Dissertations / Theses on the topic 'Via NF-κB'

L’infection par le VIH-1 est un problème majeur de la santé publique qui touche plus de 35 millions de personnes à l’échelle mondiale. La réplication du VIH-1 est déclenchée par l’activation du promoteur LTR, qui contient deux sites de liaison pour le facteur de transcription NF-κB. Ces sites de liaison sont hautement conservés dans le génome du VIH-1, illustrant ainsi l’importance de NF-κB dans l’activité transcriptionelle des gènes du VIH-1 et la production de nouvelles particules virales. La withaferin A (WA) est une substance bioactive extraite de la plante Withania somnifera, qui possède des propriétés pharmacologiques non négligeables dans la régulation de la réponse immunitaire. Des études récentes ont démontré que le potentiel anti-inflammatoire de la WA est dû principalement à l’inhibition de la voie de NF-κB. Le but de ce projet est de déterminer l’effet de la WA sur la réplication du VIH-1 dans les cellules T, qui sont les cibles principales du virus. Des essais de transfections transitoires de cellules T Jurkat E6.1 avec des plasmides contenant le promoteur du VIH-1 ayant différentes constructions de NF-κB, ont démontré que la WA peut réduire l’activité du promoteur d’une manière dépendante de NF-κB. Quant à la production de particules virales, des essais d’infection avec des virus pseudotypés démontrent que la WA diminue la production virale jusqu’à 90% dans des cellules T stimulées avec PMA/PHA et TNF-α, tandis que les mutants ayant des sites de liaison défective pour NF-κB ne sont pas affectés. Des essais de retardement sur gel ainsi que des immunobuvardages de type Western ont montré que la WA altère l’habilité de NF-κB à transloquer dans le noyau, ce qui se traduit par l’inhibition de la synthèse de l’IκB-α, protéine inhibitrice de NF-κB, phénomène sous contrôle étroite de ce facteur de transcription. Ces résultats suggèrent que la WA pourrait permettre une diminution de la réplication virale d’une manière dépendante de NF-κB et ainsi empêcher la propagation du virus aux cellules T chez les individus infectés.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Orf virus (ORFV), the type member of the genus Parapoxvirus of the family Poxviridae, is the etiologic agent of orf or contagious ecthyma, a contagious and ubiquitous disease of sheep and goats. ORFV genome consists of a double stranded DNA molecule with approximately 138 Kb, and contains 131 putative genes. Among those, 15 are novel genes, unique to parapoxviruses, which lack homology to other known viral or cellular genes. In the present study we describe the functional characterization of three of these genes, ORFV024, ORFV002, and ORFV121. Results presented here demonstrate that the proteins encoded by these genes inhibit the activation of the nuclear factor-kappa B (NF-κB) signaling pathway. ORFV-encoded ORFV024 inhibits activation of the NF-κB signaling pathway in the cell cytoplasm by inhibiting phosphorylation of the IκB kinases, IKKα and IKKβ, consequently inhibiting the activation of the IKK complex. Deletion of ORFV024 from the ORFV genome had no significant effect on disease severity, progression or time to resolution in sheep, indicating that ORFV024 does not contribute to ORFV virulence. ORFV-encoded ORFV002 functions in the cell nucleus, where it interacts with the NF-κB subunit NF-κB-p65, inhibiting its acetylation, a p300-mediated modification of NF-κB-p65 which modulates its transcriptional activity. Similarly to ORFV024, deletion of ORFV002 from the ORFV genome had no significant effect on ORFV virulence and disease pathogenesis in sheep. ORFV-encoded ORFV121 functions in the cell cytoplasm, where it binds to and inhibits phosphorylation and nuclear translocation of NF-κB-p65. Deletion of ORFV121 from the ORFV genome resulted in a marked attenuated disease phenotype in sheep, indicating that ORFV121 is a determinant of virulence of ORFV in the natural host. These results indicate that ORFV, like other poxviruses, has evolved multiple strategies to modulate NF-κB, targeting different steps of the signaling pathway. Results obtained in the pathogenesis studies performed here suggest that multiple NF-κB inhibitors encoded by ORFV may exert complementary and/or redundant functions to effectively block host cell responses regulated by the NF-κB signaling pathway. Additionally, it is possible that ORFV-encoded NF-κB inhibitors modulate distinct cellular processes regulated by NF-κB in vivo. A better understanding of ORFV-host interactions may provide valuable insights for the development of improved vaccines against orf, or yet for the development of novel ORFV-based therapeutic agents and vaccine vectors with enhanced safety and efficacy, and a broader applicability.
O vírus da orf (ORFV), protótipo do gênero Parapoxvirus da família Poxviridae, é o agente etiológico da orf ou ectima contagioso, uma enfermidade contagiosa de distribuição mundial que afeta primariamente ovinos e caprinos. O genoma do ORFV consiste de uma molécula de DNA de fita dupla com aproximadamente 138 Kb, que contém presumidamente 131 genes. Dentre estes, 15 são genes novos, identificados apenas nos parapoxvírus e que não possuem homologia com outros genes de origem viral ou celular. O presente estudo descreve a caracterização funcional de três destes genes, ORFV024, ORFV002 e ORFV121. Os resultados apresentados no presente estudo demonstram que as proteínas codificadas pelos genes ORFV024, ORFV002 e ORFV121 inibem a ativação da via de sinalização do fator de transcrição nuclear-kappa B (NF-κB). O produto da ORFV024 bloqueia a ativação da via do NF-κB no citoplasma celular, inibindo a fosforilação das quinases IκB (IKK), IKKα e IKKβ e, consequentemente inibindo a ativação do complexo IKK. A deleção do gene ORFV024 do genoma do ORFV não alterou a severidade, a progressão, ou o tempo de resolução das lesões produzidas pelo ORFV em ovinos, indicando que o produto deste gene não contribui para a virulência do vírus. O gene ORFV002 codifica um inibidor do NF-κB que atua no núcleo das células. O produto do ORFV002 interage com a subunidade NF-κB-p65 do NF-κB, inibindo a sua acetilação, uma modificação pós-traducional do NF-κB-p65 mediada pela acetiltransferase p300 que regula a sua atividade transcripcional. Semelhante ao ORFV024, a deleção do gene ORFV002 do genoma do ORFV não afetou a virulência do vírus nem alterou a patogenia da enfermidade em ovinos. O produto do gene ORFV121 atua no citoplasma das células, onde esta proteína viral interage com o NF-κB-p65 inibindo sua fosforilação e translocação nuclear. A deleção do gene ORFV121 do genoma do ORFV reduziu significativamente a severidade, a progressão e o tempo de resolução da doença em ovinos, indicando que este produto viral constitui-se em um fator de virulência para o ORFV em seu hospedeiro natural. Estes resultados demonstram que, assim como outros poxvírus, o ORFV também desenvolveu múltiplas estratégias para modular a via de sinalização do NF-κB, codificando proteínas que atuam em diferentes eventos desta complexa via de sinalização intracelular. Os resultados obtidos nos estudos de patogenia sugerem que os inibidores do NF-κB codificados pelo ORFV desempenham funções complementares e/ou redundantes, provavelmente, para promover um bloqueio efficiente dos processos biológicos regulados pelo NF-κB. Além disso, estes produtos virais podem modular diferentes processos biológicos controlados pelo NF-κB in vivo. Um melhor entendimento das interações do ORFV com o seu hospedeiro pode favorecer o desenvolvimento de vacinas mais eficazes para o ectima contagioso, ou ainda, promover o desenvolvimento de vacinas vetoriais ou imunoterápicos, baseados no ORFV, mais eficazes e com uma maior espectro de aplicações.

Scrub typhus is a potentially fatal infection that threatens one billion persons in the Asia-Pacific region and is caused by the obligate intracellular bacterium, Orientia tsutsugamushi. How this organism facilitates its intracellular survival and pathogenesis is poorly understood. Intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) into the host cell to modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank libraries as well as Type 1 and Type 4 secretion systems (T1SS and T4SS), which are expressed during infection. In silico analyses of the Anks’ C-termini revealed that they possess characteristics of T1SS secretion signals. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks. In addition to infecting endothelial cells, O. tsutsugamushi infects professional phagocytes. To better understand why these innate immune cells are unable to eliminate O. tsutsugamushi, we addressed the activity of host NF-κB proinflammatory transcription factor. Screening of O. tsutsugamushi infected cells at an MOI of 1 revealed inhibition of NF-κB nuclear accumulation as early as 8 hours in HeLa and bone-marrow derived macrophage cells. When stimulating infected cells with TNF-α, IκBα degradation still occurs, however NF-κB dependent gene transcription remains downregulated. Immunofluorescence microscopic analysis of TNF-α treated cells ectopically expressing all O. tsutsugamushi Anks revealed that two nuclear trafficking Anks, Ank1 and Ank6, result in a significant decrease in NF-κB nuclear accumulation. Additionally, these Anks also significantly inhibited NF-κB dependent gene transcription. Co-immunoprecipitation experiments revealed that both Anks interact with importin-β1, exportin-1, and the p65 NF-κB subunit. Treating cells with importazole significantly reduces the nuclear accumulation of Ank1 and Ank6. Finally, treating infected cells or cells ectopically expressing Ank1 or Ank6 with leptomycin B resulted in restoration of NF-κB nuclear accumulation. With these data, we propose that O. tsutsugamushi secretes Ank1 and Ank6 to initially interact with importin-β1, which permits their nuclear entry where they then interact with NF-κB and subsequently exportin-1 to prevent NF-κB nuclear accumulation.

