Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

Made available in DSpace on 2014-12-17T14:03:31Z (GMT). No. of bitstreams: 1 MariliaSN.pdf: 1926704 bytes, checksum: da3259de4a29785c64e3b5736af19e2f (MD5) Previous issue date: 2010-02-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Several pharmacological properties have been attributed to isolated compounds from mushroom. Recently, have these compounds, especially the polysaccharides derived from mushrooms, modulate the immune system, and its antitumor, antiviral, antibiotic and antiinflammatory activities. This study assesses the possible pharmacological properties of the polysaccharides from Scleroderma nitidum mushroom. The centesimal composition of the tissue showed that this fungus is composed mainly of fibers (35.61%), ash (33.69%) and carbohydrates (25.31%). The chemical analysis of the polysaccharide fraction showed high levels of carbohydrates (94.71%) and low content of protein (5.29%). These polysaccharides are composed of glucose, galactose, mannose and fucose in the following molar ratios 0.156, 0.044, 0.025, 0.066 and the infrared analysis showed a possible polysaccharide-protein complex. The polysaccharides from Scleroderma nitidum showed antioxidant potential with concentration-dependent antioxidant activity compared to ascorbic acid. The analysis scavenging of superoxide radical and inhibition of lipid peroxidation showed that the polysaccharides from S. nitidum have an IC50 of 12.70 mg/ml and EC50 10.4 μg/ml, respectively. The antioxidant activity was confirmed by the presence of reducing potential of these polysaccharides. The effect of these polymers on the inflammatory process was tested using the carrageenan or histamine-induced paw edema model and the sodium thioglycolate or zymosan-induced model. The polysaccharides were effective in reducing edema (73% at 50 mg/kg) and cell infiltrate (37% at 10 mg/kg) in both inflammation models tested. Nitric oxide, a mediator in the inflammatory process, showed a reduction of around 26% at 10 mg/kg of body weight. Analysis of pro- and anti-inflammatory cytokines showed that in the groups treated with polysaccharides from S. nitidum there was an increase in cytokines such as IL-1ra, IL-10, and MIP-1β concomitant with the decrease in INF-γ (75%) and IL-2 (22%). We observed the influence of polysaccharides on the modulation of the expression of nuclear factor κB. Thus, polysaccharides from S. nitidum reduced the expression of NF-κB by up to 64%. The results obtained suggest that NF-κB modulation is one of the possible mechanisms that explain the anti-inflammatory effect of polysaccharides from the fungus S. nitidum. / Diversas propriedades farmacol?gicas t?m sido atribu?das aos compostos isolados de fungos. Recentemente, t?m-se referido quanto ? capacidade desses compostos, principalmente os polissacar?deos derivados de cogumelos, de modular o sistema imunol?gico, al?m de suas a??es antitumoral, antiviral, antibi?tica e antiinflamat?ria. Este estudo avalia a capacidade dos polissacar?deos do fungo Scleroderma nitidum quanto ?s suas poss?veis propriedades farmacol?gicas. A composi??o centesimal do tecido deste fungo demonstrou que este ? composto principalmente por fibras (35,61%), cinzas (33,69%) e carboidratos (25,31%). As an?lises qu?micas da fra??o polissacar?dica revelaram alto teor de carboidratos (94,71%) e baixo teor de prote?nas (5,29%). Esses polissacar?deos s?o constitu?dos por glicose, galactose, manose e fucose nas seguintes propor??es molares 0,156; 0,044; 0,025; 0,066, respectivamente e a an?lise de infravermelho demonstrou um poss?vel complexo polissacar?deo-prote?na. Os polissacar?deos de S. nitidum demonstraram potencial antioxidante com atividade relativa ao ?cido asc?rbico massa-dependente. As an?lises sobre a varredura de radicais super?xido e inibi??o da peroxida??o lip?dica demonstraram que os polissacar?deos de S. nitidum apresentam um IC50 estimado em 12,70 mg/ml e EC50 10,4 μg/ml, respectivamente. A atividade antioxidante foi confirmada pela presen?a de potencial redutor dos polissacar?deos. Este estudo tamb?m avaliou a capacidade dos polissacar?deos do fungo S. nitidum como agente antiinflamat?rio. O efeito destes pol?meros no processo inflamat?rio foi testado usando-se os modelos de edema de pata induzido por carragenana ou histamina e o modelo de peritonite induzida por tioglicolato de s?dio ou zymosan. Os polissacar?deos foram efetivos na redu??o do edema (73% a 50 mg/kg) e infiltrado celular (37% a 10 mg/kg) nos dois modelos de inflama??o testados. ?xido n?trico, um mediador do processo inflamat?rio, mostrou uma redu??o de cerca de 26% nos grupos tratados com a dose 10 mg/kg dos polissacar?deos. A an?lise de citocinas pr? e antiinflamat?rias mostrou que nos grupos tratados com os polissacar?deos de S. nitidum houve aumento de citocinas como IL-1ra (2x), IL-10 (3x) e concomitante a diminui??o de INF-γ (75%), MIP-1β (29%) e IL-2 (22%). Al?m disso, nos grupos tratados com os polissacar?deos tamb?m foi verificada uma inibi??o de cerca de 64% na express?o do NF-κB. Os resultados obtidos sugerem que a modula??o do NF-κB ? um dos poss?veis mecanismos que esclarece os efeitos anti-inflamat?rios dos polissacar?deos do fungo S. nitidum.

