Oligodendrocytes (OL)s are the dominant cell type in the white matter and are integral for synaptic transmission essential for proper neuronal communication between brain areas. Previous studies have shown that intravenous administration of the mononuclear fraction of human umbilical cord blood (HUCB) cells in rat models of stroke reduced white matter injury, gray matter injury and behavioral deficits. Yet the mechanisms used by HUCB cells remain unknown in ischemic injury. These studies will investigate both in vitro and in vivo approaches to elucidate this mechanism in OLs. When mature primary OLs were coincubated with HUCB cells, HUCB cells secreted soluble factors that reduced cell death in OLs exposed to OGD. Microarray analysis revealed that HUCB cell treatment induced OL gene changes. These changes included genes involved in cell proliferation, signaling, anti-oxidant activity, and myelination. To extend these findings, the middle cerebral artery occlusion (MCAO) model was used to assess the expression profile of protein products of gene changes observed in vitro. The in vivo data mirrored in vitro data in that metallothionein 3 (Mt3), peroxiredoxin 4 (Prdx4), myelin oligodendrocyte glycoprotein (Mog), U2AF homology kinase 1(Uhmk1), and insulin induce gene 1(Insig1) were upregulated in OLs of the white matter tract adjacent to the infarct. Furthermore, double immunofluorescence staining determined that OLs expressed these proteins. Other reports have shown that HUCB cells secrete soluble factors related to cellular protection, including interleukin 6 (IL-6), interleukin 8 (IL-8), and interleukin 10 (IL-10). Other factors are known for their proliferative actions, such as vascular endothelial growth factor (VEGF), BDNF, platelet derived growth factor B (PDGF-B), leukemia inhibitory factor (LIF), and granulocyte colony stimulating factor (GCSF) all of which converge on the Akt survival pathway. Given these findings we hypothesize that Akt activation is integral to HUCB cell mediated OL protection. In models of excitotoxicity, the addition of Akt inhibitor IV blocked HUCB cell mediated protection in OL cultures exposed to 24 hrs OGD. In vivo, HUCB cell treatment increased Akt activation, antioxidant protein expression and decreased caspase 3 cleavage in the external capsule in a time dependent manner. The next series of experiments determine whether the soluble factors secreted by HUCB cells could replace HUCB cells as treatment. LIF expression is increased in HUCB cells as compared to peripheral blood and as previously mentioned, LIF is secreted by HUCB cells. Additionally, LIF rescued OLs from spinal cord and experimental autoimmune encephalomyelitis injury. Thus LIF was investigated. LIF protected OL subjected to 24 hr OGD, increased antioxidant Prdx4 gene expression and reduced reactive oxygen species production. Additionally the inclusion of Akt inhibitor IV blocked LIF induced OL protection. Similar results were obtained when GCSF was evaluated. All these findings indicate that HUCB cell mediated OL/white matter protection is due to the soluble factors secreted by the mononuclear population of these cells. These soluble factors including LIF activate cellular machinery leading to enhanced cellular survival. Here we found a specific survival pathway activated by soluble factors released from HUCB cells, leading to Akt activation. Akt activation arrests stroke induced apoptosis and reduced the expansion of the infarct, promoting functional recovery from acute ischemic injury.

ABSTRACT A defect implantation process is the major reason for unexplained infertility. Estrogen andprogesterone are steroid hormones preparing the endometrium for implantation. They mediatetheir effect through their receptors: estrogen receptor alpha and beta and progesteronereceptor A and B, respectively. Leukemia inhibitory factor (LIF), which is also important forimplantation, mediates its effect through LIF receptor and the coreceptor, gp130, and is downregulated by suppressors of cytokine signaling 1. The aim of the study was to compare thelevels of the steroid hormone receptors and LIF related factors in the endometrium of twogroups of women with the diagnosis unexplained infertility: one that became pregnant afterassisted reproduction and one that did not become pregnant. Before treatment of thesewomen, endometrial mRNA was collected during the window of implantation in themenstrual cycle. The levels of specific mRNAs were measured with real-time PCR. Womenwho had become pregnant had a significantly higher level of steroid hormone receptors. Thus,these proteins seem to be important for a pregnancy and may be suitable as receptivitymarkers.