T. gondii interfère avec l'activation des voies de signalisation de NF-kB des cellules hôtes. Ainsi, lors de l’infection par T. gondii, 85% des gènes dépendant de NF-kB sont up-régulés. Un autre facteur de transcription dont l’expression est modulée par le parasite est UHRF1 (Ubiquitin-like,containing PHD and RING finger domains, 1). UHRF1, en se fixant sur le promoteur du gène de la cycline b, induit une répression épigénétique de ce dernier conduisant à un arrêt du cycle cellulaire des cellules infectées en phase G2 et à un arrêt de la prolifération parasitaire. L’analyse in silico du promoteur du gène uhrf1 a montré qu’il possédait 9 sites de fixation de NF-kB. Effectivement nous avons démontré que NF-kB interagit avec le promoteur du gène uhrf1 lors d’une infection par T. gondii. L’expression d’UHRF1 serait donc modulée par NF-kB dans les cellules infectées par T. gondii. Or NF-kB a une régulation différentielle en fonction de la nature de la souche infectante. Là encore, nous avons pu observer une régulation différentielle d’UHRF1 selon la nature de la souche infectante, pouvant être dues à la régulation souche dépendante de NF-kB. La détermination du rôle précis de l’activation d’UHRF1 dans les cellules infectées et l’identification du ou des facteurs parasitaires responsables pourraient permettre de mieux comprendre les mécanismes de persistance intracellulaire du parasite et de découvrir de nouveaux points d’impact thérapeutiques
T.gondii interferes with the activation of NF-kB signaling pathways. Thus, upon infection by T.gondii, 85% of genes NF-kB-dependent are up-regulated. Another transcription factor whose expression is modulated by the parasite is UHRF1 (Ubiquitin-like, Containing PHD and RINGfinger domains, 1). UHRF1, bind to the gene promoter of cyclin b and induces epigenetic repression of this gene leading to cell cycle arrest in G2 phase of infected cells and stop the proliferation in both infected cells and parasite. In silico analysis of the uhrf1 gene promoter has been shown to possess 9 binding sites of NF-kB. Our study showed that NF-kB actually interacts with the promoter of gene uhrf1 during infection with T. gondii. This suggests that the expression of UHRF1 is modulated by NF-kB in T. gondii-infected cells. In addition we observed differential regulation of UHRF1 depending on the nature of the infecting strain. These variations may also be due to already well-known differential regulation of NF-kB by different strains of T.gondii. Determining the precise role of UHRF1 activation in infected cells and the identification of the parasitic factor responsible of this activation would allow to a better understanding of the mechanisms of intracellular persistence of the parasite and allow to unravel new therapeutic trails

L'infection par vih-1 est caractérisée par une phase de latence clinique particulièrement longue. La progression de la maladie jusqu'au stade sida nécessite une forte activation de la réplication virale. Parmi les stimuli induisant cette activation, on trouve des stress cellulaires tels que choc thermique, choc oxydant ou irradiation aux uv. L'action de ces agents s'exerce sur la transcription du provirus et, pour certains, (tnfalpha, peroxyde d'hydrogene) a été corrélée à l'activation du facteur de transcription cellulaire nf-kappa b, capable de se lier en deux sites, au ltr de vih-1. Ce travail de thèse s'attache à étudier l'influence d'un stress oxydant ou thermique sur le fonctionnement du ltr de vih-1 et de nf-kappa b. Il précise le rôle des radicaux libres oxygénés (rlo) dans l'activation de nf-kappa b par un choc oxydant. Ainsi, la diminution du taux de rlo intracellulaire par surexpression de la glutathion peroxydase ou de la protéine de stress hsp27 inhibe l'activation, par un stress oxydant, de nf-kappa b et les étapes de phosphorylation et de dégradation de sa sous-unité inhibitrice i kappa b-alpha. D'autre part, il décrit le mécanisme d'activation du ltr de vih-1 par le choc thermique ; ce mécanisme fait intervenir l'activation du facteur nf-kappa b. Cette stimulation de nf-kappa b est indépendante d'une dégradation de sa sous-unité inhibitrice i kappa b-alpha, mais dépend du potentiel redox cellulaire. Enfin, il met en évidence de nouveaux inducteurs du ltr de vih-1, que sont les analogues d'acides aminés. Ces composes exercent leur effet par l'intermédiaire d'une activation de nf-kappa b, concomitante d'une dégradation de i kappa b-alpha, mais sans étape de phosphorylation préalable de la sous-unité inhibitrice ; cette activation dépend, de plus, d'une variation de l'état redox intracellulaire. Ces résultats illustrent l'avantage sélectif que nf-kappa b confère au virus vih-1, dans des conditions de stress cellulaire.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Introdução: A sobrecarga de pressão causada pela hipertensão arterial sistêmica (HAS) pode gerar mudança na arquitetura do colágeno, favorecer a fibrose, bem como o desbalanço entre a produção de espécies reativas de oxigênio (ERO) e a capacidade antioxidante. Aumento das ERO pode gerar ativação de vias sinalizadoras como a do fator nuclear kappa B (NF-kB) e das proteínas quinases ativadas por mitógenos (MAPK). Alterações dessas vias contribuem para o processo de remodelamento cardíaco causado pela HAS. O exercício físico desempenha importante papel na atenuação dos fatores de risco cardiovascular como a HAS. Dessa forma, o objetivo desse estudo foi avaliar a influência do treinamento físico sobre o remodelamento cardíaco de ratos espontaneamente hipertensos (SHR) na fase que antecede o desenvolvimento de insuficiência cardíaca. Métodos: Foram constituídos quatro grupos experimentais de ratos: normotensos Wistar (W) sedentários (W-SED, n=27); W exercitados (W-EX, n=31); SHR sedentários (SHR-SED, n=27); e SHR exercitados (SHR-EX, n=32). A partir de 13 meses de idade, os animais dos grupos exercitados foram submetidos a protocolo de exercício em esteira, cinco dias por semana, durante quatro meses. A avaliação estrutural e funcional in vivo do coração foi realizada por ecocardiograma. A função miocárdica in vitro foi avaliada em preparações de músculo papilar isolado do ventrículo esquerdo (VE). Amostras de tecido do VE foram obtidas para análises bioquímicas, histológicas e moleculares. A avaliação do colágeno miocárdico total foi realizada pela histologia e por quantificação de hidroxiprolina. O tamanho dos miócitos foi medido em cortes histológicos do VE. A atividade das enzimas antioxidantes foi quantificada por espectrofotometria. A atividade da NADPH oxidase foi avaliada pela redução da lucigenina. A quantificação proteica dos colágenos I e III, lisil oxidase, vias MAPK e NF-kB, e inibidores teciduais 1 e 2 das metaloproteinases foi realizada por Western blot. A atividade das metaloproteinases foi realizada por zimografia. As comparações entre os grupos foram realizadas por análise de variância (ANOVA) complementada pelo teste de Bonferroni (distribuição normal), ou o teste de Kruskal-Wallis complementado pelo teste de Dunn (distribuição não normal). Resultados: A pressão arterial sistólica foi maior nos grupos SHR. Os grupos exercitados apresentaram maior capacidade física. Os sinais de insuficiência cardíaca foram maiores nos grupos hipertensos em relação aos controles, e o grupo SHR-EX apresentou menor prevalência de derrame pleural e taquipneia em comparação ao SHR-SED. O ecocardiograma mostrou reduções da espessura da parede do VE, espessura relativa do VE, diâmetro do átrio esquerdo e melhora do relaxamento no grupo SHR-EX vs. SHR-SED. O estudo da função miocárdica in vitro mostrou melhor performance no grupo SHR-EX (derivada positiva da tensão desenvolvida) vs. SHR-SED. O grupo SHR-EX mostrou maior atividade das enzimas antioxidantes em comparação SHR-SED. A produção de hidroperóxido de lipídeo, diâmetros dos miócitos, expressões proteicas da JNK fosforilada e da IkB total foram maiores nos grupos hipertensos. A quantificação de hidroxiprolina, malondialdeído, atividade da NADPH oxidase, expressões proteicas do colágeno III, lisil oxidase, TIMP-1, JNK total, p38 fosforilada, p65 fosforilada e total e IkB fosforilada não apresentaram diferença entre os grupos. A fração colágena intersticial, a atividade da MMP-2 e a expressão proteica da p38 total, ERK total e fosforilada foram maiores no SHR-SED em comparação com controle. O exercício causou redução da atividade da MMP-2 e da expressão da ERK fosforilada nos ratos hipertensos. Conclusão: O exercício físico em ratos espontaneamente hipertensos atenua o remodelamento cardíaco que está associado à melhora da tolerância ao esforço físico e redução da frequência de sinais de insuficiência cardíaca. Além disso, associa-se ao aumento da atividade das enzimas antioxidantes, diminuição da fosforilação da ERK e da atividade da MMP-2, e atenuação da expressão proteica da ERK total.
Introduction: The pressure overload caused by systemic arterial hypertension (SAH) may change the collagen architecture, induce fibrosis, as well as imbalance between the reactive oxygen species (ROS) production and antioxidant capacity. Increased ROS leads to activation of signaling pathways such as nuclear factor kappa B (NF-kB) and mitogen-activated protein kinases (MAPK). Alterations in these pathways contribute to cardiac remodeling process induced by SAH. Physical exercise plays an important role in mitigating cardiovascular risk factors such as hypertension. Therefore, the aim of this study was to evaluate the influence of physical training, started before clinical evidence of heart failure, on cardiac remodeling in spontaneously hypertensive rats (SHR). Methods: Four experimental groups were used: sedentary (W-SED n=27) and trained (W-EX, n=31) normotensive Wistar rats, and sedentary (SHR-SED, n=27) and exercised (SHR-EX, n=32) hypertensive rats. Rats of the exercise groups underwent a protocol of treadmill exercise five days a week, for four months; exercise started at 13 months of age. Echocardiogram was performed to evaluate in vivo cardiac structures and function. In vitro myocardial function was analyzed in left ventricular (LV) papillary muscle preparations. LV tissue samples were obtained for biochemical, histological, and molecular analysis. Total myocardial collagen was assessed by histology and hydroxyproline quantification. Cardiomyocyte size was measured in LV histological sections. Antioxidant enzymes activity was quantified by spectrophotometry. NADPH oxidase activity was analyzed by reduction of lucigenin. Protein expression of collagen I and III, lysyl oxidase, MAPK and NF-kB, and metalloproteinases tissue inhibitors 1 and 2 was quantified by Western blot. The activity of metalloproteinases was evaluated by zymography. Comparisons between groups were performed by two factors analysis of variance (ANOVA), complemented with the Bonferroni test (normal distribution), or Kruskal-Wallis complemented with Dunn test (non-normal distribution). Results: Systolic blood pressure was higher in the SHR groups. The exercised groups showed greater physical capacity. Prevalence of heart failure signs was higher in the hypertensive groups compared to controls, and the SHR-EX group showed lower prevalence of pleural effusion and tachypnea compared to SHR-SED. Echocardiogram showed lower LV wall thickness, LV relative wall thickness, left atrium diameter, and relaxation time in the SHR-EX group vs. SHR-SED. Myocardial functional study showed better performance in the SHR-EX group (positive derivative of the developed tension) vs. SHR-SED. The SHR-EX group showed higher antioxidant enzymes activity compared to SHR-SED. Lipid hydroperoxide production, myocyte diameters, and phosphorylated JNK and total IkB protein expression were higher in the hypertensive groups. Quantification of hydroxyproline, malondialdehyde, NADPH oxidase activity, and protein expression of collagen III, lysyl oxidase, TIMP-1, total JNK, phosphorylated p38, phosphorylated and total p65, and phosphorylated IkB did not differ between groups. The interstitial collagen fraction, MMP-2 activity, protein expression of total p38, and total and phosphorylated ERK were higher in the SHR-SED group compared to normotensive control. Physical exercise reduced the MMP-2 activity and the phosphorylated ERK expression in hypertensive rats. Conclusion: Physical exercise in spontaneously hypertensive rats attenuates cardiac remodeling associated with improved physical capacity and reduced prevalence of heart failure signs. In addition, it is associated with increased antioxidant enzymes activity, decreased ERK phosphorylation and MMP-2 activity, and attenuation of total ERK protein expression.
FAPESP: 2014/00747-1