Orientador: Eduardo Melani Rocha / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T17:43:11Z (GMT). No. of bitstreams: 1 Alves_MonicadeCassia_D.pdf: 5666644 bytes, checksum: 2d8f41985c6b80374f8fd6d042fc9ff0 (MD5) Previous issue date: 2006 / Resumo: Este estudo avaliou as possíveis vias comuns na fisiopatogênese da síndrome do olho seco no Diabetes Mellitus (DM) e no envelhecimento, envolvendo o acúmulo dos produtos de glicosilação ("Advanced Glycation End-products" - AGEs), seu receptor RAGE e a ativação do Fator Nuclear-?B (NF-?B) na glândula lacrimal (GL) e alterações do filme lacrimal nessas condições. Modelos animais de DM induzido com estreptozotocina e animais senis (24 meses de vida) foram comparados a animais controle tratados com tampão citrato e adultos jovens (2 meses de vida). Foram avaliadas vias de sinalização, envolvendo AGEs, RAGE e a ativação do NF-?B na GL e alterações no filme lacrimal em ratos Wistar de todos os grupos. A análise do filme lacrimal foi realizada através de medidas de volume de secreção basal e dosagem de citocinas como a Interleucina-1 ß (IL-1 ß) e Fator de Necrose Tumoral - a (TNF- a). A capacidade secretória da GL foi avaliada através de medidas da atividade de peroxidase. Técnicas de "western blot" foram utilizadas para avaliar a expressão e ativação do NF-?B na GL. A expressão de AGE, RAGE e NF-?B na GL e córnea nos grupos estudados, foi avaliada através de microscopia confocal com imunofluorescência. O volume lacrimal foi significativamente menor nos animais diabéticos e senis (P=0,02 e 0,016, respectivamente). Concentrações de IL-1ß e TNF-a na lágrima foram mais altas nos ratos diabéticos e senis do que nos controles e adultos jovens (P=0,007 e 0,02 nos diabéticos e P= 0,007 e P= 0,05 nos senis). A atividade de peroxidase foi significativamente mais baixa no grupo senil (P=0,016), mas não nos experimentos, comparando animais diabéticos e controles (P=0,34). A expressão de AGE, RAGE e NF-?B na GL foi aumentada nos animais diabéticos e senis. O DM e a senilidade induzem alterações estruturais e secretórias significativas na GL e nos tecidos da superfície ocular e associa-se a maior incidência de olho seco. A expressão aumentada de AGE, RAGE e NF-?B na GL sugere a participação desses fatores nas vias de sinalização e subseqüentes alterações inflamatórias relacionadas ao olho seco nessas condições. / Abstract: The present study evaluates the dry eye syndrome related to diabetes and aging through the involvement of the Advanced Glycation End-product (AGEs), the Advanced Glycation End-product Receptor (RAGE) axis and Nuclear Factor-?B (NF-?B) activation in lacrimal gland (LG) and tear film dysfunction in these conditions. To evaluate whether AGE, RAGE and NF-?B signaling in LG are altered in diabetes and aging, streptozotocin-induced diabetic, normoglicaemic, aging (24 month-old) and young adults (2 month-old) male Wistar rats were compared. Tear film alterations and the expression of AGE, RAGE and NF-?B in ocular tissues were evaluated in all considered groups. Tear secretion parameters as basal tear secretion volume, Interleukin-1 ß (IL-1 ß) and Tumor Necrosis Factor- a (TNF- a) levels and LG and peroxidase activity in LG tissue were measured. NF-?B expression and activation was evaluated in LG by western blot. Immunohistochemistry with confocal microscopy was used to assess AGE, RAGE and NF-?B expression in LG of all groups. Tear volume was significantly lower in diabetic and aging rats (P=0.02 and 0.016, respectively). IL-1ß and TNF-a concentrations in tears were higher in diabetic and aging than in control and young rats (P=0.007 and 0.02 in diabetic and P= 0.007 and P= 0.05 in aging). Peroxidase activity was significantly lower in the aging group (P=0.016) but not the assays with diabetic rats (P=0.34). AGE, RAGE and NF-?B expressions were increased in LG of diabetic and aging rats. Diabetes and aging induce significant alterations in rat LG structure and secretion. The higher expression of AGE, RAGE and NF-?B in LG may suggest that these factors are involved in signalling and in subsequent inflammatory alterations related to dry eye related to these conditions. / Doutorado / Clinica Medica / Doutor em Clínica Médica

