level.

Transwell inserts with microporous membranes, available from multiple commercial sources, have been widely used for various mammalian cell culture applications, including the reduction of cell culture mixing. In this study, we examined the feasibility and functionality of using this technology for separating human embryonic stem cells (hESCs) from their respective feeder cells. We found that when hESCs were propagated on transwell inserts positioned directly above feeder cells grown in a separate dish, the hESCs could be maintained in an undifferentiated state for over 10 passages with no change in their basic pluripotent characteristics. In parallel with our transwell insert experiments, we also evaluated the ability of a new defined, xeno-free medium, HEScGRO™, to enhance the animal-free characteristics of the transwell insert-based culture system. Results from our studies demonstrate that HEScGRO™ medium assists in maintaining the pluripotent characteristics of hESCs propagated in the transwell insert- based culture system. These combined results represent a significant development in properly segregating stem cells from their feeders, thus eliminating cell mixing, contamination, and providing the cells with a superior environment for nourishment and controlled self-renewal. Overall, this development in hESC propagation could have wide-reaching applications for self-renewal and differentiation studies within the field of stem cell biology.

La période de réceptivité endométriale chez l’humain coïncide avec la différentiation des cellules stromales de l’endomètre en cellules hautement spécifiques, les cellules déciduales, durant le processus dit de décidualisation. Or, on sait qu’une transformation anormale des cellules endométriales peut être à l’origine de pertes récurrentes de grossesses. LRH-1 est un récepteur nucléaire orphelin et un facteur de transcription régulant de nombreux évènements relatif à la reproduction et comme tout récepteur, son activation promouvoit l’activité transcriptionnelle de ses gènes cibles. Nous avons déjà montré que LRH-1 et son activité sont essentiels pour la décidualisation au niveau de l’utérus chez la souris et nous savons qu’il est présent dans l’utérus chez l’humain au moment de la phase de prolifération mais aussi de sécrétion du cycle menstruel, et que son expression augmente dans des conditions de décidualisation in vitro. Notre hypothèse est alors la suivante : LRH-1 est indispensable à la décidualisation du stroma endométrial, agissant par le biais de la régulation transcriptionnelle de gènes requis pour la transformation de cellules stromales en cellules déciduales. Afin d’explorer le mécanisme moléculaire impliqué dans la régulation transcriptionnelle effectuée par l’intermédiaire de ce récepteur, nous avons mis en place un modèle de décidualisation in vitro utilisant une lignée de cellules stromales de l’endomètre, cellules humaines et immortelles (hESC). Notre modèle de surexpression développé en transfectant les dites cellules avec un plasmide exprimant LRH-1, résulte en l’augmentation, d’un facteur 5, de l’abondance du transcriptome de gènes marqueurs de la décidualisation que sont la prolactine (PRL) et l’insulin-like growth factor binding protein-1 (IGFBP-1). En outre, la sous-régulation de ce récepteur par l’intermédiaire de petits ARN interférents (shRNA) abolit la réaction déciduale, d’un point de vue morphologique mais aussi en terme d’expression des deux gènes marqueurs cités ci-dessus. Une analyse par Chromatin ImmunoPrécipitation (ou ChIP) a démontré que LRH-1 se lie à des régions génomiques se trouvant en aval de certains gènes importants pour la décidualisation comme PRL, WNT 4, WNT 5, CDKN1A ou encore IL-24, et dans chacun de ces cas cités, cette capacité de liaison augmente dans le cadre de la décidualisation in vitro. Par ailleurs, des études structurelles ont identifié les phospholipides comme des ligands potentiels pour LRH-1. Nous avons donc choisi d’orienter notre travail de façon à explorer les effets sur les ligands liés à LRH-1 de traitements