Le facteur de transcription NF-kappaB constitue une famille de cinq protéines qui régule l'expression d'un grand nombre de gènes impliqués dans diverses fonctions biologiques, notamment l'immunité, l'inflammation, le développement et l'apoptose. Les membres de la famille NF-kappaB comprennent les protéines p65, RelB, c-Rel, p50 et p52 qui forment des homo- et des hétérodimères. Le facteur de transcription NF-kappaB est normalement séquestré dans le cytoplasme, en association avec les membres de la famille IkB (inhibitor of kappa B). La phosphorylation et l'ubiquitination médiée par IKK (IkB Kinase) conduisent à la dégradation de IkB et à la translocation de NF-kappaB dans le noyau où la régulation transcriptionnelle des gènes cibles a lieu. L'activation de IKK est tributaire entre autres des protéines adaptatrices TRAF et RIP. Ainsi la voie de signalisation NF-kappaB se compose des dimères NF-kappaB, des protéines IkB, du complexe IKK et des protéines adaptatrices. L'activation de la voie de signalisation NF-kappaB est fréquente au cours des infections virale. NF-kappaB étant un composant essentiel de la réponse immunitaire innée antivirale, il participe à la réaction de l'hôte contre les agents pathogènes. Les virus ont développé des stratégies pour moduler la voie de signalisation NF-kappaB en leur faveur notamment pour faciliter leur réplication, empêcher l'apoptose des cellules infectées et favoriser l'échappement à la réponse immunitaire. Par ailleurs, les virus ont incorporé des sites de fixation de NF-kappaB dans leurs promotteurs. Ainsi l'activation de NF-kappaB résulte en une transactivation des promoteurs viraux et en une transcription virale augmentée. En effet de nombreux virus et certaines protéines virales activent ou inhibent la voie de signalisation NF-kappaB pour créer un environnement favorable au développement du cycle viral dans la cellule hôte. Dans la première partie de notre étude, nous nous sommes intéressés au rôle du facteur de transcription NF-kappaB dans la transcription du cytomégalovirus humain (HCMV) en infectant des macrophages dérivés de monocytes sanguins (MDMs). Les macrophages jouent un rôle important dans la pathogenèse liée à HCMV et l'effet de l'infection virale sur la signalisation cellulaire dans ces cellules reste peu documenté. Nous avons montré que les souches virales AD169 (souche de laboratoire) et HCMV-DB (souche issue d'un isolat clinique) pouvaient se multiplier dans des cultures de cellules primaires MDMs mais que le titre viral demeurait inférieur à celui observé dans des cellules plus permissives telles que les fibroblastes de la lignée MRC5. Etant donné que le facteur de transcription NF-kappaB joue un rôle essentiel dans la réplication virale notamment par la transactivation du promoteur viral très précoce IE et du gène tardif viral, nous avons étudié l'activation et la composition du complexe NF-kappaB dans des cellules MDMs et des fibroblastes MRC5 infectés par HCMV. Par des techniques de retard sur gel (EMSA) et de colorimétrie (Microwell colorimetric NF-kappaB assay), nous avons montré que l'infection par HCMV entraînait une activation du complexe p52/Bcl-3 dans les cellules MDMs alors qu'il activait le complexe classique NF-kappaB p50/p65 dans les fibroblastes de la lignée MRC5. L'utilisation des cellules monocytoïdes U937 transfectées par un vecteur pCMV-Luc, vecteur d'expression de la luciférase sous la dépendance du promoteur viral du gène très précoce EA (MIEP), nous a permis de montrer que le complexe p52/Bcl-3 activait le promoteur viral MIEP. Par la technique d'immunoprécipitation de chromatine (technique ChIP), nous avons mis en évidence l'interaction entre le complexe p52/Bcl-3 et le MIEP dans les cellules MDMs infectées par le HCMV. Ainsi, l'activation du complexe NF-kappaB p52/Bcl-3 dans les cellules MDMs pourrait être à l'origine de la réplication limitée d'HCMV dans ces cellules. Dans la deuxième partie de notre étude, nous avons étudié le rôle de NF-kappaB dans la transcription du VIH-1 dans les cellules MDMs lors de la co-infection par le virus de l'hépatite C (VHC) et le VIH-1. L'infection par VIH favorise l'évolution de l'hépatite C chronique et augmente la charge virale VHC, mais le rôle deu VHC sur la réplication virale de VIH reste mal connu. Les cellules mononuclées périphériques (PBMC) sont permissives aux virus VIH et VHC et peuvent constituer un réservoir extra-hépatique de virions. Il a été montré que la transcription de NF-kappaB est activée lors de l'infection par VHC et VIH-1. Nous nous sommes proposés d'étudier le rôle de NF-kappaB sur la transcription du VIH-1 dans les cellules MDMs lors d'une co-infection VHC/VIH-1. Des études préliminaires conduites dans notre laboratoire ont montré que la charge virale VIH-1 était plus élevée dans les cellules PBMC et MDMs provenant de sujets co-infectés que chez les sujets mono-infectés. Nous avons observé, in vitro, que les protéines Nef du VIH-1 et Core du VHC activent le complexe p50/p65 de NF-kappaB dans les cellules MDM. Grâce à une technique reposant sur l'expression de la luciférase, nous avons montré que les deux protéines activaient la transcription du LTR du VIH-1. Nous avons également démontré que les protéines Nef et Core activent la réplication du VIH-1 dans les cellules promonocytaires chroniquement infectés U1. De plus, ces protéines stimulaient également la réplication du VIH-1 dans des macrophages primaires infectés par le virus. Par conséquent, les deux protéines Nef et Core pourraient favoriser la formation de réservoirs cellulaires contenant les deux virus. L'ensemble de ces études nous a permis de mieux comprendre le rôle de NF-kappaB dans la transcription virale au sein des macrophages
The mammalian nuclear factor-kB (NF-kB) is a family of live DNA-binding proteins that regulate expression of a large number of genes involved in diverse biological functions including immunity, inflammation, development and apoptosis. Members of NF-kappaB family include p65 (RelA), RelB, c-Rel, p50 (NF-kappaB) and p52 (NF-kappaB2), which are found as homo-and heterodimers. The transcription factor NF-kappaB is normally sequestred in the cytoplasm in association with the members of the inhibitor of kappa B (IkB) family. IkB Kinase (IKK) mediated phosphorylation, ubiquitination and degradation of IkB frees NF-kappaB to translocate to the nucleus to regulate the transcription of target genes. Activation of IKK is dependent upon intracellular adapter proteins such as TRAF and RIP. Thus NF-kappaB pathway consists of NF-kappaB dimers, IkB proteins, IKK complex and intracellular adapter proteins. Activation of NF-kappaB is a common feature during viral infections. NF-kappaB is an essential component of innate antiviral immune response and is a part of the protective reaction of the host against pathogens. Viruses have evolved strategies to modulate NF-kappaB signaling pathway for their own benefit especially to facilitate their replication, prevent apoptosis of infected cells and evasion of immune responses. In addition a number of viruses contain NF-kappaB binding sites in their promoters. Thus activation of NF-kappaB results in the transactivation of viral promoters, thus enhancing viral transcription and replication. In fact, several viruses and a number of viral proteins have been reported either to stimulate or inhibit NF-kappaB activation to create an environment for successful viral life cycle in the host cell. In the first part of our study we studied the role of NF-kappaB in the transcription and replication of HCMV in primary human monocyte-derived macrophages (MDMs). Monocytes/macrophages are key cells in the pathogenesis of human cytomegalovirus (HCMV) infection, but the in vitro rate of viral production in MDMs is considerably lower than in fibroblasts. Considering that the NF-kappaB signaling pathway is potentially involved in the replication strategy of HCMV through efficient transactivation of the major immediate-early promoter (MIEP), efficient viral replication, and late gene expression, we investigated the composition of the NF-kappaB complex in HCMV-infected MDMs and fibroblasts. Preliminary studies showed that HCMV could grow in primary MDMs culture but that the viral titer in culture supernatants was lower than that observed in the supernatants of more permissive MRC5 fibroblasts. EMSA and microwell colorimetric NF-kappaB assay demonstrated that HCMV infection of MDMs increased p52 binding activity without activating the canonical p50/p65 complex. Moreover, Bcl-3 was up-regulated and was demonstrated to associate with p52, indicating p52/Bcl-3 complexes as the major component of the NF-kappaB complex in MDMs. Luciferase assays in promonocytic U937 cells transfected with an MIEP-luciferase reporte construct demonstrated MIEP activation in response to p52 and Bcl-3 overexpression. Chromatin immunoprecipitation assay demonstrated that p52 and Bcl-3 bind the MIEP in acutely HCMV-infected MDMs. In contrast, HCMV infection of MRC5 fibroblasts resulted in activation of p50/p65 heterodimers. Thus, activation of p52/Bcl-3 complexes in MDMs and p50/p65 heterodimers in fibroblasts in response to HCMV infection might explain the low-level growth of the virus in MDMs vs efficient growth in fibroblasts. In the second part of our study, we studied the role of NF-kappaB in the transcription and replication of HIV-1 in macrophages during HIV-1/hepatitis C virus (HCV) coinfection. HIV infection favors the progression of HCV disease and enhances the viral load of HCV but the effect of HCV on replication of HIV-1 is not well studied. Macrophages are permissive to HCV and HIV-1 infection so can constitute extra-hepatic reservoir of these viruses. As transcription factor NF-kappaB is activated during HIV-1 and HCV infection we studied the role of NF-kappaB in the transcription of HIV-1 in MDMs. Preliminary studies from our laboratory demonstrate higher levels of HIV-1 viral load in MDMs isolated from the peripheral blood of HIV-1/HCV coinfected subjects in comparison with HIV-1 monoinfected patients. To assess the potential role of HIV-1 Nef and HCV Core proteins in this phenomenon, we studied their respective role regarding NF-kappaB activation and HIV-1 replication in primary macrophages. Following the treatment of MDMs with exogenous HIV-1 Nef and HCV Core proteins, we observed activation of NF-kappaB which consist of p50/p65. Consistently, degradation of NF-kappaB, and phosphorylation of IKKalpha, IKK beta was observed in response to both HIV-1 Nef and HCV Core proteins. In addition, HIV-1 Nef and HCV Core proteins stimulated synergistically the HIV-1 long terminal repeat (LTR), and subsequently enhanced HIV-1 replication in both chronically infected promonocytic U1 cells and acutely HIV-1 infected MDMs. Therefore, our results indicate that HIV-1 Nef and HCV Core proteins synergise to enhance NF-kappaB activation and HIV-1 replication in primary macrophages and thereby could fuel the progression of the HIV-1 disease in HIV/HCV coinfected patients. All together our results have important implication in terms of viral persistence and formation of viral reservoirs in macrophages during chronic viral infections