O sistema NADPH oxidase é um complexo enzimático gerador de superóxido. O NF-kB é um fator de transcrição envolvido no controle da expressão de diversos genes ligados à resposta inflamatória. Defeitos no eixo IL-12/23-IFN-g resultam em infecções recorrentes e à susceptibilidade mendeliana a micobacterioses, podendo diminuir a expressão do componente gp91-phox da NADPH oxidase. Estudamos qual é a relação direta do NF-kB e de defeitos no eixo IL-12/23-IFN-g na regulação dos genes CYBA, NCF1, NCF2 e NCF4 do sistema NADPH oxidase humano em células U937, células B EBV transformadas provenientes de pacientes com EDA-ID, DGC, ou de pacientes com defeitos no eixo IL-12/23-IFN-g. A expressão dos genes NCF1 e NCF2 foi diminuída em células com defeitos no eixo (IFNGR1 e INFGR2) e em células U937 IkB S32A/S36A. A expressão do gene NCF1 também foi diminuída em células EDA-ID S32I e em células EDA-ID NEMO/IKKg W420X. O NF-kB e os IFNGR1 e INFGR2 são necessários para a expressão dos genes NCF1 e NCF2 e para a ativação do sistema NADPH oxidase humano neste sistema modelo. / The NADPH oxidase system is an enzymatic complex that generates superoxide. The NF-kB is a transcriptional factor involved in the expression of several genes related to the inflammatory response. The IL-12/23-IFN-g axis defects lead to recurrent infections and to the mendelian susceptibility of mycobacterial disease (MSMD), and they can decrease the gp91-phox expression (a NADPH oxidase component). We studied the NF-kB and the IL-12/23-IFN-g axis defects consequences on the regulation of CYBA, NCF1, NCF2 and NCF4 genes of the human NADPH oxidase system in U937 cells, and in B EBV cells from patients with EDA-ID, DGC, or patients with IL-12/23-IFN-g axis defects. The NCF1 and NCF2 gene expression was decreased in IL-12/23-IFN-g axis defects cells (IFNGR1 and INFGR2) and in U937 IkB S32A/S36A cells. NCF1 gene expression was decreased in EDA-ID S32I and in EDA-ID NEMO/IKKg W420X cell lineages. The NF-kB and the IFNGR1 and INFGR2 are necessary for NCF1 and NCF2 gene expression and activation of the human NADPH oxidase in this model system.

A tuberculose (TB) é um grave problema de saúde pública. Somente no ano de 2004, cerca de 9 milhões de pessoas desenvolveram TB ativa e mais de 2 milhões de pessoas morreram da doença. O desenvolvimento de novos modelos experimentais de TB seriam de grande utilidade para para elucidar mecanismos fisiopatológicos da doença e testar esquemas terapêuticos para a prevenção e contenção da doença. Além disso, o desenvolvimento de novas vacinas torna-se indispensável como ferramenta de prevenção e controle da TB. A TB no sistema nervoso central (SNC), assim como em outros tecidos do organismo, promove a ativação de células inflamatórias. No SNC a micróglia desempenha este papel, sendo capaz de produzir ou ser influenciada por mediadores solúveis. Vários mediadores estão envolvidos nos mecanismos moleculares decorrentes da infecção e inflamação causados pela TB, entre eles: NFB, iNOS e VEGF. A ativação do NFB, um fator de transcrição citoplasmático que sob estímulo migra para o núcleo celular, tem íntima relação com a indução da iNOS e de VEGF. A resistência intracelular a patógenos, inclusive ao Mycobacterium tuberculosis, parece estar associada a expressão de iNOS em macrófagos. O óxido nítrico (NO) tem papel importante na comunicação intercelular, estimulando a síntese de mediadores inflamatórios, como as citocinas, e regulando sua própria produção endógena. Estas citocinas por sua vez também podem induzir a atividade do NFB e a expressão da iNOS e VEGF. O VEGF é um potente ativador de permeabilidade vascular e de angiogênese, envolvido na ruptura da barreira hemato-encefálica. Neste estudo, mostramos a caracterização morfológica e imuno-histoquímica de um modelo murino de TB no SNC, com a indução da doença pela inoculação de BCG. Com este modelo experimental obtivemos importantes resultados que podem esclarecer mecanismos envolvidos na fisiopatologia da neuro-TB humana. A indução de meningite e tuberculomas foi possível através da inoculação de 104 cfu de BCG no cerebelo de camundongos, por estereotaxia, e esta indução foi dependente do tempo. A confirmação do diagnóstico foi feita pela detecção de bacilos álcool-ácido resistentes (BAAR), nas lesões tuberculosas. Observamos, ao longo do tempo (1 a 6 dias; 1, 2, 4 e 8 semanas) o recrutamento de diferentes populações gliais (micróglia e astrócitos) no sítio de injeção. Houve aumento de produção