No description available.

Orientadores: Liliana Aparecida Lucci de Angelo Andrade, Fatima Aparecida Böttcher-Luiz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T02:59:06Z (GMT). No. of bitstreams: 1 Pietro_Luciana_D.pdf: 3492137 bytes, checksum: 723d823e8ddb16925da7aa8f48f22ea1 (MD5) Previous issue date: 2013 / Resumo: A invasão do endométrio pelo trofoblasto extraviloso é fundamental no desenvolvimento do feto e da placenta, processo este controlado por fatores ligados à atividade imunológica e hormonal que, quando alterada, pode resultar em interrupção da gestação e/ou geração das chamadas doenças trofoblásticas gestacionais. Em algumas situações, pode haver evolução para o coriocarcinoma, neoplasia maligna do trofoblasto, em que há evidências da atuação das moléculas ligadas ao processo de fusão celular e inflamação. Porém, os estudos neste tema são incipientes e inconclusivos. Considerando essas informações, o objetivo deste trabalho é estudar de forma comparativa a expressão das citocinas LIF, IL-6 e do ativador de transcrição STAT-3, além da telomerase, em material de aborto, de placenta normal a termo e de coriocarcinoma. Métodos: a expressão destas moléculas foi avaliada pelos métodos: imunoistoquímica (IHQ), imunofluorescência (IF), Western Blotting (WB) e Real-Time PCR (RT-PCR), em amostras de material de aborto, placenta normal a termo e coriocarcinoma (N=12 cada um). Os ensaios de WB e Real-Time PCR empregaram material a fresco de placenta normal a termo e seu cultivo celular e cultura da linhagem BeWo. Resultados: no material de aborto, as reações de IHQs evidenciaram expressão moderada de IL-6 em 58,4% dos casos e intensa de STAT-3 em 33,3%. Na placenta normal, observou-se intensa marcação de IL-6 em 50% e de STAT- 3 em 16,7% dos casos, enquanto que, no coriocarcinoma, houve expressão intensa de IL-6 em 50% e de STAT-3 em 75% dos casos. Por outro lado, as reações para LIF tiveram expressão nula em todos os três grupos. Pelo WB houve expressão proteica de IL-6 apenas no material fresco de placenta normal e ausência de expressão na sua cultura primária e na linhagem BeWo; LIF não foi expresso em todos os grupos estudados. STAT-3 foi detectado no citoplasma em todos os grupos, entretanto, a expressão nuclear da STAT-3 fosforilada (pSTAT-3) não foi observada na IF e nem pelo WB. Na análise gênica pelo RTPCR houve forte expressão de IL-6 e STAT-3 no material fresco de placenta normal e expressão muito fraca na cultura primária de placenta normal e na linhagem BeWo; a expressão de LIF foi muito fraca em todos os grupos. Apenas a linhagem BeWo demonstrou forte expressão gênica da telomerase, contrastando com a completa falta de expressão no material fresco de placenta normal e em sua cultura primária. Conclusão: A intensa expressão IHQ de IL-6 e STAT-3 no coriocarcinoma indica a atuação de ambas na carcinogênese. A expressão proteica de IL-6 no material fresco de placenta normal e sua ausência no material de cultura primária e na linhagem BeWo pode ser ocasionado pelo contato célula-a-célula nas culturas aderentes, inibindo o crescimento celular e, consequentemente, as vias de sinalização. A falta de expressão da pSTAT-3 tanto na IF como por WB demonstra que a via JAK-STAT está sendo desativada. A ausência de expressão de LIF, em todos os métodos estudados, sugere que esta citocina poderia estar sendo inibida por meio de proteínas SOCS3 ou, atuando, de modo indireto, na proliferação celular do coriocarcinoma. O aumento da atividade da telomerase nas células BeWo reforça sua relação com o fenótipo maligno e a aponta como um bom marcador para progressão da doença / Abstract: The invasion of the endometrium by extravillous trophoblast is a fundamental process in the growth of the fetus and placenta. The process is controlled by factors related to the immune and hormonal activity that, when changed, may result in termination of pregnancy and development of so-called gestational trophoblastic diseases. In some cases, changes can result in malignancy, in which some molecules play a role in cell fusion process and inflammation, although studies in this area are inconclusive. Considering this information, the study had the aim