碩士
中國醫藥大學
臨床醫學研究所碩士班
97
Lung cancer is the leading cause of cancer-related mortality worldwide. It frequently results in distal metastases, including, brain, liver and bone marrow etc while it is diagnosed. Investigation of the factors and mechanism affecting tumor metastases is important in treatment of the disease. Chemokine plays a crucial role in the inflammatory response and the migration and metastasis of human cancer cells. CCL5 (previously called RANTES) is in the CC-chemokine family. Previous studies show it related with activation of T-cell and metastases of breast tumor cells. Besides, integrins are the major adhesive molecules in mammalian cells and important in the migration of cells. In this study, we examine the effect of CCL5 on integrin expression and migration activity in human non-small cell lung cancer cells. Here we found CCL5 increased the migration and cell surface expression of αvβ3 integrin in human lung cancer cells (A549 cells, H929 and H1299). The CCR5 of lung cancer cells have high expression than in lung epithelium cells (HBE-E6/E7 and BEAS-2B). Furthermore, we found CCL5 stimulation increased phosphorylation of the p85α subunit of phosphatidylinositol 3-kinase (PI3K) and serine 473 of Akt. Also, PI3K inhibitor (Ly294002) or Akt inhibitor suppressed CCL5-induced migration activities and integrin expression of A549 cells. Transfection of cells with p85 or Akt mutant also reduced CCL5-mediated cancer migration. In addition, treatment of A549 cells with CCL5 induced IκB kinase α/β?}??(IKK α/β) phosphorylation, IκB phosphorylation, p65 Ser536 phosphorylation, and κB-luciferase activity. Furthermore, the CCL5-mediated increases in p65 Ser536 phosphorylation were inhibited by Ly294002 and Akt inhibitor. Taken together, our results suggest that CCL5 acts through PI3K/Akt, which in turn activates IKK?悈/β and NF-κB, resulting in the activation of αvβ3 integrin and contributing to the migration of human lung cancer cells.

碩士
中興大學
生命科學院碩士在職專班
95
Obesity, the excessive accumulation of fat, is a risk factor for development of metabolic syndrome. The adipose tissue itself has proven to be an important endocrine organ, secreting several hormones and cytokines, usually referred as adipocytokines or adipokines. Visfatin can bind to and activate the insulin receptor, exerting insulin mimetic effects both in vitro and in vivo. In addition, visfatin, found to identical to pre-B-cell colony-enhancing factor (PBEF), a novel inflammatory cytokine that plays a requisite role in the delayed neutrophil apoptosis , and it levels in blood levels have been reported higher in subjects with obesity and/or type 2 diabetes mellitus. It is currently unclear the pathophysiological role of visfatin and it association with endothelial dysfunction related inflammatory and adhesion molecule expression has been largely unexplored. Primary human umbilical vein endothelial cells (HUVECs) pretreated with visfatin (1, 10, 50 ng/ml) were used to study the relationship between visfatin and endothelium dysfunction. Expression of cytokine (IL-6 and IL-8) and adhesion molecules (VCAM-1 and E-selectin) affected by visfatin were investigated by real-time PCR and ELISA. Activity of NF-κB was examined by electrophoretic mobility shift assay (EMSA).At a visfatin concentration of 50 ng/ml, significant increases in IL-6 (2.72 folds), IL-8 (1.93 folds), ICAM-1 (2.96 folds), VCAM-1 (3.97 folds) and E-selectin (2.80 folds) gene expression (all p<0.05) along with increased IL-6 (1.76 folds), IL-8 (1.23 folds) and sE-selectin (1.78 folds) protein levels (all p<0.05) in the conditioned medium were detected. Visfatin significantly increased ICAM-1 (1.11 folds) expression on 10 ng/ml, and VCAM-1 (1.06 folds) expression on 50 ng/ml detected by flow cytometry. Results from EMSA confirmed that visfatin (10, 50 ng/ml) increased DNA binding activity of NF-κB (1.56 folds, 1.38 folds respectively) (p<0.05). In addition, increased human monocyte cell line THP-1 attaching to HUVECs when pretreated with visfatin (10, 50 ng/ml) was also demonstrated(1.22 folds, 1.22 folds respectively) (p<0.05). We demonstrated that visfatin increased inflammatory and adhesion molecule expression, at least partly via up-regulated of NF-κB activity, that might contribute to increased endothelial dysfunction. In addition, visfatin enhanced THP-1 adherence to HUVECs. Our findings provide direct evidence that visfatin causes endothelial dysfunction, which may help establish a link between obesity and cardiovascular disease, as well as a novel approach to target the development of new therapeutic strategies.