e ativação NFB nas lesões tuberculosas, caracterizada pela translocação da molécula do citoplasma para o núcleo celular. Houve expressão de iNOS restrita às lesões tuberculosas, além do aumento de expressão de VEGF nestas lesões. Além disso, camundongos imunizados com a vacina gênica hsp65, contra a TB, não expressam VEGF em suas lesões. Esta vacina parece conferir um efeito protetor em nosso modelo experimental, reduzindo a expressão de VEGF, e consequentemente reduzindo seu efeito angiogênico decorrente do processo inflamatório. O recrutamento glial, e a produção de mediadores solúveis (NFB, iNOS e VEGF) pelo hospedeiro, em resposta à invasão do patógeno no SNC, parecem estar envolvidos na fisiopatologia da neurotuberculose, como demonstrado neste modelo experimental. Nosso modelo permitirá investigar fatores possivelmente responsáveis pelo desenvolvimento e manutenção de lesões tuberculosas no SNC. O objetivo final seria elucidar a fisiopatologia desta grave doença e compreender eventos moleculares envolvidos na produção de lesões. O conhecimento gerado poderá permitir o delineamento de terapias específicas e efetivas. / Tuberculosis (TB) is a serious public health problem; in 2004, 9 million people developed active TB and the disease killed 2 million patients. Development of experimental models and new vaccines are essential both to elucidate physiopathological mechanisms and to control the disease. This infection in the central nervous system (CNS), as in other tissues of the organism, activates inflammatory cells. In CNS, this role is performed by the microglia, which is capable of producing or be influenced by soluble mediators. Several mediators are involved in the molecular mechanisms of the infection and inflammation by mycobacteria , such as NFB, iNOS and VEGF. NFB activation, a cytoplasmic transcriptional factor that migrates to the cellular nucleus under stimuli, is involved with the iNOS and VEGF induction of expression. The intracellular resistance to Mycobacterium tuberculosis has been associated with iNOS expression in macrophage cells. Nitric oxide (NO) is crucial in intercellular communication, modulating the synthesis of mediators of inflammation, such as cytokines, and modulation itself. These cytokines induces NFB activity, and induces iNOS and VEGF expression. VEGF is a potent activator of vascular permeability and of angiogenesis and it is a factor involved in the breakdown of the blood brain-barrier in tuberculous meningitis. In this study, we showed the morphologic and immunohistochemistry characterization of an experimental model of TB in the CNS, with inoculation of BCG in mice. In this model we elicited important outcome that can elucidate mechanisms involved in the physiopathology of human neuron-TB. Induction of meningitis and tuberculomas were possible with stereotaxic inoculation of 104 cfu of BCG in mice cerebellum, in a time-dependent way. Diagnostic was confirmed by detection of alcohol-acid resistant bacilli (BAAR), in tuberculous lesions. We observed, the time-course (1 to 6 days; 1, 2, 4 e 8 weeks) of the recruitment of different glial populations (microglia and astrocytes) in the injection site. There was increased production and activation of NFB in the tuberculous lesions, it was characterized by its nuclear translocation from cytoplasm. There was iNOS expression only in the tuberculous lesions, and expression increased of VEGF in these lesions. Furthermore, mice immunizated with vaccine DNA-hsp65 there was no expression of VEGF in its lesions. This vaccine seems confer a protector effect in our experimental model, reducing the expression of VEGF, and then reducing its angiogenic effect derived from inflammatory process. Glial recruitment, and the soluble mediators production (NFB, iNOS e VEGF) by the host, producing in response to invasion of the pathogen in the CNS, has been involved in the pathophysiology of the neuro-TB, such as demonstrated in this experimental model. Our model will allow investigate possible factors responsible for the development and maintenance of tuberculous lesions in the CNS. The final aim is to elucidate the physiopathology of this serious illness and understand the molecular events involved in the production of the lesions. The knowledge created may permit to pave the way to delineate specific and effective therapies.