of investigating the expression of cytokines LIF, IL-6, STAT- 3 and the function of telomerase to understand their participation in abortion, in normal at term placenta and choriocarcinoma. Methods: The expression of the molecules was assessed by immunohistochemical assay (IHC), immunofluorescence (IF), Western Blotting (WB) and Real-Time PCR (RT - PCR) using fixed material from biopsies of abortions, normal at term placentas and choriocarcinoma along with fresh tissue of normal at term placenta and their primary culture and BeWo cell line. Paraffin embedded material used in IHC and IF assays were obtained from the Department of Pathology files. Tests of WB and Real-Time PCR employed fresh material, obtained from cell cultures of normal at term placenta and the BeWo line. Results: IHC reactions to abortion biopsies showed moderate staining for IL-6 in 58.4% of cases and intense for STAT-3 in 33.3 % of cases. In biopsies of normal placenta, there was intense reaction for IL-6 in 50% of cases, intense for STAT-3 in 16.7%; choriocarcinoma showed intense staining for IL- 6 in 50% of cases and also for STAT-3 in 75% of cases. On the other hand, LIF expression was missing in all three groups. WB analyses showed IL-6 protein in fresh material from normal placentas, but no expression in placenta primary cultures and BeWo line. LIF was absent in all groups. Cytoplasmic STAT-3 was observed in all groups, while the nuclear expression of phosphorylated STAT-3 was absent. On gene analyses a strong expression of IL-6 and STAT- 3 was observed from fresh normal placenta, but very weak expression in primary cultures of normal placenta and BeWo cell line. LIF expression was very weak in all groups. In regard to the gene expression of telomerase, it was strong in the BeWo line which contrasted with its complete lack of expression in fresh normal placenta and its primary culture. Conclusion: The high expression of IL-6 and STAT-3 in biopsies of choriocarcinoma indicates the role of both in tumor progression. Regarding protein expression, the presence of IL-6 in the material from fresh normal placenta, and its absence in primary culture and BeWo line may be caused by the cell-to-cell contact cultures by inhibiting cell growth and thus signaling pathways. However, the lack of expression of phosphorylated STAT-3 whether through IF or WB shows that its JAK-STAT pathway is inhibited. Lack of expression of the LIF suggests that it might be involved indirectly in choriocarcinoma cell proliferation or be inhibited by SOCS3 protein. Moreover, the increased telomerase activity of BeWo cells enhances their relation to the malignant phenotype and indicates a good marker for disease progression / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

STAT5A et B sont des facteurs de transcription qui constituent le point de convergence de nombreux signaux extracellulaires. Parmi leurs fonctions biologiques, ils sont connus pour leur rôle dans le développement et la différentiation des cellules hématopoïétiques. Cependant, un taux d’activation et/ou d’expression élevé de ces protéines aboutit à une prolifération incontrôlée des cellules aboutissant ainsi à une leucémogenèse. Ce présent travail vise à caractériser des aptamères ADN (Apta1 et Apta2) sélectionnés préalablement au sein de notre laboratoire contre STAT5B afin de réguler son activité dans le contexte leucémique. Les aptamères ADN sont des oligonucléotides simple brin qui adoptent une structure 3D et interagissent de manière spécifique avec leurs cibles. Contrairement aux anticorps, ils sont peu immunogènes ; ils possèdent alors un potentiel thérapeutique intéressant. La première partie de ce projet se focalise sur l’étude de la capacité d’Apta1 et Apta2 à interagir avec la forme cellulaire et recombinante de STAT5B par pull down et calorimétrie à titrage isotherme. La seconde partie concerne l’évaluation de l’activité d’Apta2 par l’étude de son effet sur la viabilité d’un modèle de leucémie myéloïde chronique et sur sa capacité à perturber la voie de signalisation impliquant STAT5. / STAT5A and B are common transcription factors that constitute a convergent point for many cellular pathways. Among their multiple