博士
中山醫學大學
醫學分子毒理學研究所
98
Apurinic endonuclease 1 (Ape1) is not only involved in base excision repair, but also activate some transcriptional factors via its redox activity. However, which subcellular localization of Ape1 involved in the activation of transcriptional factor remains unclear. We first observed that Cox-2 expression was associated with cytoplasmic Ape1 expression in lung tumors and cancer cell lines. We thus hypothesize that NF-κB is activated by cytoplasmic Ape1 to cause Cox-2 expression. Herein, we generated cytoplasmic and nuclear Ape1 in Ape1-knockdown lung cancer cells by exogenous expression of Ape1 containing various deletions and/or mutations of the nuclear localization sequence. It was observed that cytoplasmic Ape1, but not nuclear Ape1, induced Cox-2 expression via NF-κB activation. NF-κB activation by cytoplasmic Ape1 was diminished by the Ape1 redox activity inhibitor resveratrol. Cells expressing cytoplasmic Ape1 exhibited tumor progression and metastasis in vitro and in vivo as xenografts, but cells expressing nuclear Ape1 not. Patients with tumors containing elevated cytoplasmic Ape1 had poor prognosis and 3.722-fold risk of tumor recurrence and/or metastasis. Cytoplasmic Ape1 may therefore enhance lung tumor malignancy via NF-κB activation, suggesting that combination of cisplatin and specific redox inhibitor may improve chemotherapeutic response in patients with tumors containing elevated cytoplasmic Ape1. Among 111 lung tumors, we further observed that HPV16/18 E6-positive lung tumors had higher cytoplasmic Ape1 expression than in HPV16/18-negative lung tumors (P = 0.037). To verify whether E6 could enhance subcellular localization of Ape1 via increased Ape1 transcription, HPV16 E6-positive TL-1 and –negative TL-4 lung cancer cells were used for knockdown and overexpression of E6 by shE6 and E6 vector, respectively. Our data showed that Ape1 mRNA and protein expression was increased by E6 overexpression in TL-4 cells and decreased by E6-knockdown in TL-1 cells. Meanwhile, increased Cox-2 expression was observed in E6-overexpressed TL-4 cells via activation of NF-κB pathway. By contrast, Cox-2 expression was decreased in E6-knockdown TL-1 cells via blocking NF-κB activation. ChIP analysis further showed that ROS induced by E6 may be linked with Ape1 transcription via increased CREB and AP-1 binding on Ape1 promoter. To further verify whether Ape1 nuclear export promoted by E6 is via increased S-nitrosation of Ape1, FL-Ape1 and E6 vector were co-transfected into shApe1#7 H157 stable clones and then the cells were treated with or without NO scavenger (carboxyl-PTIO). Our data indicated that cytoplasmic Ape1 expression in shApe1#7 stable clones with FL-Ape1 was markedly increased by E6 transfection, but the increase of cytoplasmic Ape1 expression was diminished by the treatment of carboxyl-PTIO. To further verify whether cytoplasmic Ape1 increased by E6 is via S-nitrosation of Ape1, FL-Ape1 was replaced by C93S/C310S mutant Ape1 to be transfected into E6-transfected shApe1#7 stable clones. The increase of cytoplasmic Ape1 was failed in E6-positive stable clones when co-transfected with mutant Ape1. These results strongly suggest that cytoplasmic Ape1 increased by E6 is predominately mediated through S-nitrosation of Ape1. More interestingly, the increase of cytoplamic Ape1 by E6 enhanced significantly cell invasion ability and increased Cox-2 expression. In summary, subcellular localization of Ape1 may be partially responsible for lung tumor progression and malignancy via activation of NF-κB signaling pathway, particularly in HPV E6-positive lung tumors.

碩士
中山醫學大學
醫學分子毒理學研究所
99
ZAK is a novel MLK family of mixed lineage kinase-like protein. The molecular weight of this protein is 92 kDa. The protein structure of ZAK contains three main functional motifs, including kinase domain (KD), leucine zipper (LZ) and sterile-alpha motif (SAM) domain, but its function is similar to MAP kinase kinase kinase (MAP3K). Our previous studies show that overexpression of ZAK gene enhances AP-1 activity via the activation of ERK and JNK, thereby making the cell growth slowed. In this study, we found that ZAK gene in migration and invasion of lung cancer cells plays an important role. In vitro, experiments proved that ZAK overexpression in human lung cancer cells H460 cells reduced the ability of migration and invasion. It increased the tissue inhibitor of metalloproteinase-1 (TIMP-1) expression , and thus inhibited matrix metalloproteinase -9 (MMP9) activity. At the same time, ZAK decreased protein expression downstream of Wnt / β-catenin pathway. From the experimental results also can be found when the presence of ZAK gene increased the cell adhesion ability. Lung cancer cells treated with different doses of TNF-α, the transcription capability of NF-κB into the nucleus was enhanced. Next, in order to prove its specificity, we used IKKα and IKKβ to affect the NF-κB entering the nuclear capability. We demonstrated that ZAK gene can suppess lung cancer cell growth and metastasis by reducing NF-κB transcription factor. In vivo, experiment also proved that lung cancer cells overexpression ZAK gene decreased the number of tumor migration in mouse lungs. Moreover, from the experimental results, we also found that the antiapoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1, survivin, cIAP-2 and XIAP were decreased significantly in ZAK overexpressed cells. But the proapoptotic protein BAX,BAK and BAD, the amount of protein expressions were increased. From the experimental results, regardless of flow cytometry analysis or DAPI fluorescence staining, TRAIL can enhance apoptosis in ZAK overexpression cells. Taken together, ZAK gene for the H460 human lung cancer cells not only inhibited the growth and metastasis of cancer cells, while using TRAIL also increased the apoptosis of tumor cells and these results have been an important discovery in treatment of lung cancer.

碩士
中國醫藥大學
基礎醫學研究所
98
Nephroblastoma overexpressed (Nov; CCN3), from the CCN gene family, which is involved in many cellular activities such as growth, differentiation, cell motility, adhesion and division. However, the effect of CCN3 on migration activity in human chandrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 through the αvβ3 and αvβ5 integrin receptor in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 and αvβ5 monoclonal antibody but not RAD peptide inhibitor inhibited the CCN3-induced increase migration and MMP-13 expression. Activations of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt and NF-κB pathways after CCN3 treatment was demonstrated, and CCN3-induced expression of MMP-13 and migration activity was inhibited by the specific inhibitor of PI3K, Akt and NF-κB cascades. Transfection of cells with FAK, p85, Akt, IKKα and IKKβ mutant also reduced CCN3-induced cancer migration. Taken together, our results suggest that CCN3 acts through FAK/PI3K/Akt, which in turn activates NF-κB, resulting in the activation of MMP-13 and contributing to the migration of human chondrosarcoma cells.

碩士
國立陽明大學
生物醫學影像暨放射科學系暨研究所
98
Purpose: Colorectal carcinoma is common cancer and the first became the most common cause of cancer since 2009 in Taiwan. Radiation therapy plays an important role in neoadjuvant therapy of colorectal cancer. However, only 20% of patients achieved complete response due to radioresistance of the tumor. Several studies suggest that radiation-induced nuclear factor kappa B (NF-κB) expression is related to tumor tolerance and poor prognosis. Sorafenib is a multikinase inhibitor that can block tumor proliferation and induce apoptosis by inhibition of MAPK pathways. Both mechanisms have been shown to be linked to NF-κB pathway. In this study, we investigated whether sorafenib caused the enhance radiosensitivity of colorectal carcinoma, and elucidated the underlying mechanism. Materials & Methods: We established the stable clone, HT29/tk-luc, and analyzed the mechanism of radioresistance by colony formation assay, flow cytometry and western blotting assay. For in vivo study, HT29/tk-luc was subcutaneously implanted into the right gluteal back region of NOD/SCID mice. The mice were randomly divided into four groups: control, sorafenib alone (15mg/kg/day), radiation alone (single dose 12Gy), and combination (sorafenib15mg/kg/day + radiation 12 Gy). Bioluminescent imaging (BLI) was performed weekly and micoSPECT was used on the 35th day. Mice were then sacrificed with immunohistostaining performed. Results: Sorafenib combined with radiation significantly inhibited the proliferation of tumor growth as compared to sorafenib or radiation alone (p&lt;0.05). Radiation could induce transient elevation of NF-κB activity, whereas sorafenib suppressed the radiation-induced Bcl-2, Mcl-1, XIAP, cyclin D1 and MMP-9 through inhibition of NF-κB expression. BLI and microSPECT/CT further confirmed that the best therapeutic efficacy could be by the combination therapy. Conclusions: Our results suggest that sorafenib potentiates the antitumor effects of radiation in colorectal cancer via suppression radiation-induced NF-κB expression and NF-κB regulated gene products. Combination treatment of sorafenib and radiation exerted synergistic effect both in vitro and in vivo as shown in these studies.

碩士
中山醫學大學
生化暨生物科技研究所
102
Chinese women in the top ten causes of death in cancer, cervical cancer is seventh, 10 years standardized mortality rates of cervical cancer in females with the largest decline, should be attributed to the Republican National Health Board since 1995, providing Pap smear screening after the inspection, so that the mortality rate declined by about Liu Cheng. However, cervical cancer remains the first female reproductive tract cancers. Berberine is a plant alkaloid with a long history of medicinal use in Chinese medicine. The berberine alkaloid can be found in the roots, rhizomes, and stem bark of the plants.Berberine extracts and decoctions have demonstrated significant antimicrobial activity and has several anti-inflammation and anti-cancer biological effects. However, its mechanism of action on the cell migration and invasion of human cervical cancer cells is not fully understood. Human cervical cancer cells were treated with berberine, subjected to invasion assay, motility assay and MTT assay, significantly inhibits invasive, motility in human cervical cancer cells. Zymography and promoter luciferase analysis revealed that berberine inhibits the proteinase and transcription activities of MMP-2 and u-PA, respectively.We also use NF-κB inhibitor to find that berberine inhibited NF-κB which then led to the inhibition MMP-2 expression. Overall, berberine on transcriptionally regulation of MMP-2 expression via the down-regulation of NF-κB followed by the inhibition of migration and invasion in human cervical cancer.

