Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-08-30T20:48:41Z No. of bitstreams: 2 Dissertação - Pollyana Guimarães de Oliveira - 2014.pdf: 1550661 bytes, checksum: 2c614da0d23de4cac81d9260ef077071 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-08-30T20:48:58Z (GMT) No. of bitstreams: 2 Dissertação - Pollyana Guimarães de Oliveira - 2014.pdf: 1550661 bytes, checksum: 2c614da0d23de4cac81d9260ef077071 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-30T20:48:58Z (GMT). No. of bitstreams: 2 Dissertação - Pollyana Guimarães de Oliveira - 2014.pdf: 1550661 bytes, checksum: 2c614da0d23de4cac81d9260ef077071 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-10-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Dendritic cells (DCs) are a heterogeneous group of cells and the major activators of naive T lymphocytes. During antigen presentation, different types of cytokines are produced and they will interfere with the immune response profile generated. IL-12p70 promotes the differentiation of Th1 phenotype and IL-23 p enhances the Th17 response. Given the major role of IL-12 and IL-23 on the stabilization of the acquired immune response, the production of these cytokines is highly controlled and it is an issue for several studies. The objective of this work was to characterize a new lineage of DC, described as AP284, based on their surface markers and evaluate the ability of these cells to produce IL-12p40, IL-12p70 and IL-23 after different stimulus in vitro. Additionally it will be evaluate their phagocytic capacity and their ability to induce a Th1 and Th17 response in vivo. The expression of the markers was analyzed by flow cytometry and the quantitation of IL-12 and IL23 was performed by ELISA after stimulation of cells AP284 in vitro with lipopolysaccharide (LPS) and Escherichia coli. AP284 cells express MHC class II, CD11c and 33D1 on its surface, which are characteristic markers of DCs. After stimulus with LPS or E.coli, AP284 cells produce large amounts of IL-12p40 and IL-23 but do not produce IL-12p70. The production of IL-12p70 was not detected even when IFN-γ is added to prime cultures, moreover, priming the cells with IFN-γ promoted inhibition of IL-12p40 and IL-23. Our data sugest that the AP284 is a DC17 lineage. Thus, AP284 can be an important tool to study the mechanisms of induction or regulation of IL-23 production and a possible tool to study generation and maintenance of a Th17 profile. / As células dendríticas (DCs) constituem um grupo heterogêneo de células e são as principais ativadoras de linfócitos T virgens. Durante a apresentação de antígenos são produzidos diferentes tipos de citocinas que interferem com o perfil de resposta imune que será gerado. A produção de IL-12p70 favorece a diferenciação do perfil Th1 e a produção de IL-23 potencializa o perfil de resposta Th17. Devido à importância da IL-12 e IL-23 na estabilização de uma resposta imune adquirida, a produção destas citocinas é altamente controlada e alvo de diversas pesquisas. O objetivo deste projeto foi de caracterizar uma nova linhagem de DC AP284 a partir dos seus marcadores de superfícies e avaliar a capacidade destas células em produzir IL-12p40 e IL-23 após diferentes estímulos in vitro, além de avaliar a sua capacidade fagocítica e indução de um perfil de resposta Th1 ou Th17 in vivo. A expressão dos marcadores foi analisada por citometria de fluxo e a dosagem de IL-12 e IL23 por ELISA após o estímulo de células AP284 in vitro com lipopolisssacáride (LPS) e Escherichia coli. Células AP284 expressam MHC classe II, CD11c e 33D1 em sua superfície, sendo estes marcadores característicos de populações de DCs. Após estímulos com LPS e E.coli, células AP284 produzem grande quantidade de IL-12p40 e IL-23, mas não produzem IL-12p70. A produção de IL-12p70 não foi detectada mesmo após a adição de IFN-γ às culturas, além disso, a primagem das células com IFN-γ promoveu a inibição de IL-12p40 e IL-23. Neste trabalho foi demonstrado que células AP284 são DCs, pois além de expressarem MHC classe II expressam marcadores que são característicos deste grupo celular. Nossos dados ainda suportam a ideia de que a linhagem AP284 seja uma linhagem de DC17, pois produziram grandes quantidades de IL-12p40 e IL-23, mas nenhuma quantidade de Il-12p70. Assim, AP284 pode ser uma ferramenta importante para o estudo dos mecanismos de indução e de regulação da produção da IL-23 e uma possível ferramenta para o estudo da geração e manutenção de um perfil de resposta Th17.