impliquant des agonistes et antagonistes à notre récepteur nucléaire. Les analyses par q-PCR et Western blot ont montré que la modulation de l’activité de LRH-1 par ses ligands influait aussi sur la réaction déciduale. Enfin, des études récentes de Salker et al (Salker, Teklenburg et al. 2010) ont mis en évidence que les cellules stromales humaines décidualisées sont de véritables biocapteurs de la qualité embryonnaire et qu’elles ont la capacité de migrer en direction de l’embryon. La série d’expériences que nous avons réalisée à l’aide de cellules hESC placées en co-culture avec des embryons de souris confirme que la migration cellulaire est bien dirigée vers les embryons. Cette propriété quant à l’orientation de la migration cellulaire est notoirement diminuée dans le cas où l’expression de LRH-1 est déplétée par shRNA dans les hESC. Nos données prouvent donc que LRH-1 régule non seulement la transcription d’un ensemble de gènes impliqués dans le processus de décidualisation mais agit aussi sur la motilité directionnelle de ces cellules hESC décidualisées in vitro. / The period of endometrial receptivity in humans coincides with the differentiation of endometrial stromal cells into highly specialized decidual cells through a process known as decidualization. This transformation of endometrial cells is abnormal in recurrent pregnancy loss patients. Liver homolog receptor 1 (LHR-1, NR5A2) is an orphan nuclear receptor and a transcription factor that regulates many reproductive events. The activation of this receptor leads to transcriptional activation of its target genes. We have previously shown that it is essential for decidualization in the mouse uterus. LRH-1 is expressed in the human uterus in both proliferative and secretory phases of the menstrual cycle and its expression increases during in vitro decidualization. We hypothesize that LRH-1 is indispensable for proper decidualization of the endometrial stroma, acting through the transcriptional regulation of genes required for transformation of stromal cells into decidual cells. To explore the molecular mechanism of transcriptional regulation mediated by this receptor, we established an in vitro model of decidualization, using an immortal human endometrial stromal cell line (hESC). An overexpression model developed by transfecting the cells with a plasmid constitutively expressing Lrh-1, resulted in 5 fold increases in abundance of transcripts for the decidualization marker genes prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Furthermore, the downregulation of the receptor using short hairpin RNA (shRNA) abrogates the decidual reaction, from both a morphological point of view and in terms of expression of the two marker genes. Chromatin immunoprecipitation (ChIP) analysis showed that LRH-1 binds to genomic regions upstream of genes important for decidualization such as PRL, wingless-type MMTV integration site family, member 4 (WNT4), wingless-type MMTV integration site family, member 5 (WNT5), cyclin-dependent kinase inhibitor 1A (p21, CDKN1A) and interleukin-24(IL-24). For each of these genes, the binding increased during in vitro decidualization. Structural studies have identified phospholipids as potential LRH-1 ligands. We therefore explored the effect of ligand treatment on LRH-1 with an agonist and an inverse agonist for the nuclear receptor. Analysis by quantitative polymerase chain reaction (qPCR) and Western blot demonstrated that the modulation of LRH-1 activity by its ligands also affects the decidual reaction. Recent studies have shown that decidualized human stromal cells are biosensors of embryo quality and that they have the capacity to migrate towards the embryo. Our time-lapse evaluation of hESC cells co-cultured with mouse embryos indicates directed migration of the cells toward the embryo. This effect is markedly diminished when LRH-1 is depleted by shRNA in hESC. Our data provide evidence that LRH-1 regulates not only the transcription of a set of genes involved in decidualization but also the directional motility of these cells in vitro.