biological functions, they are well known in promoting immune cell development and differentiation. When some oncogenic mutations occur, STAT5A and B are highly activated leading to uncontrolled proliferation and then to leukemia. Thus, they constitute a prime target to therapeutic intervention. In this work, we characterize new DNA aptamers (Apta1 and Apta2) selected previously by our laboratory against STAT5B. DNA aptamers are single stranded DNA molecules that can adopt 3D structures and recognize specific targets. Unlike antibodies, they fail to induce the immune response: they emerge as potentiel therapeutic molecules. In the first part of this work, the selected aptamers were assessed on their ability to interact with the cellular and recombinant form of STAT5B by using pull down assay and Isothermal Titration Calorimetry. In the second part, we focused on evaluating the effect of Apta2 on chronic myeloid leukemia cell line. For this purpose, cell viability, apoptosis process and JAK-STAT5 signaling pathway were depicted when cells are treated with Apta2.

Evidências epidemiológicas associam diferentes fatores ambientais, tais como poluição e ingestão de alimentos contaminados, com desfechos gestacionais negativos e fertilidade diminuída em humanos. Não há duvidas de que a poluição do ar nos grandes centros urbanos é capaz de provocar desfechos negativos sobre a gestação: baixo peso ao nascer, prematuridade, perda gestacional, entre outros. Entretanto, poucos estudos foram conduzidos para avaliar um possível efeito da exposição à poluição ambiental particulada do ar sobre a saúde reprodutiva feminina. O objetivo deste trabalho foi avaliar se a exposição subcrônica a poluição atmosférica particulada da cidade de São Paulo é capaz de alterar a receptividade uterina à implantação embrionária. Para tanto, foram avaliados 3 grupos de fêmeas de camundongos (n=10), expostas cronicamente desde o período de desmame (PND21) até atingirem a idade reprodutiva (PND60) à duas concentrações de MP2,5 (600?g/m3 ou 1200ug/m3) ou ar filtrado. Diferentes parâmetros relacionados à fertilidade e a receptividade uterina foram avaliados. Nossos achados mostram que a exposição ao material particulado de origem veicular provoca alterações na ciclicidade estral prévia ao acasalamento, bem como um aumento no peso dos ovários. Avaliação da reserva folicular indica que há um aumento na quantidade de folículos médios associado à exposição a menor concentração de MP (p=0,04). A avaliação histopatológica do tecido uterino revelou que há aumentos na fração de volume das glândulas uterinas (600ug/m3; p=0,01); o epitélio glandular (p=0,001) e luminal (p=0,03) estão espessados e o diâmetro médio das glândulas uterinas foi maior nos grupos expostos ao MP (p=0,004). A análise qualitativa da distribuição de pinopódios no epitélio luminal por microscopia eletrônica de varredura e transmissão indica que há uma redução na presença destas estruturas. A avaliação da expressão de LIF por imunomarcação mostrou-se reduzida no epitélio luminal (p<0,001), nas glândulas (p<0,001) e estroma (p=0,004) nas fêmeas expostas ao MP, porém nenhuma diferença foi observada na expressão de MUC-1 (mucina). Entretanto quando avaliadas a expressão gênica de MUC-1 e LIF no tecido uterino e os níveis de IL-1beta e IL-6 no fluído uterino nenhuma diferença foi observada entre os grupos. Com base em nossos achados conclui-se que a exposição à poluição particulada do ar de origem veicular pode estar envolvida no aumento das perdas gestacionais e/ou implantacionais pelo comprometimento da receptividade uterina provavelmente pelo prejuízo do remodelamento uterino necessário a implantação / Epidemiological evidences have shown that environmental factors, such as environmental pollution and ingestion of contaminated food, are associated with negative gestational outcomes and decreased fertility in human. There is no doubt that exposure to air pollution in large urban areas are capable of impairing health (e.g. hypertension) and of aggravating preexisting diseases (e.g asthma). However, the effects of air pollution exposures on female reproductive health are lesser known. Previous experimental studies