博士
國立陽明大學
生物醫學影像暨放射科學系
100
Invasion through activation of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) were via the regulation of nuclear factor-kappaB (NF-κB). Sorafenib can improve the overall survival in patients with advanced HCC and its inhibitory mechanism associated with the inactivation of NF-κB remains unclear. Here, Huh7 cells transfected with NF-κB-luc2 vector were used to study the effects of sorafenib on NF-κB activity and expressions of MMP-9 and VEGF induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) with bioluminescent imaging, Western blotting, reverse-transcription polymerase chain reaction, electrophoretic mobility shift and gelatin zymography assays. TPA increased significantly NF-κB activity and expressions of MMP-9 and VEGF, which was suppressed by sorafenib, in a dose-dependent manner. Similar results were reproduced by using PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Furthermore, HCC cells transfected with IκBα mutant vector (p-IκBαM) exhibited decreased TPA-induced MMP-9 and VEGF mRNA expressions. Together, sorafenib inhibits TPA-induced MMP-9 and VEGF expressions via the suppression of ERK/NF-κB pathway in HCC cells.

碩士
高雄醫學大學
藥理學研究所
101
Neuropathic pain occurs as a result of injury to the sensory transmission after a primary lesion or dysfunction in the nervous system. Peripheral nerve injury may produce chronic pain states characterized by hyperalgesia, allodynia and spontaneous pain. Patients with neuropathic pain have higher pain scores and lower quality of life than other chronic pain patients. Chronic pain is categorized into inflammatory pain, induced by peripheral tissue inflammation, and causes damage and malfunction of the nerve system. Many reports have been shown that peripheral nerve injury could induce inflammatory states, resulting in the genesis and maintenance of neuropathic pain. Despite the large number of approved analgesic drugs, effective treatment of chronic pain is still unsatisfactory because of the severe side effects from these drugs. In the present study, we tried to investigate whether KMUP-1 could attenuate pain hypersensitivity and inflammatory mediators, and to explore its possible mechanisms in the dorsal horn of spinal cord of rats after chronic constriction injury-induced neuropathic pain. Male SD rats were randomly assigned to four groups: sham, sham plus KMUP-1, chronic constriction injury (CCI) of bilateral sciatic nerve and CCI plus KMUP-1 groups. After the CCI model was established, KMUP-1 (5 mg/kg) was administrated intraperitoneally once daily for 3, 7 and 14 days. Measurements of mechanical withdrawal threshoulds using von Frey filaments and thermal withdrawal latency using radiant heat applied to the plantar surface, were made daily before and after the injection of each dose. CCI of the sciatic nerve induced mechanical allodynia and thermal hyperalgesia and this effect was accompanied by increased levels of inflammatory mediators. The dorsal horn of spinal cord was divided into ipsilateral and contralateral parts for western blots and enzyme-linked immunosorbent assay to analyze the levels of inflammatory proteins and cytokines, respectively. We demonstrated that chronic intraperitoneally KMUP-1 has beneficial effects on the behaviors of both thermal and mechanical stimulations in the neuropathic pain animals. Furthermore, KMUP-1 reduced the expression of inflammatory protein and cytokines in the spinal dorsal horn on Day 3, 7, 14 for the model rats of CCI. In conclusion, KMUP-1 has anti-inflammatory and anti-pain hypersensitivity properties through inhibition of ERK and NF-κB activation in the development of pain hypersensitivity after nerve injury. Therefore, KMUP-1 could be a potential pharmacotherapeutic agent for the treatment of neuropathic pain.

碩士
國立成功大學
微生物及免疫學研究所
90
Streptococcal pyrogenic exotoxin A (SPEA) is produced by invasive Streptococcus pyogenes isolated from streptococcal toxic-shock syndrome (STSS) with the severe and frequently fatal illness. The present study provides the first evidence that SPEA acts through the PI3K-Akt and nuclear factor-kappa B (NF-kB)-related mechanism in human peripheral blood mononuclear cells (PBMC) to stimulate the production of pyrogenic cytokines which induced a febrile response in rabbits. The levels of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in the supernatant fluids of SPEA-treated PBMC started to rise at 12 h, reached its plateau levels at 72 h, and displayed dose-dependent manner. Western blot analysis and electrophoretic mobility shift assay (EMSA) demonstrated that SPEA induced the translocation of RelA/p65 into its nucleus and DNA binding activity of NF-kB were also in a dose- and time-dependent manner. The increased levels of TNF and IL-1 in the supernatant fluids and NF-kB nuclear translocation of SPEA-treated PBMC were attenuated by PI3K inhibitor, wortmannin, or Akt inhibitor, 1L-6-hydroxymethyl- chiro-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). However, they did not affected by 5-lipoxygenase (5-LOX) inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA), or 5-LOX activating protein (FLAP) inhibitor, MK886. Moreover, Western blot analysis demonstrated that wortmannin and HIMO, but not ETYA and MK886, inhibited SPEA-mediated phosphorylation of Akt, IKKα/β, and IκBα, and nuclear translocation of NF-kB in PBMC. Furthermore, intravenous administration of supernatant fluids obtained from SPEA-treated PBMC caused the fever in rabbits, and were also attenuated by wortmannin and HIMO, but not ETYA and MK886. Taken together, it appears that the activation of NF-kB increases the production of the pyrogenic cytokines in SPEA-treated PBMC may act through PI3K-Akt pathway.

博士
國立陽明大學
生物醫學影像暨放射科學系
102
Purpose: Hepatocellular carcinoma (HCC) is the third most common cancer and accounts for a significant amount of deaths each year in Taiwan. Treatment outcome highly depends on the liver cancer stage at diagnosis. Resistance of cancer cells to chemotherapy and/or radiotherapy is a major challenge to current anticancer treatment. Although the mechanism involved in the radioresistance or chemoresistance in HCC is not fully understood, several studies reported that the transcription factor nuclear factor-κB (NF-κB) plays an important role. Radiotherapy is one of the treatments for patients with unresectable hepatocellular carcinoma (HCC); however, some studies have shown that there are disappointing outcomes due to the developed radioresistance of the tumor. Otherwise, some studies show that histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), could induce cell-cycle arrest, and apoptosis in HCC. In addition, NF-κB inhibitor was shown to enhance the cytotoxicity of HDAC inhibitor or radiation in several cancer cell types. In our previous study, we found that sorafenib could inhibit tumor growth in HCC via inhibition of ERK/NF-κB pathway. Though sorafeinb could suppress NF-κB activity in hepatoma cells, the clinical data showed that sorafenib only prolonged the lifespan of HCC patients by 2.8 months. However, whether sorafenib could increase the anti-tumor activity of SAHA or radiation via suppression of ERK/NF-κB pathway in HCC has not been elucidated. Here, we demonstrated the therapeutic efficacy and mechanism of sorafenib combined with radiation or SAHA in human HCC both in vitro and in vivo. Materials and Methods: A human HCC Huh7 cell line transfected with NF-κB responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, was renamed as Huh7/NF-κB-tk-luc2/rfp cells, and monitored by bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to evaluate the effect of sorafenib combined with radiation or SAHA on NF-kB activation and tumor growth inhibition. Cytotoxic mechanism of sorafenib combined with radiation or SAHA on HCC was also elucidated. Results: Tumor growth inhibition by SAHA or radiation was significantly enhanced by sorafenib, which suppressed radiation-induced and SAHA-induced NF-κB activity in HCC both in vitro and in vivo. On the other hand, our results showed that sorafenib decreased radiation-induced and SAHA-induced NF-κB activity via ERK dephosphoryaltion. NF-κB inhibition could increase radiation-induced and SAHA-induced cell cytotoxicity and apoptosis in HCC. Our results showed that sorafenib could sensitize human HCC to radiation or to SAHA via suppression of ERK/NF-κB pathway. Conclusion: Combination of sorafenib and SAHA could improve the therapeutic efficacy of human HCC through suppression of ERK/NF-κB pathway and NF-κB regulated downstream effector proteins. This NF-κB promoter combined with triple reporter genes system is a useful system for evaluation of therapeutic efficacy of newly developed NF-κB related pharmaceuticals.

碩士
中國醫藥大學
基礎醫學研究所碩士班
99
Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver. Chemoresistance is the major problem affecting HCC therapy, the solution of HCC Chemoresistant is the most important issue today. Our previous research indicated that β-catenin play a key role in metastasis in HA22T Hepatocellular cell line and HCC patients. Apicidin is a novel HDAC inhibitor derived from a fungal metabolite, and it’s treatment resistant in HCC remains to be elucidated. To establish a stable liver cancer cell lines chronically resistant to apicidin, HA22T cells were exposed to gradually increasing concentrations of apicidin. We observed that Apicidin-resistant (AR) HA22T cells were highly increased in β-catenin nuclear accumulation and significantly decreased in GSK-3-β protein level than HA22T cells, results also showed that AR cells abundantly increased in Tbx3, a downstream target of the Wnt pathway which implicated in liver tumorigenesis metastasis. In addition, the epithelial-mesenchymal transition (EMT) determining factor, matrix metalloproteinase (MMP)-2 was also highly up-regulated in AR cells. Moreover, we identified the extraordinarily up-regulation of MMP-2 and Wnt signaling pathway, individually via IGF-IR/PI3K/Akt and Ikkαβ/NF-κB pathway. Therefore, our results suggest that AR cells highly potentiate the aggressive behavior of EMT and metastasis effect, and further suggest that β-catenin and MMP-2 gene knockdown or nature herbal extraction candidates might overcome apicidin drug resistance. Our finding might lead to develop the novel therapeutic strategies, and improve the overall survival rate of Chemoresistant HCC patients.