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MicroRNAs (miRNAs) sind endogenene kurze RNA-Moleküle, die zentrale Aufgaben bei der Regulation der eukaryotischen Zellhomöostase erfüllen. MiRNAs wurden bereits als potente Immunregulatoren beschrieben. Trotz dieser Erkenntnisse blieb die Rolle dieser kurzen RNA Moleküle in Infektionen mit Mycobacterium tuberculosis weitgehend unerforscht. Im Rahmen dieser Arbeit wurde ein miRNA-Expressionsprofil von Makrophagen generiert, die mit Mycobacterium tuberculosis infiziert waren. Dies ermöglichte die Identifizierung von miRNAs, welche bei der Infektion differenziell reguliert waren. Anhand eines ex-vivo-Modells von Todesrezeptor-induzierter Apoptose konnte gezeigt werden, dass miRNA-133b apoptoseresitente Zellen empfindlich gegen Tumornekrosefaktor-alpha (TNFalpha), TNF-related apoptosis-inducing ligand (TRAIL) oder CD95 ligand (Fas/APO1 ligand) induzierte Zytotoxizität machte. Eine umfassende Studie führte zur Identifizierung der anti-apoptotischen Proteine Fas apoptosis inhibitory molecule (FAIM) und glutathione-S-transferase pi (GSTP1) als direkte Zielgene für miRNA-133b. Desweiteren zeigte sich die Expression von Osteoprotegerin (OPG) und Fettsäuresynthase (FASN), als miRNA-133b abhängig. Dies unterstrich die pleiotrope Art der pro-apoptotischen Aktivität dieser miRNA. Die Expression von miRNA-133b wurde durch Mitglieder der Toll-like Rezeptor (TLR)-Familie aktiviert. MiRNA-133b Transfektion führte zu einer verstärkten Aktivierung des Transkriptionsfaktors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). Diese resultierte in erhöhten Mengen an Interleukinen 6 und 8 (IL6/8). Diese Ergebnisse stellen die erste detaillierte Charakterisierung von miRNA-133b im Zusammenhang der Todesrezeptor-vermittelten Apoptose und der angeborenen Immunität dar. Die erforschten molekularen Wechselwirkungen ergänzen und bereichern das Verständnis über die regulatorischen molekularen Mechanismen, die mit der Tumorentstehung und Entzündung verbunden sind. / MicroRNAs (miRNAs) are endogenous short RNA molecules which perform essential tasks in the regulation of eukaryotic cell homeostasis. During the past few years miRNAs have emerged as very potent immune regulators. Despite the consequences of this discovery for our understanding of immune response regulation hitherto virtually nothing is known about miRNA function during innate immunity to Mycobacterium tuberculosis. Herein, a miRNA expression profile of human macrophages infected with Mycobacterium tuberculosis was generated. This led to the identification of miRNAs being differentially regulated during infection. By using an experimental ex-vivo model of death receptor (DR)-induced apoptosis it could be demonstrated that miRNA-133b rendered apoptosis-resistant cells sensitive to tumor necrosis factor-alpha (TNFalpha)-, TNF-related apoptosis-inducing ligand (TRAIL)- or CD95 ligand (Fas/APO1 ligand)-activated cytotoxicity. Comprehensive analysis led to the discovery of the anti-apoptotic proteins Fas apoptosis inhibitory molecule (FAIM) and glutathione-S-transferase pi (GSTP1) as direct miRNA-133b targets. Moreover, underlining the pleiotropic and synergistic nature of miRNA activity, the expression of osteoprotegerin (OPG) and fatty acid synthase (FASN) could be further proven as miRNA-133b dependent. Expression of miRNA-133b increased following innate immune activation by members of the Toll-like receptor (TLR) family. MiRNA-133b enhanced the activity of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). This translated into increased levels of the pro-inflammatory interleukins 6 and 8 (IL6/8). The results presented in this work represent the first detailed characterization of miRNA-133b in the context of DR-mediated apoptosis and innate immunity. The molecular interactions dissected herein improve the understanding of the regulatory processes associated with tumorigenesis and the immune response.

INTRODUÇÃO: Perda óssea é um achado comum em pacientes com Nefrite Lúpica (NL), mesmo naqueles com diagnóstico recente. Algumas evidências indicam um aumento na osteoclastogênese como um dos distúrbios principais no processo de remodelamento ósseo. O objetivo deste estudo foi investigar algumas vias de sinalização (RANKL/OPG, Wnt/Beta-catenin and Th17/IL-17) possivelmente envolvidas na osteoclastogênese anormal detectada em mulheres jovens ao diagnóstico de nefrite lúpica ativa, assim como avaliar a ação da vitamina D (VitD) nesse cenário e sua correlação com fatores inflamatórios. MÉTODOS: Realizamos culturas com a linhagem de células osteoblásticas humanas hFOB 1.19 (ATCC) e as dividimos em um grupo suplementado com soro de pacientes lúpicas (NL) (n=15) e em um grupo com soro de controles saudáveis (CS) (n=15) em vez de soro fetal bovino (SFB). Em seguida, adicionamos 1,25-dihidroxivitamina D3 (1,25(OH)2D3) em dois subgrupos nas concentrações 10-9M e 10-7M, resultando em 6 grupos: CS, CS+vitD 10-9M, CS+vitD 10-7M, NL, NL+vitD 10-9M, NL+vitD 10-7M). Após 