O carcinoma hepatocelular é um dos tipos de cânceres mais comuns em adultos com sua incidência aumentando mundialmente a cada ano. O tratamento curativo é o transplante de fígado, mas as muitas dificuldades encontradas para o procedimento faz com que a quimioterapia seja amplamente utilizada. Além disso, pode ocorrer quimiorresistência e muitos efeitos adversos, o que impulsiona a busca por novos compostos terapêuticos. L-aminoácido oxidases (LAAO) isoladas de peçonhas de serpentes têm demonstrado bons resultados de citotoxicidade em linhagens celulares tumorais, no entanto estes resultados representam sistemas in vitro em monocultura e estudos recentes demonstram que o microambiente tumoral tem um importante papel na transformação neoplásica, no crescimento e invasão do tumor e na resistência quimioterápica. Assim, avaliou-se uma LAAO purificada da peçonha de Bothrops jararacussu (BjussuLAAO-II) em células de carcinoma hepatocelular (HepG2) em monocultura e em cocultura com células endoteliais de veia umbilical humana (HUVEC) com o objetivo de simular o microambiente tumoral, onde, normalmente, é observado mais de um tipo celular. A atividade citotóxica foi avaliada por meio do ensaio do MTT e do ensaio de sobrevivência clonogênica, a atividade genotóxica por meio do ensaio do cometa, a produção de espécies reativas de oxigênio por meio de fluorescência e os danos ao cromossomo por meio do ensaio do micronúcleo. Em células HepG2, em monocultura, todas as concentrações testadas (0,25 - 5,00 ?g/mL) foram citotóxicas e aumentaram os níveis intracelulares de espécies reativas de oxigênio. Verificou-se dano genotóxico na concentração de 5,00 ?g/mL e não houve dano cromossômico. Quando cultivada em cocultura com células HUVEC, as concentrações de 1,00 e 5,00 ?g/mL de BjussuLAAO-II foram citotóxicas às células HepG2 e aumentaram os níveis de espécies reativas de oxigênio. A concentração de 5,00 ?g/mL induziu danos ao DNA e não houve danos aos cromossomos. Estes efeitos podem ser correlacionados ao aumento de espécies reativas de oxigênio intracelulares e as diferenças entre os resultados de mono e cocultura devido à simulação de parte do microambiente tumoral. Em monocultura de células HUVEC, todas as concentrações testadas foram citotóxicas, houve dano genotóxico nas concentrações de 1,00 e 5,00 ?g/mL e nenhuma concentração testada induziu danos cromossômicos. Como há elevada citotoxicidade, danos ao DNA e indução de efeitos pró-oxidantes em células HepG2, BjussuLAAO-II representa um composto promissor no desenvolvimento de novos fármacos antitumorais / Hepatocellular carcinoma is one of the most common types of cancers in adults whose incidence increases worldwide each year. Liver chirurgic and transplantation is the best choice of treatment, but the many difficulties encountered in the procedure cause chemotherapy to be widely used. In addition, chemo resistance and many adverse effects can occur, which improves the search for new therapeutic molecules. L-amino acid oxidases (LAAO) isolated from snake venoms have demonstrated good cytotoxicity results in tumor cell lines, however these results represent in vitro monoculture systems and recent studies have shown that the tumor microenvironment plays an important role in neoplastic transformation in tumor growth and invasion and chemotherapeutic resistance. A LAAO purified from Bothrops jararacussu venom (BjussuLAA-IIO) venom was evaluated in hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with human umbilical vein endothelial cells (HUVEC) with the aim of simulating the tumor microenvironment, where more than one cell type is normally observed. Cytotoxic activity was assessed by the MTT assay and clonogenic survival assay, genotoxic activity by comet assay, reactive oxygen species production by fluorescence, and chromosome damage by the micronucleus assay. In HepG2 cells, in monoculture, all concentrations tested (0.25 - 5.00 ?g/mL) were cytotoxic and increased intracellular levels of reactive oxygen species. Genotoxic damage at the concentration of 5.00 ?g/mL was observed and there was no chromosomal damage. When cultured with HUVEC cells, concentrations