碩士
中國醫藥大學
生物科技學系碩士班
101
Deguelin is a rotenoid of the flavonoid family with chemopreventive activities which is able to decrease tumor incidence for colon, mammary and skin carcinogenesis through AKT inhibition. However, it is still unclear whether deguelin effectively inhibits lung cancer cell metastasis. The anti-metastasis effect of deguelin was initially evaluated on human non-small cell lung cancer (NSCLC) NCI-H292 cells using matrigel invasion assay and wound healing assay. The suppressive effects of matrix metalloproteinase -2 (MMP-2) gelatinolytic activity and AKT, c-Jun N-terminal kinase 1/2, NF-κB activations by deguelin treatment on NCI-H292 cells were further determined. In the present studies, we found deguelin suppressed NCI-H292 cell proliferation and cell adhesion ability but no effect on cell death at the concentration of 0.5, 1, 1.5, 2, 2.5 μM by using MTT assay, flow cytometry and collagen adhesion assays. Deguelin was identified to display the anti-metastasis effect in association with a dose-dependent reduction in MMP-1, -9 protein expressions and activity. The AKT, JNK phospholations and NF-κB were also decreased by deguelin. In addition, deguelin was found to inhibit protein expression of Ras, Rho A, SOS 1…etc. but it did not phosphorylate protein levels of extracellular regulated protein kinase 1/2 and p38. Taking together, deguelin is an inhibitor of NCI-H292 cell for metastasis, and the molecular mechanism involves at least in part the down-regulation of MMPs activations and expressions by targeting the AKT/JNK/NF-κB activation. In the future, this class of naturally occurring small molecules thus may have potential to use as suppresses tumor metastatic treatments.

碩士
國立中興大學
動物科學系所
105
Antrodia cinnamomea, a precious and unique medical fungus existing exclusively in Taiwan, exhibits antioxidant and immunomodulatory properties. This study was divided into two parts, the preliminary study employed a commercial solid-state cultured Antrodia cinnamomea mycelial powder (ACP); and the second part applied the same strain of A. cinnamomea (AC) as the first part to wheat bran (WB) by solid-state fermentation for 16 days (FAC). Both experiments aimed to evaluate the beneficial effects of AC on chickens, and to further illuminate its underlying antioxidant and immunomodulation molecular mechanisms in broilers. The functional compounds of ACP and FAC - crude triterpenoids, crude polysaccharides and total phenolic content - were both assayed at first to evaluate the possible effects of these materials. In the first animal trial, 240 d-old broiler chickens (Ross 308) were assigned to 4 treatment groups receiving diet supplemented with ACP at 0%, 0.1%, 0.2% and 0.4% for 35 days. Each group had four replicate pens, with 15 birds per pen. For the second trial, 400 d-old broiler chickens were allotted into 5 treatment groups fed control diet, and control diet replaced with 5% WB, 10% WB, 5% FAC, and 10% FAC respectively. Regarding the entire experimental period, chickens in the ACP-supplemented groups demonstrated increased body weight gain compared to those had control diet. 5% and 10% FAC inclusion in diet had birds optimal weight gain than those in WB groups. Moreover, cecal and ileal coliform count were decreased in both the 0.1% and 0.2% ACP groups; and cecal coliform and lactic acid bacteria were diminished and increased respectively while diet replaced with FAC. Blood antioxidant potentiality - SOD activity, increased in birds fed ACP supplemented diet at both 21 and 35 day, accompanied by higher CAT activity at 21 day; yet for FAC inclusion in diet, SOD activity rather increased at 35 day only, with CAT elevated at 21 and 35 day. Oxidative species, in terms of H2O2 and NO levels, induced by LPS and AAPH in chicken peripheral blood mononuclear cells (cPBMCs) were compromised in chickens received FAC containing diet. Furthermore, in 35-d-old birds, PGE2 production in cPBMCs was also suppressed while offering ACP and FAC in chicken diet. mRNA expressions were detected in cPBMCs and liver for the first and the second trial respectively. Antioxidant genes dominated by Nrf2, such as HO-1, GCLC, were up-regulated in 35 day-old birds given ACP supplemented diets and mostly in 5% and 10% FAC groups. On the other hand, inflammatory-related genes, like IL-1β and IL-6, ruled mainly by NF-κB, were rather down-regulated by 0.2% ACP addition at 21 and 35 day; for the second trial, these patterns were pronounced at 35 d. Protein expression levels of Nrf2 and NF-κB in chicken liver supported the mRNA results, demonstrating that all the ACP-supplemented groups showed significantly higher Nrf2 expression, whereas the NF-κB was inhibited. Particularly, promoted Nrf2 and inhibited NF-κB nuclear translocation in chicken liver were found in FAC containing groups. In conclusion, preferable microbial balance may indicate the improvement of immunomodulatory capacity by ACP and FAC. Furthermore, ACP and FAC could induce the Nrf2-dependent pathway and decrease NF-κB dominated inflammatory signaling pathway. Antioxidant and immune capacity in terms of antioxidant enzymes and cell tolerance were also elevated by ACP and FAC. Concomitantly, body weight increased by ACP supplementation and shown commensurate by FAC replacement as comparing with the corresponding control group further implied the promising effects exerted by ACP and FAC.