48h da adição de 1,25(OH)2D3 ao meio de cultura, células hFOB foram tripsinizadas e separado o lisato celular de cada grupo. Ensaios de citometria de fluxo e multiplex foram realizados para quantificação das seguintes proteínas do lisato cellular: CD166, CD54, fosfatase alcalina, RANKL, OPG, CD14, TLR4, NF-KappaB, SOST, DKK-1, Beta-catenina, IL-1-beta, IL-2, IL-6, TNF-alfa, IL-17A, IL-17F, IL-21 and IL-22. RT-PCR foi empregado para quantificação de mRNA dos genes RANKL, SOST, OPG e Beta-catenina. RESULTADOS: Pacientes com NL evidenciaram maiores níveis séricos de DKK-1 (2802,04 ± 1380,06 x 696,30 ± 421,22pg/ml, p < 0,001), OPG (560,12 ± 333,56 x 340,24 ± 102,08pg/ml, p=0,0212), TNF-alfa (9,63 ± 14,49 x 1,27 ± 0,35pg/ml, p=0,0337), IL-6 (15,58±39,08 x 8,02±3,49, p= 0,0053) and IL-2 (3,36 ± 3,06 x 1,54 ± 0,9pg/ml, p=0,0353) do que CS. Após exposição ao meio enriquecido com soro de pacientes com NL, células hFOB 1.19 apresentaram maior nível de mRNA de RANKL (p=0,045)) e menor nível de proteína OPG (178,81 ± 66,40 x 298,76 ± 114,94pg/mg, p=0,0016). Suplementação com 1,25(OH)2D3 aumentou a diferença da expressão das proteínas DKK-1 (673,03 ± 171,93 x 456,69 ± 234,53pg/mg, p=0,0215), IL-6 (0,80 ± 0,25pg/mg x 0,66 ± 0,18, p=0,0417) and IL-2 (4,97 ± 2,2 x 3,90 ± 1,66pg/mg, p=0,042) entre hFOB NL comparados com hFOB CS, enquanto diminuiu o nível de mRNA de Beta-catenina em células do grupo NL. DISCUSSÃO: Dentro das limitações deste estudo, os resultados sugerem que os maiores níveis séricos de citocinas pró-inflamatórias, como TNF-alfa, IL-6 e talvez IL-2, detectadas em pacientes com NL pode ter induzido a maior expressão osteoblástica de RANKL, representada pelo maior nível de mRNA RANKL em células do grupo NL, e suprimido OPG, conforme a diminuição observada na quantificação proteica de OPG nos lisatos celulares, o que pode ter contribuído para a aumentada osteoclastogênese evidenciada pela biópsia óssea dessas pacientes. A adição de 1,25(OH)2D3 não preveniu os efeitos inflamatórios do soro de pacientes com NL ativa em células hFOB 1.19 neste estudo / INTRODUCTION: Bone loss is a common finding in Lupus Nephritis (LN) patients even in those recently diagnosed. Some evidences indicate an increased osteoclastogenesis as the main disturb of the bone remodeling process. The aim of this study was to investigate some pathways (RANK-L/OPG, Wnt/?-catenin and Th17/IL-17) possibly involved in the abnormal osteoclastogenesis detected in women at the diagnosis of proliferative LN as well as evaluating the action of vitamin D (vitD) in this scenario and their correlation with inflammatory factors. METHODS: We cultured the human osteoblastic cell line hFOB 1.19 (ATCC), and divided cultures into those supplemented with serum from healthy controls (HC) (n=15) and LN patients (n=15) instead of fetal bovine serum (FBS). Then 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was added in two subgroups at the concentrations of 10-9M e 10-7M while vitD was absent in one subgroup in both HC and LN cultures (HC, HC+vitD 10-9M, HC+vitD 10-7M, LN, LN+vitD 10-9M, LN+vitD 10-7M) . After 48h of vitD addition, hFOB cultures were trypsinized. Flow cytometry and multiplex assays were performed to test CD166, CD54, alkaline phosphatase, RANKL, OPG, CD14, TLR4, NF-KappaB, SOST, DKK-1, ?-catenin, IL-1Beta, IL-2, IL-6, TNF-alfa, IL-17A, IL-17F, IL-21 and IL-22 concentrations in the cell lysates. Polymerase reaction chain (RT-PCR) assays analyzed the expression of RANKL, SOST, OPG and Beta-catenin mRNA. RESULTS: LN patients showed higher serum levels of DKK-1 (2802.04 ± 1380.06 x 696.3 ± 421.22pg/ml, p < 0.001), OPG (560.12 ± 333.56 x 340.24 ± 102.08pg/ml, p=0.0212), TNF-alfa (9.63 ± 14.49 x 1.27 ± 0.35pg/ml, p=0.0337), IL-6 (15.58±39.08 x 8.02±3.49, 0.0053) and IL-2 (3.36 ± 3.06 x 1.54 ± 0.9pg/ml, p=0.0353) than HCs. After exposure to medium enriched with LN serum, osteoblasts expressed higher RANKL mRNA (fold change 1.573, p=0.045) and lower OPG protein (178.81 ± 66.40 x 298.76 ± 114.94pg/mg, p=0.0016). 1,25(OH)2D3 supplementation increased the difference between LN and HC expression of DKK-1 (673.03 ± 171.93 x 456.69 ± 234.53pg/mg, p=0.0215), IL-6 (0.80 ± 0.25pg/mg x 0.66 ± 0.18, p=0.0417) and IL-2 (4.97 ± 2.2 x 3.90 ± 1,66pg/mg, p=0.042) proteins and diminished Beta-catenin mRNA in LN cells. DISCUSSION: Within the limitations of this study, the results suggest that the higher serum levels of proinflammatory cytokines, such as TNF-alfa, IL-6 and perhaps IL-2, detected in LN patients would possibly have induced RANKL genes, as demonstrated by an enhanced RANKL mRNA expression in LN osteoblasts, and suppressed OPG protein in cell lysates, which would have contributed to the increased osteoclastogenesis detected in bone biopsies of women with new onset of LN. 1,25(OH)2D3 addition to osteoblast cultures did not prevent the effects of inflammatory LN serum in vitro