of 1.00 and 5.00 ?g/mL of BjussuLAA-II were cytotoxic to HepG2 cells and increased levels of reactive oxygen species. The concentration of 5.00 ?g/mL induced DNA damage and there was no damage to the chromosomes. These effects can be correlated to the increase of intracellular reactive oxygen species and the differences between the mono and co-culture results due to the simulation of part of the tumor microenvironment. In HUVEC monoculture, all concentrations tested were cytotoxic, genotoxic damage at concentrations of 1.00 and 5.00 ?g/mL, and no concentration tested induced chromosome damage. As there is high cytotoxicity, DNA damage and induction of pro-oxidant effects in HepG2 cells, BjussuLAAO-II represents a promising compound in the development of novel antitumor drugs

Observações feitas pelo próprio autor sugerem potencial ação do Líquido da Castanha de Caju (LCC) na pigmentação da pele, ação esta semelhante a da hidroquinona. O LCC é um líquido contido na casca da castanha de caju, possui característica de resina líquida, bastante viscosa e de odor forte, sua coloração varia de marrom claro, escuro a preto, dependendo do método de extração utilizado, podendo ser denominado de Natural ou Técnico. Este estudo propôs cultivar melanócitos e queratinócitos em cocultura e posteriormente tratálos com LCC. A L-DOPA, agente estimulador da melanogênese, via da produção de melanina, responsável pela pigmentação da pele, foi utilizada na cocultura para avaliar a ação do LCC. A hidroquinona, conhecido inibidor desta via, foi utilizada na cocultura como controle positivo para o LCC, visto que este poderia apresentar ação semelhante a da hidroquinona. Para a utilização do LCC na cocultura, testes de solubilidade do mesmo para posterior dispersão no meio de cultura, foram necessários, bem como a identificação de seu potencial cito e fototóxico in vitro. Para a realização do teste de fototoxicidade foi construída uma câmara específica, atendendo as normas exigidas pelos guias ©ECVAM DB-ALM: INVITTOX protocol e OECD TG-432, sendo esta qualificada por método validado. Os testes realizados com o LCC (natural e técnico) indicaram potencial ação destes na pigmentação da pele, estimulando a proliferação de melanócitos em cocultura. Este perfil apresentado, pelos extratos de LCC, é contrário ao da hidroquinona, e ao esperado inicialmente, sendo necessário aprofundar estes estudos. No entanto, estes resultados são promissores, sugerindo a descoberta de um novo tratamento para hipocromias. / Observations, made by the author, suggest potential action of Cashew Nut Shell Liquid (CNSL) in skin pigmentation, action similar to hydroquinone. The CNSL is a liquid contained inside the shell of the cashew nut, it has features of liquid resin, quite viscous and strong smell. Its color varies from clear or dark brown, to black, depending on the extraction method used, which may be called Natural or Technical. This study aimed to cultivate melanocytes and keratinocytes in co-culture and thus treat them with CNSL. The L-DOPA, stimulating agent of melanogenesis, melanin production pathway, responsible for skin pigmentation, was used to assess the CNSL action in the co-culture. Hydroquinone, known inhibitor of this pathway, was used as positive control for CNSL in the co-culture, since this could provide a similar action to hydroquinone. For use of CNSL in the co-culture, solubility tests, for subsequent dispersion in the culture medium, were necessary, as well as the identification of its cytototoxic and phototoxic potential in vitro. To achievement of the phototoxicity tests a specific chamber was built according to the standards required by the guides ©ECVAM DB-ALM: INVITTOX protocol and OECD TG-432, being qualified by validated method. Tests conducted with the CNSL (natural and technical) indicated their potential actions in skin pigmentation, stimulating the proliferation of melanocytes in co-culture. This behavior presented by the CNSL extracts, is opposite to the hydroquinone, and to the initially expected, being required further studies. However, these results are promising, suggesting the discovery of a new treatment for hypochromia.