Made available in DSpace on 2014-06-12T23:13:59Z (GMT). No. of bitstreams: 2 arquivo7066_1.pdf: 2312624 bytes, checksum: ed1cc8ee13566ce4ab19fe42c69b1f42 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O derivado tiazolidínico (LPSF/AC-23) sintetizado pelo Laboratório de Planejamento e Síntese de Fármacos da Universidade Federal de Pernambuco apresentou importante atividade antitumoral em camundongos albinos Swiss. Tal resultado despertou o interesse e a necessidade do desenvolvimento e validação de um método bioanalítico para determinação do LPSF/AC-23 em fluidos biológicos para possibilitar a posterior determinação dos parâmetros farmacocinéticos desta molécula candidata a fármaco. Neste contexto, um método bioanalítico sensível e seletivo foi desenvolvido e validado utilizando a técnica de Cromatografia Líquida de Alta Eficiência acoplada a um detector ultravioleta (CLAE-UV) para quantificação desta molécula em plasma de ratos. O método envolveu a preparação da amostra utilizando extração por precipitação protéica com acetonitrila e uma tiazolidinadiona (LPSF/GQ 113-B) foi utilizada como padrão interno (PI). A separação e quantificação do LPSF/AC-23 e PI foi realizada utilizando uma fase móvel composta por uma mistura de acetonitila/metanol/tampão fosfato 5 mM (pH 6,0) (55:30:15) eluida de forma isocrática através de uma coluna analítica Phenomenex® (C18, 5μm, 150mm x 4.6mm) a uma temperatura de 40°C e fluxo de 1mL/min. O comprimento de onda para detecção foi de 249 nm. A curva de calibração foi linear na faixa de 100-10000 ng/mL. A precisão intra e inter-dia apresentou valores de Desvio Padrão Relativo preconizados pela ANVISA, e a exatidão expressa pelo erro relativo (ER) variou de 3,49 a 7,67%. A recuperação foi de 92,25% para o analito e 89,67% para o PI. Desta forma, o método proposto pode ser aplicado para determinação quantitativa do LPSF/AC-23 em plasma de ratos em estudos farmacológicos, toxicológicos, farmacocinéticos e de biodisponibilidade

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In recent years, the subsets of mammalian CD4+ T cells and their repertoire of effector cytokines has expanded beyond the original Th1/Th2 paradigm, to include natural (n) and inducible (i) regulatory T cells (Treg), Th17, Th9, Th22 and follicular T helper (Tfh) cells. Whilst Th1, Th2 and nTreg immune responses have been described in the chicken, the existence of other Th cell subsets is yet to be determined. To investigate Th17 immune responses in the chicken, the mammalian components of these responses currently unannotated in the chicken genome, IL-23 p19 and the IL-23R, were identified and cDNAs cloned. A chicken IL-23 flexiconstruct, containing IL-23 p19 and p40 joined by a linker, was designed. Recombinant chicken IL-23 protein (rchIL-23) was expressed and purified. Bioactivity of rchIL-23 was demonstrated by increased mRNA expression of chIL- 17F and chIL-22 in rchIL-23-stimulated splenocytes. Monoclonal antibodies which identify chIL-12/chIL-23 p40 also recognised purified rchIL-23. Further, chIL-23 p19 mRNA levels were measured and detected in a wide range of tissues but was not up-regulated in stimulated splenocytes, thymocytes or bursal cells. Messenger RNA (mRNA) expression levels of Th17 cytokines (chIL-17A, chIL-17F, chIL-21, chIL-22 and chIL-23) were measured in a chicken tissue panel, in stimulated splenocytes, thymocytes and bursal cells, as well as during infections previously described as initiating typical Th1 or Th2 adaptive immune responses in the chicken. Chicken IL-17A mRNA expression levels were up-regulated in susceptible chickens during infection with Marek’s disease virus (a disease which typically drives a Th1 immune response), but were down-regulated in resistant birds. Chicken CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS) and recombinant Th17-associated cytokines used to attempt to drive the cells towards a Th17 phenotype, as measured by expression of mRNA for chIL-17A and chIL-23R. The sorted chicken CD4+ cells failed to proliferate or respond to Th17 cytokine stimulation. ChIL-23R was also correctly identified and cloned as cDNA, and its mRNA expression measured in a panel of unstimulated and stimulated tissues and cells. The chIL-23R mRNA levels were detected in a wide range of tissues as well as stimulated splenocytes, thymocytes and bursal cells. Future work would seek to positively identify Th17 cells in the chicken and determine the role of Th17 immune responses against avian diseases.

Monocyte-derived macrophages (MDMs) from HIV-infected patients and MDMs infected in vitro with HIV manifest inhibition of various cytokines including IL 12. Recently, IL-27 was shown to inhibit HIV replication in macrophages. Whether HIV infection or HIV regulatory proteins such as tat, impact IL-23 or IL-27 production in macrophages remains unknown. I have demonstrated that intracellular HIV-tat expression as well as HIV-tat basic domain peptides inhibited LPS-induced IL-23 and IL-27 proteins and their subunits in MDMs. First I investigated the signalling pathways involved in the regulation of LPS-induced IL-23 and IL-27 production in MDMs infected with control pLXIN retrovirus-infected MDMs. The p38 MAPK, SHP-1 and PI3K signalling molecules positively regulated LPS-induced IL-23 expression. In contrast, Src kinases and JNK MAPK negatively regulated LPS-induced IL-23 production. On the other hand, LPS-induced IL-27 production was positively regulated by the PI3K, p38 MAPKs and SHP-1 and Src kinases. Src kinases positively regulated LPS-induced IL-27 production whereas Src kinases and JNK negatively regulated LPS-induced IL-23 production. HIV-Tat significantly inhibited p38 MAPK and PI3K which were implicated in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. Even though HIV-Tat inhibited ERK and JNK MAPK activation, these kinases were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. While SHP-1 regulated LPS-induced IL-23 and IL-27 production, HIV-Tat did not inhibit SHP-1 and therefore were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. HIV-Tat did not inhibit Src kinases and hence were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-27 production. Furthermore, HIV-Tat did not inhibit the expression of upstream TLR4-activated signaling molecules including TRAF3, TRIF, MyD88, IRAK1, IRAK3, IRAK4, TRAF-1, TRAF-2, cIAP-1, cIAP-2 and, xIAP. These results suggest association of IL-23 and IL-27 inhibition by HIV with decreased HIV-specific immune responses, and increased viral replication. These results further suggest novel strategies to improve cellular immune responses and inhibition of HIV replication.