Die Phospholipid-Hydroperoxid Glutathion-Peroxidase (phGPx) ist ein monomeres Selenoprotein, welches innerhalb der Familie der Glutathion-Peroxidasen aufgrund seiner breiten Substratspezifität und der Fähigkeit Proteinthiole zu modifizieren eine Sonderstellung einnimmt. Vom Gen der phGPx werden nach heutigem Kenntnisstand drei verschiedene Protein-Isoformen gebildet. Die mitochondriale Isoform enthält am N-Terminus ein mitochondriales Insertionssignal und wird bevorzugt im Testis exprimiert. Von einem im Leserahmen stromabwärts liegenden Startkodon wird die kürzere, ubiquitär exprimierte zytosolische Isoform synthetisiert. Eine dritte phGPx-Isoform besitzt eine N-terminale nukleäre Lokalisationssequenz (kodiert von einem alternativen Exon 1) und wird vornehmlich in den Kernen post-meiotischer Zellen der Spermatogenese gefunden. Aufgabe dieser Arbeit war es, die molekularen Mechanismen zu untersuchen, die am Zustandekommen des vielfältigen Expressionsmusters der phGPx-Isoformen beteiligt sind. Im ersten Teil der Arbeit wurden transkriptionelle Regulationsmechanismen der phGPx-Expression untersucht. Im proximalen Promotorbereich (-100 bp – +228 bp) des phGPx-Gens wurden unter in vitro (Supershift-Assay) und in vivo (Chromatin-Immunopräzipitation) Bedingungen die Transkriptionsfaktoren Sp1 und NF-Y identifiziert, die an drei GC-reiche Motive beziehungsweise zwei inverse CCAAT-Boxen binden. Darüber hinaus konnten in kompetetiven Gelshift-Assays im proximalen Promotorbereich zwei Bindungssequenzen identifiziert werden, die von Faktoren der Smad-Familie gebunden werden. Funktionelle in vitro Promotorstudien mit mutierten Promotorkonstrukten zeigten, dass die Mutagenesen der Sp1- und NF-Y Bindestellen einen starken Einfluss auf die Reportergenaktivität hatten. Im zweiten Teil der Arbeit wurden durch Untersuchungen von Protein-RNA-Interaktionen post-transkriptionelle Mechanismen der Expressionsregulation studiert. Mit Hilfe des in vivo Ansatzes des Hefe Drei-Hybrid Systems wurde der Guanin-reiche Sequenz bindende Faktor 1 (GRSF1) identifiziert, der in der 5’-untranslatierte Region der mitochondrialen phGPx-mRNA bindet. In RNA Gelshift-Assays wurde die Spezifität dieser Interaktion bestätigt und näher charakterisiert. Schließlich wurden für GRSF1 und die phGPx Expressionsprofile in murinen Gewebe erstellt sowie die zeitabhängige Expression beider Proteine während der Embryogenese verfolgt. Die auffällig ähnlichen Expressionsmuster lassen ähnliche Regulationsmechanismen vermuten. Die in dieser Arbeit identifizierten trans-regulatorischen Proteine Sp1, NF-Y, Smad und GRSF1 sollten an der differentiellen Expression der phGPx-Isoformen beteiligt sein. / The Phospholipid Hydroperoxide Glutathione Peroxidase (phGPx) is a monomeric selenoprotein that is unique in the family of Glutathione Peroxidases due to its low substrate specificity and its ability to oxidise protein thiols. Three different isoforms are known to derive from one common gene. The mitochondrial Isoform contains an N-terminal mitochondrial insertion sequence and is preferentially expressed in postpubertal testis. The shorter, ubiquitously expressed, cytosolic isoform is expressed from an in-frame start codon. A third isoform contains an N-terminal nuclear localization signal coded for by an alternative exon 1 and is preferentially expressed in the nuclei of post-meiotic spermatides. The aim of the present study is to investigate the molecular mechanisms leading to the different isoforms and causing their tissue specific expression pattern. In the first part of this work transcriptional regulatory mechanisms will be analysed. Within the proximal promoter region (-100 to +228 bp) of the phGPx gene the transcription factors SP1 and NF-Y were identified to bind to three GC-boxes and two CCAAT-boxes respectively using in vitro methods (Supershift Assays) and in vivo methods (Chromatine immunoprecipitation). Moreover, performing competitive gel shift assays two binding elements for the smad family of transcription factors could be identified. Functional in vitro reporter gene assays provided evidence that the mutagenesis of the binding sequences for NF-Y and Sp1 has a strong impact on promoter activity. In the second part of this work post-transcriptional events in the expression regulation of the phGPx were analysed on the basis of protein/RNA interactions. Applying the in vivo approach of the yeast three hybrid system the Guanin-riche sequence binding factor 1 (GRSF1) could be identified binding to the 5’-untranslated region of the mitochondrial phGPx messenger. RNA mobility shift assays were performed to further characterize the specificity of this protein/RNA interaction. Eventually, the tissue distribution of GRSF1 and phGPx was studied in murine tissues and their expression kinetics were followed during murine embryogenesis. The obvious parallel expression kinetics for mitochondrial phGPx and GRSF1 suggest common regulatory mechanisms for these two genes. All the identified trans-regulatory elements are very likely to be involved in the differential expression regulation of the phGPx isoforms.