O sucesso no desenvolvimento de tumores não dependente somente de mutações, mas também é influenciado pelo microambiente do tumor; nele ocorre a interação entre as células tumorais e o estroma. Essa interação pode ser mediada por vesículas liberadas por essas células para o meio extracelular. Essas vesículas atuam na comunicação celular que pode influenciar a progressão tumoral. O objetivo deste estudo foi analisar as interações mediadas por vesículas entre células tumorais e fibroblastos normais. As células tumorais foram plaqueadas sobre a monocamada de fibroblastos e carregadas com diferentes corantes vitais. Nossos resultados evidenciaram a presença e a troca de vesículas entre as células em co-cultura. Vesículas isoladas mostraram tamanhos heterogêneos. Células tumorais possuem mais vesículas que as células normais. As vesículas são compostas pelas proteínas AHNAK e Anexinas. AHNAK foi detectada em vesículas trocadas e estava aumentada em tumores. AHNAK é molécula estrutural das vesículas extracelulares que pode influenciar a biologia dos tumores de mama. / The successful development of tumors is not only dependent on cell mutations, but also driven by the tissue microenvironment; relies on interaction of cells and their surrounding stroma. Some cell types release vesicular structures into the extracellular space that would be involved in cellular communication and tumor progression. The aim of this study was to analyze vesicle-mediated interactions between tumor cells and normal fibroblasts. Tumor cells were plated above fibroblasts monolayer and both loaded with different vital dyes. Our results evidenciated presence and exchange of vesicles between breast tumor cells and fibroblasts in co-culture. Vesicles isolated showed heterogeneous sizes. Tumor cell showed more vesicles than normal cells. These vesicles were composed of AHNAK and Annexins proteins. The protein AHNAK was detected in exchanged vesicles and was increased in tumors when compared to normal breast tissues. AHNAK could represent a vesicle structural molecule that would influence breast tumor biology.

Bostrychia tenella, proveniente dos costões rochosos da Praia Dura, no Estado de São Paulo. Para atingir este objetivo, foram realizados culturas isoladas dos microrganismos e a co-cultura entre o fungo T68 e a cianobactéria CENA139; técnicas de extração por partição volume/volume; métodos cromatográficos como cromatografia em camada delgada (CCD) e cromatografia líquida de alta eficiência (CLAE), bem como técnicas espectroscópicas e espectrométricas de RMN 1-D e 2-D, CL-EM, CG-EM, além de desreplicação (utilizando o banco de dados MarinLit® e o Dicionário de Produtos Naturais. Foram isoladas três substâncias do fungo estudado: Esterigmatocistina, T68ARf60_a e T68ARf60_b. A micotoxina esterigmatocistina foi isolada também a partir da co-cultura entre o fungo e a cianobactéria. Este trabalho mostrou que houve interações ecológicas competitivas na cultura mista entre Cyanobium sp. CENA139 e o fungo T68, resultando na prevalência da cultura fúngica. Para finalizar, este trabalho avaliou a atividade biológica dos extratos brutos das culturas isoladas e co-cultura, não apresentando atividade antimicrobiana. Entretanto, o extrato bruto T68 mostrou-se fototóxico nos ensaios de fototoxicidade e a substância isolada esterigmatocistina foi citotóxica e fotoxóxica no mesmo ensaio, além de exercer citotoxicidade frente à linhagem tumoral HepG2 no ensaio de citotoxicidade MTT. / This study had as main objective the evaluation of the chemical and biological profiles of two strains of microorganisms, the cyanobacteria Cyanobium sp. CENA139, from Ilha do Cardoso mangroves, São Paulo State, and the endophytic fungus T68, Xylariaceae family, isolated from the red algae Bostrychia tenella, from the rocky shores at Praia Dura, São Paulo State. To accomplish this objective, we performed isolated cultures of the microorganisms and the co-culture between the fungus T68 and the cyanobacteria CENA139; partition extraction techniques; chromatographic methods such as thin layer chromatography and high performance liquid chromatography. Also, we performed spectroscopic and spectrometric techniques of NMR 1-D and 2-D, LC-MS, GC-MS, besides dereplication using data sources as MarinLit® and the Dictionary of Natural Products. Three compounds were isolated from the endophytic fungus culture: Sterigmatocystin, T68ARf60_a and T68ARf60_b. The mycotoxin sterigmatocystin was also isolated from the co-culture. This study showed that there was competitive ecological interactions between Cyanobium sp. CENA139 and the fungus T68 in the co-culture, resulting in the prevalence of the fungal culture. Finally, this study evaluated the biological potential of the crude extract from both isolated cultures and the co-culture, not exerting antimicrobial activity. Nevertheless, the T68 crude extract was shown to be phototoxic on the phototoxicity assays and the isolated sterigmatocystin was both cytotoxic and phototoxic in the same assay, besides exerting cytotoxicity on the tumor cell line HepG2 in the MTT assay.