have shown that low birth weights are reduced and embryonic implantational index are reduced in animals exposed to ambient levels of air pollution. The aim of this study was to evaluate if sub chronic exposures to particulate air pollution before pregnancy and during the initial stages is capable to alter the uterine receptivity of mice. To test this, 3 groups of female mice were continually exposed from 21st to 60th postnatal day to either filtered or two different doses of concentrated ambient particles (MP2,5 - 600ug/m3 or 1200ug/m3) with the aid of a Ambient Particle Concentrator and different parameters associated with fertility and uterine receptivity were evaluated. Or data have shown that exposures to particulate air pollution from vehicular origin are associated to changes in estrous ciclicity, cycles are shorter and the number of days in estrous reduced. Evaluation of the follicular reserve also indicates that animals exposed to MP present an increased number of ovarian medium follicles (p=0.04). Histopathological evaluation of the uterine tissue revealed increases in the volume fraction of uterine glands (p= 0.01) of those animals exposed to 600ug/m3. The luminal (p= 0.03) and glandular epithelium (p= 0,001) are thicker and the uterine glands diameters (p=0,004) were greater in exposed animals. Qualitative analysis by transmission and scanning electron microscopy indicates that there is a reduction in the presence of pinopódios in the luminal epithelium of PM exposed females. The expressions of LIF assessed by immunohistochemistry in those females exposed to PM were reduced in the luminal epithelium (p<0,001), and in the glandular (p<0,001) and stromal compartments as well. However no differences in the expression of MUC-1 were seen. Gene expression of LIF and MUC-1 in the whole endometrium (qPCR) and the expression of IL-6 and IL-1beta in the uterine fluid did not show significant difference between the groups tested. In conclusion, our data have shown that exposures to ambient air particulate pollution can be associated with increased rates of implantational losses due to changes in the uterine receptivity related to factors involved in uterine remodeling for pregnancy

Evidências epidemiológicas associam diferentes fatores ambientais, tais como poluição e ingestão de alimentos contaminados, com desfechos gestacionais negativos e fertilidade diminuída em humanos. Não há duvidas de que a poluição do ar nos grandes centros urbanos é capaz de provocar desfechos negativos sobre a gestação: baixo peso ao nascer, prematuridade, perda gestacional, entre outros. Entretanto, poucos estudos foram conduzidos para avaliar um possível efeito da exposição à poluição ambiental particulada do ar sobre a saúde reprodutiva feminina. O objetivo deste trabalho foi avaliar se a exposição subcrônica a poluição atmosférica particulada da cidade de São Paulo é capaz de alterar a receptividade uterina à implantação embrionária. Para tanto, foram avaliados 3 grupos de fêmeas de camundongos (n=10), expostas cronicamente desde o período de desmame (PND21) até atingirem a idade reprodutiva (PND60) à duas concentrações de MP2,5 (600?g/m3 ou 1200ug/m3) ou ar filtrado. Diferentes parâmetros relacionados à fertilidade e a receptividade uterina foram avaliados. Nossos achados mostram que a exposição ao material particulado de origem veicular provoca alterações na ciclicidade estral prévia ao acasalamento, bem como um aumento no peso dos ovários. Avaliação da reserva folicular indica que há um aumento na quantidade de folículos médios associado à exposição a menor concentração de MP (p=0,04). A avaliação histopatológica do tecido uterino revelou que há aumentos na fração de volume das glândulas uterinas (600ug/m3; p=0,01); o epitélio glandular (p=0,001) e luminal (p=0,03) estão espessados e o diâmetro médio das glândulas uterinas foi maior nos grupos expostos ao MP (p=0,004). A análise qualitativa da distribuição de pinopódios no epitélio luminal por microscopia eletrônica de varredura e transmissão indica que há uma redução na presença destas estruturas. A avaliação da expressão de LIF por imunomarcação mostrou-se reduzida no epitélio luminal (p<0,001), nas