Los actinomicetos nocardioformes que contienene ácidos micólicos (mycolata) pertenecen al suborden Corynebacterinea, orden Actinomycetales. Este grupo de microorganismos contiene especies filamentosas que causan problemas de formación de espumas biológicas ("foaming") en plantas de tratamiento de aguas residuales con sistemas de fangos activos. .Estas espumas interfieren en el proceso de desputación de aguas residuales, ya que retienen hasta el 40% de los sólidos en suspensión. La presencia de especies patógenas en este grupo de microorganismos implica un riesgo para la salud pública y por lo tanto la necesitad de detectar estas especies. Para caracterizar mycolata se han utilizando los sistemas miniaturizados API,ZYM, API ID32 C y MicroLog que permiten la realizaciçón de muchas pruebas bioquímicas en poco tiempo. Las pruebas bioquímicas obtenidas por los sistemas API en ocasiones no diferencias todas las especies de este grupo. el único sistema miniaturizado que permite diferenciar todas las cepas de referencia es el sistema Micro-Log. De todos los caracteres quimiotaxonómicos el más importante es la detección de áccidos micólicos, ya que los mycolta son los únicos que contienen estos compuestos. Con la técnica de la reacción en cadena de la polimerasa (PCR) se han caracterizado cepas de Nocardia de referencia, así como aislados de muestras de fango utilizando iniciadores previamente descritos. Para caracterizar especies de Gordonia aisladas de fangos se han diseñado unos iniciadores específicos que amplifican todas las cepas Gordonia de referencia. Los iniciadores diseñados para Rhodococcus amplifican todas las especies de referencia de este género. La detección de la especia patógena R. equi se realiza con los iniciadores diseñados previametne para esta especie. El rendimiento en el aislamiento de mycolata se ha mejorado gracias a los trtamientos de descontaminación aplicados a las muestras de fango que reducen la microbiota acompañante de rápido crecimient / Cuesta Amat, G. (2004). Detección y caracterización por métodos fenotípicos y moleculares de mycolata formadores de espumas en estaciones depuradoras de aguas residuales domésticas con sistemas de fangos activos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/2665 / Palancia

Indiana University-Purdue University Indianapolis (IUPUI) / Fibroblast growth factor-23 (FGF23) is a hormone produced in bone that acts upon the kidney to control blood phosphate and 1,25-(OH)2 vitamin D concentrations. Chronic kidney disease-mineral bone disorder (CKD-MBD) is a major public health problem, affecting 1 in 8 individuals. These patients can have markedly elevated FGF23 at end stage disease which is associated with metabolic bone anomalies, left ventricular hypertrophy, as well as increased mortality (>6-fold). The FGF23 co-receptor αKlotho (αKL) is a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as a cleavage product of mKL (‘cleaved’, or cKL). Previously, a patient with increased plasma cKL from a balanced translocation between chromosomes 9 and 13 in the KLOTHO gene presented with metabolic bone disease and a complex endocrine profile, despite hypophosphatemia. The lack of a reliable cell model in which to study potential FGF23-cKL interactions is a major hurdle for the field of phosphate metabolism. The goal of the present studies was to test and characterize bone cell lines that may respond to FGF23 and/or cKL, permitting study of novel aspects of phosphate handling and control of FGF23 expression. It was confirmed that stable delivery of cKL via AAV2/8 to wild type (WT) and KL-KO mice resulted in highly elevated bone FGF23 mRNA. MC3T3 (mouse) and ROS (rat) osteoblastic cell lines were tested for p-ERK1/2 responses to control FGFs, as well as FGF23 and cKL, alone or in combination. Importantly, both cell lines demonstrated responsiveness to FGF23+cKL only, and not the individual factors. To test responsiveness at the cell level, EGR1 mRNA was tested as an index of FGFR activity and showed modest increases with the same treatments, supporting that other factors may be required for full transcriptional effects. The present studies show that MC3T3 have FGF-dependent signaling capabilities, and that the combination of FGF23+cKL is required for efficient MAPK signaling. These results demonstrated that cKL provision is permissive for efficient FGF23 signaling in bone, and revealed important implications for the regulation of FGF23 and cKL in Mendelian, and common, genetic disorders of phosphate handling and biomineralization.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21634号 / 医博第4440号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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No description available.