INTRODUÇÃO: O hiperparatireoidismo secundário (HPTS) é uma complicação da doença renal crônica que compromete a integridade do esqueleto. Pacientes com HPS submetidos à paratireoidectomia (PTX) passam de uma condição de níveis séricos de paratormônio (PTH) muito elevados para outra, onde esses níveis hormonais caem drasticamente. Os efeitos da PTX no tecido ósseo são mal compreendidos, especialmente no que se refere às proteínas expressas por osteócitos, como o fator de crescimento de fibroblastos 23 (FGF23), dentin matrix protein 1 (DMP-1), fosfoglicoproteína de matriz extracelular (MEPE), esclerostina, Fator nuclear Kappa beta ligante (RANKL) e osteoprotegerina (OPG), que regulam a remodelação e a mineralização óssea. OBJETIVOS: Caracterizar a expressão óssea dessas proteínas por imuno-histoquímica e estabelecer relações com os dados da histomorfometria do tecido ósseo em pacientes com HPS, antes e após a PTX. MÉTODOS: Estudamos biópsias ósseas obtidas de um banco de biópsias de 23 pacientes com DRC e HPTS, que foram realizadas antes e 12 meses após a PTX. RESULTADOS: A avaliação dos parâmetros histomorfométricos demonstrou uma melhora da microarquitetura óssea, porém com um maior retardo em sua mineralização após a PTX. A análise da expressão das proteínas osteocíticas revelou um aumento significativo na expressão da esclerostina e da OPG e uma diminuição da relação RANKL/OPG após a PTX, sugerindo a participação dessas proteínas na melhora das lesões ósseas decorrentes do HPTS. Observamos um aumento significativo na expressão da OPG no grupo de pacientes que evoluiu com defeito de mineralização somente após a cirurgia, sugerindo a participação dessa proteína no retardo de mineralização óssea desses pacientes. A expressão das proteínas osteocíticas que participam da formação e mineralização óssea apresentou correlação com parâmetros envolvidos na remodelação óssea. CONCLUSÕES: Mudanças significativas na expressão óssea de proteínas osteocíticas que podem potencialmente regular a remodelação e a mineralização óssea foram observadas após a PTX / INTRODUCTION: Secondary hyperparathyroidism (SHPT) is a complication of chronic kidney disease that compromises skeletal integrity. Patients with SHPT undergoing parathyroidectomy (PTX) go from a very high serum parathyroid hormone (PTH) condition to another, where these hormonal levels dramatically fall. The effects of PTX on bone tissue are poorly understood, especially as regards proteins expressed by osteocytes, such as fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP-1), extracellular matrix phosphoglycoprotein (MEPE), sclerostin, Kappa beta ligand nuclear factor (RANKL) and osteoprotegerin (OPG), which regulate bone remodeling and mineralization. OBJECTIVES: Characterize bone expression of these proteins by immunohistochemistry and establish relations with bone tissue histomorphometry data in SHPT patients, before and after PTX. METHODS: We studied bone biopsies obtained from a biopsy database of 23 patients with CKD and SHPT, which were performed before PTX and 12 months after PTX. RESULTS: Evaluation of histomorphometric parameters showed improvement of bone microarchitecture, but with longer delay in mineralization after PTX. Analysis of osteocyte protein expression revealed significant increase in sclerostin and OPG expression and decrease in RANKL/OPG ratio after PTX, suggesting participation of these proteins in improvement of bone lesions due to SHPT. We observed significant increase in OPG expression in the group of patients who evolved with mineralization defect only after surgery, suggesting participation of this protein in bone mineralization delay of these patients. Expression of osteocyte proteins that participate in bone formation and mineralization correlated with parameters involved in bone remodeling. CONCLUSIONS: Significant changes in bone expression of osteocyte proteins that can potentially regulate bone remodeling and mineralization were observed after PTX