glândulas (p<0,001) e estroma (p=0,004) nas fêmeas expostas ao MP, porém nenhuma diferença foi observada na expressão de MUC-1 (mucina). Entretanto quando avaliadas a expressão gênica de MUC-1 e LIF no tecido uterino e os níveis de IL-1beta e IL-6 no fluído uterino nenhuma diferença foi observada entre os grupos. Com base em nossos achados conclui-se que a exposição à poluição particulada do ar de origem veicular pode estar envolvida no aumento das perdas gestacionais e/ou implantacionais pelo comprometimento da receptividade uterina provavelmente pelo prejuízo do remodelamento uterino necessário a implantação / Epidemiological evidences have shown that environmental factors, such as environmental pollution and ingestion of contaminated food, are associated with negative gestational outcomes and decreased fertility in human. There is no doubt that exposure to air pollution in large urban areas are capable of impairing health (e.g. hypertension) and of aggravating preexisting diseases (e.g asthma). However, the effects of air pollution exposures on female reproductive health are lesser known. Previous experimental studies have shown that low birth weights are reduced and embryonic implantational index are reduced in animals exposed to ambient levels of air pollution. The aim of this study was to evaluate if sub chronic exposures to particulate air pollution before pregnancy and during the initial stages is capable to alter the uterine receptivity of mice. To test this, 3 groups of female mice were continually exposed from 21st to 60th postnatal day to either filtered or two different doses of concentrated ambient particles (MP2,5 - 600ug/m3 or 1200ug/m3) with the aid of a Ambient Particle Concentrator and different parameters associated with fertility and uterine receptivity were evaluated. Or data have shown that exposures to particulate air pollution from vehicular origin are associated to changes in estrous ciclicity, cycles are shorter and the number of days in estrous reduced. Evaluation of the follicular reserve also indicates that animals exposed to MP present an increased number of ovarian medium follicles (p=0.04). Histopathological evaluation of the uterine tissue revealed increases in the volume fraction of uterine glands (p= 0.01) of those animals exposed to 600ug/m3. The luminal (p= 0.03) and glandular epithelium (p= 0,001) are thicker and the uterine glands diameters (p=0,004) were greater in exposed animals. Qualitative analysis by transmission and scanning electron microscopy indicates that there is a reduction in the presence of pinopódios in the luminal epithelium of PM exposed females. The expressions of LIF assessed by immunohistochemistry in those females exposed to PM were reduced in the luminal epithelium (p<0,001), and in the glandular (p<0,001) and stromal compartments as well. However no differences in the expression of MUC-1 were seen. Gene expression of LIF and MUC-1 in the whole endometrium (qPCR) and the expression of IL-6 and IL-1beta in the uterine fluid did not show significant difference between the groups tested. In conclusion, our data have shown that exposures to ambient air particulate pollution can be associated with increased rates of implantational losses due to changes in the uterine receptivity related to factors involved in uterine remodeling for pregnancy

Les cancers, qu’il s’agisse de leucémies ou de tumeurs solides, sont le résultat de proliférations cellulaires anormales et non contrôlées au sein des tissus. Ces proliférations anarchiques sont le reflet d’une surexpression et/ou sur-activation de protéines intracellulaires engendrées par un événement oncogénique. Aujourd’hui encore il est donc nécessaire de trouver de nouvelles molécules à usage thérapeutique ciblant spécifiquement ces protéines. C’est dans ce contexte que les facteurs de transcription STAT5 constituent de véritables cibles de choix puisque ces protéines participent activement à la leucogénèse. L’implication directe des protéines STAT5 dans la génèse des leucémies a été démontrée par l’utilisation de formes mutées constitutivement active de STAT5. Les facteurs de transcription STAT5 jouent un rôle essentiel dans la