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018
As células cancerígenas são o resultado de um processo gradual e complexo chamado oncogénese. Durante este processo, as células normais transformam-se progressivamente em células cancerígenas através da acumulação de diversas alterações genéticas, que eventualmente culminam numa ou mais características definidas como ‘’hallmarks of cancer’’. Estas características foram definidas como propriedades essenciais ao desenvolvimento cancerígeno por Hanahan and Weinberg e correspondem a: sustentar sinalização proliferativa, escapar aos supressores tumorais, resistir à morte celular, possibilitar imortalidade replicativa, induzir angiogénese e ativar processos de invasão e metastastização. Este conjunto de características foi mais tarde alargado, considerando igualmente a importância da instabilidade genómica e da inflamação, bem como da reprogramação do metabolismo e do escape à vigilância imunitária no desenvolvimento dos processos tumorais. O carcinoma da tiroide é a neoplasia maligna mais frequente do sistema endócrino e a sua incidência tem vindo a aumentar ao longo dos últimos anos. De acordo com as suas características histológicas e morfológicas, o carcinoma da tiroide pode ser subdividido em quatro subtipos principais: carcinoma medular da tiroide (MTC, medullary thyroid cancer), carcinoma papilar da tiroide (PTC, papillary thyroid cancer), carcinoma folicular da tiroide (FTC, follicular thyroid cancer) e carcinoma anaplástico da tiroide (ATC, anaplastic thyroid cancer). Os subtipos PTC, FTC e ATC desenvolvem-se a partir das células epiteliais foliculares da glândula da tiroide, enquanto que o subtipo MTC deriva das células parafoliculares. Para além disso, dentro dos grupos que se desenvolvem a partir das células foliculares, os subtipos papilar e folicular são considerados carcinomas da tiroide bem diferenciados (WDTC, well-differentiated thyroid cancer), enquanto que o subtipo anaplástico corresponde a um tipo de carcinoma da tiroide indiferenciado. O carcinoma papilar da tiroide é o subtipo mais frequentemente diagnosticado, correspondendo a cerca de 80% dos casos dos carcinomas da tiroide. Normalmente, os doentes com estas formas apresentam um prognóstico favorável após remoção total ou parcial da glândula da tiroide e, quando se justifique, terapia com iodo radioativo. No entanto, existe um subconjunto de doentes que apresentam formas agressivas da doença, frequentemente associadas a resistência à radioterapia com iodo e para os quais não existem alternativas terapêuticas eficazes, sendo por isto essencial o desenvolvimento de novas estratégicas terapêuticas. As alterações genéticas mais frequentemente associadas ao carcinoma papilar da tiroide incluem mutações pontuais no gene BRAF ou rearranjos RET/PTC. Uma vez que estas alterações promovem a ativação constitutiva da via de sinalização MAPK (mitogen activated protein kinase), esta por sua vez é considerada essencial ao desenvolvimento do cancro da tiroide. Por outro lado, mutações pontuais no gene RAS também podem ser encontradas em doentes com o subtipo papilar. À semelhança dos rearranjos RET/PTC, as mutações em RAS têm a capacidade de ativar tanto a via de sinalização das MAPK, como a via PI3K/Akt/mTOR. Neste sentido, também a via de sinalização PI3K/Akt/mTOR tem vindo a ser considerada um elemento importante durante o desenvolvimento e progressão do cancro da tiroide. Sendo o carcinoma papilar da tiroide, um cancro que envolve frequentemente a ativação constitutiva da via MAPK, uma terapêutica dirigida à inibição da mesma poderia ser uma opção. No entanto, efeitos secundários indesejados associados ao uso de inibidores desta via, têm vindo a ser reportados em doentes com diferentes formas de carcinoma da tiroide, bem como o escape à terapêutica após longos períodos de tratamento. Desta forma, a compreensão dos mecanismos moleculares subjacentes à oncogénese do subtipo papilar e, em particular, da interação entre diferentes vias de sinalização implicadas, poderá ser uma mais valia no desenvolvimento de novas terapias dirigidas aos doentes com as variantes agressivas. A via de sinalização PI3K/Akt/mTOR é umas das vias mais estudadas no contexto da tumorigénese, devido ao seu papel determinante na proliferação e sobrevivência celular. No carcinoma da tiroide, mutações que afetam esta via costumam ser mais comuns nos tipos foliculares e anaplásticos. No entanto, pensa-se que esta via tem um papel importante na progressão de PTC para formas mais agressivas. Para além disso, como algumas das mutações associadas ao carcinoma papilar da tiroide também têm a capacidade de promover uma ativação da via de sinalização PI3K/Akt/mTOR, também esta via acaba por representar um alvo apelativo ao desenvolvimento de novas terapêuticas dirigidas, visando as formas agressivas. O NF-κB é um fator de transcrição, cuja desregulação pode facilmente promover condições favoráveis ao desenvolvimento cancerígeno, devido ao controlo que exerce sob diversas funções biológicas, tais como na inflamação ou em mecanismos associados à apoptose, crescimento e proliferação celular. No contexto do cancro da tiroide, este fator de transcrição tem sido descrito como um elemento envolvido na resistência à terapêutica, o que leva a suspeitar da presença de algum tipo de interação entre a via de sinalização do NF-κB e as vias de sinalização mais relevantes ao processo oncogénico da tiroide. De facto, uma relação entre a via de sinalização MAPK e a via canónica do NF-κB, foi já descrita por vários autores em diferentes modelos de carcinoma da tiroide, incluindo o subtipo papilar. No entanto, uma interação entre as vias NF-κB e PI3K/Akt/mTOR não se encontra ainda descrita no contexto das neoplasias da tiroide. O principal objetivo deste trabalho foi investigar esta interação em modelos celulares de carcinoma papilar da tiroide. Neste sentido, foram estabelecidas três abordagens experimentais que consistiam na avaliação da atividade do NF-κB: i) na presença de inibidores químicos da via PI3K/Akt/mTOR, ii) na presença de inibidores químicos da via PI3K/Akt/mTOR e com estimulação exógena da via canónica do NF-κB e iii) na presença combinada de inibidores químicos da via PI3K/Akt/mTOR e da via canónica do NF-κB. Os efeitos observados foram ainda comparados entre modelos celulares de PTC com diferentes contextos genéticos. A nível da análise da atividade transcricional do NF-κB, foi verificado um aumento da expressão de um alvo transcricional, em resposta à inibição química da via de sinalização PI3K/Akt/mTOR. Curiosamente, o mesmo não se verifica na presença de estimulação exógena da via canónica do NF-κB, onde a inibição da via PI3K/Akt/mTOR parece não ter impacto na atividade transcricional do NF-κB. Foi no entanto observada uma aparente inconsistência entre a avaliação da ativação de NF-κB com base na sua atividade transcricional e a avaliada através da análise da translocação nuclear da subunidade p65 deste fator de transcrição. Nesta última situação, os resultados indicam um decréscimo da translocação nuclear da subunidade p65 do NF-κB, em resposta à inibição da via de sinalização PI3K/Akt/mTOR. Este fenómeno ocorre tanto na ausência de estímulos exógenos da via canónica do NF-κB, como na presença dos mesmos. No seu conjunto, os resultados deste trabalho sugerem que a via de sinalização PI3K/Akt/mTOR poderá influenciar o estado de ativação do fator de transcrição NF-κB. No entanto, devido à aparente inconsistência entre a atividade transcricional e a translocação nuclear do NF-κB, não foi possível esclarecer se o resultado final do impacto da via de sinalização PI3K/Akt/mTOR no estado de ativação deste fator de transcrição é no sentido de inibir ou estimular a sua atividade. Assim, experiências futuras serão necessárias de forma a compreender e clarificar esta interação, bem como as suas implicações biológicas no contexto do cancro da tiroide. Compreender as possíveis interações entre diferentes vias de sinalização envolvidas na tumorigénese da tiroide será uma mais valia para o desenvolvimento e adequação de terapêuticas dirigidas, particularmente relevante na gestão de doentes com formas agressivas da doença.
Thyroid cancer is the most frequent endocrine malignancy and its incidence has been rising over the past few years. Accounting for more than 80% of the cases, the papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer. In general, PTC patients have a good prognosis after surgery which, in specific cases, is followed by radioiodine therapy. However, a subset of patients present advanced forms of the disease, with lesions that are frequently unresectable or unresponsive to radioiodine therapy. For these patients, no effective alternative treatment exists and new therapeutic options are needed in order to increase patients’ survival rate and lifespan. Throughout cancer development, several genetic changes occur that deregulate different signalling pathways controlling cancer survival, progression and invasion. The most common genetic alterations involved in papillary thyroid cancer include BRAFV600E point mutation and RET/PTC rearrangements, affecting positively the activity of the pro-tumorigenic MAPK pathway. Nonetheless, RET/PTC rearrangements can also activate the PI3K/Akt/mTOR pathway. Besides, RAS activating mutations have been detected in PTC patients and, similar to RET/PTC, can signal through both MAPK and PI3K/Akt/mTOR pathways. Thus, despite MAPK being considered the main signalling pathway involved in thyroid cancer oncogenesis, PI3K/Akt/mTOR can be expected to play an important role during this process. Therefore, targeting the PI3K/Akt/mTOR pathway becomes an attractive therapeutic option, also in the context of thyroid cancer. NF-κB transcription factor has been described as an important anti-apoptotic factor in thyroid carcinomas as well as being involved in acquired resistance to therapy. The interplay of NF-κB with both MAPK and PI3K/Akt/mTOR pathways has been described in several cancers. Considering that in thyroid carcinomas, an interplay between NF-κB and MAPK has been described it may also be relevant to analyse a possible crosstalk between NF-κB and PI3K/Akt/mTOR pathways. Thus, aiming to address this potential crosstalk, the impact of PI3K/Akt/mTOR in NF-κB activation status was analysed in PTC cellular models. NF-κB activity was evaluated in three different conditions: i) upon inhibition of PI3K signalling; ii) upon inhibition of PI3K signalling in the presence of exogenous stimulation of the NF-κB canonical pathway and iii) upon inhibition of both PI3K and NF-κB signalling. Altogether our results suggest the existence of a crosstalk between NF-κB and PI3K/Akt/mTOR signalling. However, whether PI3K/Akt/mTOR pathway exerts a positive or negative impact in the overall NF-κB activation status as well as the molecular mechanisms behind this interplay and its biological significance, require further clarification.

Dissertação de Mestrado, Oncobiologia, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016
A leucemia linfoblástica aguda de células T (LLA-T) é uma doença maligna agressiva, afetando principalmente crianças de 2 até 5 anos de idade. Esta doença manifesta-se no sangue e na medula óssea, podendo afetar vários órgãos do corpo, sendo fatal, na falta de um diagnóstico precoce e terapia apropriada. O RANKL é uma protéina membranar ou secretada que se demontrou ter capacidade estimuladora de metástases, através do aumento da motilidade celular de alguns tipos de células cancerosas. Este ligando tem a capacidade de ativar o recetor RANK e é expresso principalmente em timócitos e células T ativadas. O objetivo deste estudo foi de compreender o envolvimento da via de sinalização RANK/RANKL, na LLA-T. A estirpe de murganhos transgénicos TEL-JAK2, que desenvolve espontaneamente leucemia semelhante à LLA-T, foi utilizada para este estudo. Ao realizar RT-PCR quantitativo confirmámos que o gene que codifica a proteína RANKL é mais expresso em células leucémicas de murganhos transgénicos TEL-JAK2 que em timócitos normais. Para além disso, a análise da expressão do RANKL, por citometria de fluxo, em timócitos leucémicos TEL-JAK2, mostrou ser mais elevada no interior das células do que na sua superfície. Contrariamente, a linha celular de linfoma tímico EL4.2 apresentou expressão elevada de RANKL à superfície. A análise por citometria de fluxo a células colocadas em cultura com compostos indutores da via de sinalização do recetor de células T (TCR), mostrou que o forbol 12-miristato 13-acetato (PMA) e a Ionomicina em conjunto induzem um aumento da expressão do RANKL, tanto no interior como à superfície de células leucémicas TEL-JAK2, assim como à superfície de células EL4.2. A utilização de um inibidor da quinase IKK e o inibidor de transcrição, Actinomicina D, levaram à diminuição da indução do RANKL causada pelo tratamento com PMA e Ionomicina em células leucémicas TEL-JAK2. Neste estudo foram usados murganhos knockout condicionais para o gene Rank. Descobrimos que quando o recetor RANK está ausente de células epiteliais do timo, a expressão do RANKL aumenta em timócitos normais. Contudo, quando células leucémicas foram injetadas nesta estirpe, não se verificaram alterações significativas na expressão do RANKL naquelas células. Com estas experiências verificámos que a via de sinalização RANK/RANKL pode potencialmente contribuir para o desenvolvimento da LLA-T. No entanto, é necessário compreender como a possível interferência com o par recetor/ligando pode afetar o desenvolvimento da LLA-T antes de considerar a utilização desta via como potencial alvo terapêutico.