voie de signalisation JAK/STAT. Cette voie aboutit à la régulation de grandes fonctions biologiques telles que la prolifération cellulaire, la différenciation cellulaire ou encore l’apoptose. L’objectif de ce projet consiste donc à cibler spécifiquement les protéines STAT5 dans le but de rétablir le processus de mort cellulaire et empêcher la prolifération des cellules cancéreuses. Les inhibiteurs spécifiques des protéines STAT5 sont sélectionnés selon la méthode SELEX qui permet d’isoler des ligands structurés de forte affinité pour la protéine. L’affinité et la spécificité de ces inhibiteurs, appelés aptamères, sont caractérisées à partir de modèles cellulaires de leucémies dépendant de l’activité des facteurs de transcription STAT5. Les aptamères sont aujourd’hui de véritables outils thérapeutiques en pleine évolution. / Leukemias are due to abnormal cell proliferation, which is the result of intracellular over-expression or excessive activation of protein due to oncogenic event. Still today, it is necessary to find new therapeutic molecules, which specifically target these proteins. STAT5, via the JAK/STAT signaling pathway, controls fundamental cellular processes, including .cell survival, proliferation and differentiation. To struggle against tumorigenesis, JAK/STAT signaling pathway has to be inhibited. The aim of this project is to target specifically STAT5 factors to restore healthy signal transduction. We generated aptamers by an iterative in vitro selection. Aptamers are short-structured single strand DNAs or RNAs that bind with high affinity and specificity to their target. Once STAT5B recombinant proteins are produced, they are subjected to SELEX process. The number of rounds depends on various parameters. After seven rounds, two sequences are retrieved. The specificity and affinity of these aptamers are assessed by fluorescent immunoassays. Binding affinity and kinetics of interaction are characterized by SPR. Aptamer anti proliferative effects are determined by evaluation of the growth of cells depending on STAT5. Finally, we developed several .assays aiming at understanding the mechanism of an aptamer action on STAT5B such as phosphorylation measurement and EMSA. Aptamers are now emerging therapeutic tools; they exhibit significant advantages relative to protein therapeutics.

Ciliary neurotrophic factor (CNTF) is produced by astrocytes which have been implicated in regulating stress responses. We found that CNTF in the medial amygdala (MeA) promotes despair or passive coping, i.e., immobility in an acute forced swim stress, in female mice, while having no effect in males. Neutralizing CNTF antibody injected into the MeA of wildtype females reduced activation of downstream STAT3 (Y705) 24 and 48 h later. In concert, the antibody reduced immobility in the swim test in females and only after MeA injection, but not when injected in the central or basolateral amygdala. Antibody injected into the male MeA did not affect immobility. These data reveal a unique role of CNTF in female MeA in promoting despair or passive coping behavior. Moreover, 4 weeks of chronic unpredictable stress (CUS) increased immobility in the swim test and reduced sucrose preference in wildtype CNTF+/+, but not CNTF-/- littermate, females. Following CUS, 10 min of restraint stress increased plasma corticosterone levels only in CNTF+/+ females. In males, the CUS effects were present in both genotypes. Further, CUS increased CNTF expression in the MeA of female, but not male, mice. CUS did not alter CNTF in the female hippocampus, hypothalamus and bed nucleus of stria terminalis. This suggests that MeA CNTF has a female-specific role in promoting CUS-induced despair or passive coping, behavioral anhedonia and neuroendocrine responses. Compared to CNTF+/+ mice, CNTF-/- mice did not show differences in CUS-induced anxiety-like behavior and sensorimotor gating function as measured by elevated T-Maze, open field and pre-pulse inhibition of the acoustic startle response. Together, this study reveals a novel CNTF-mediated female-specific mechanism in stress responses and points to opportunities for developing treatments for stress-related disorders in women.