Les cavéoles sont des invaginations en forme de coupelle à la membrane plasmique. Ces organelles multifonctionnelles jouent entre autres, un rôle clé dans la mécano-protection et la signalisation cellulaire. En effet, les cavéoles ont la faculté de s’aplanir en réponse à l’augmentation de la tension membranaire, afin de protéger la cellule des contraintes mécaniques. Les cavéoles jouant un rôle clé dans la signalisation cellulaire, nous avions émis l’hypothèse que le cycle mécano-dépendent de désassemblage/réassemblage des cavéoles constitue un interrupteur mécanique de certaines voies de signalisation. Ce projet consiste à élucider le mécanisme moléculaire responsable du contrôle de la voie de signalisation JAK-STAT par la mécanique des cavéoles. Dans ces travaux, nous avons pu démontré que la cavéoline-1 (Cav1), un constitutant essentiel des cavéoles est libérée et devient hautement mobile au niveau de la membrane plasmique. Considérant les propriétés de signalisation de Cav1, Nous avons testé l’effet du désassemblage des cavéoles sur la signalisation cellulaire. Un criblage à haut débit, nous a permis identifié la voie de signalisation JAK- STAT stimulée par l’IFN-α comme voie modèle pour cette étude. En effet, la transduction du signal JAK-STAT induit par l’IFN-α est modulée par la mécanique des cavéoles. Afin de disséquer le mécanisme moléculaire responsable du contrôle de la signalisation JAK-STAT par la mécanique des cavéoles, nous avons déterminé le rôle de Cav1 dans ce contrôle. Nous avons observé que Cav1 est un régulateur négatif de la phosphorylation de STAT3 dépendante de la kinase JAK1. De plus, nous avons démontré que Cav1 interagit avec JAK1 en fonction de la tension membranaire. Nous avons également démontré que cette interaction Cav1-JAK1 fait intervenir le « scaffolding domain » de Cav1 (CSD), et que celui-ci est responsable de l’abolition de l’activité kinase de JAK1. Par conséquent, l’interaction de Cav1 avec JAK1 empêche l’activation de STAT3 par la kinase JAK1. Ces résultats démontrent que les cavéoles sont des organelles de mécano-signalisation, qui, lors d’un stress mécanique, libèrent de la Cav1 non cavéolaire capable d’inactiver la kinase JAK1, empêchant ainsi, la transduction du signal JAK-STAT. / Caveolae are small cup-shaped plasma membrane invaginations. These multifunctional organelles play a key role in cell mechanoprotection and cell signaling. Indeed our laboratory reported that caveolae have the ability to flatten out upon membrane tension increase, protecting cells from mechanical strains. Since caveolae play a key role in cell signaling we hypothesized that the mechano-dependent cycle of caveolae disassembly/reassembly may constitute a mechanical switch for signaling pathways. In this project, we elucidated the molecular mechanism underlying the control of JAK-STAT signaling by caveolae mechanics. We showed that caveolin-1 (Cav1), an essential caveolar component is released and become highly mobile at the plasma membrane under mechanical stress. Considering that caveolae are important signaling hubs at the plasma membrane, we addressed the effects of the mechanical release of Cav1 on cell signaling. Using high throughput screening, we identified the JAK-STAT signaling pathway as a candidate. To further dissect the molecular mechanism underlying the control of JAK-STAT signaling by caveolae mechanics, we addressed the role of Cav1 in the control of JAK-STAT signaling stimulated by IFN-α. We found that Cav1 was a specific negative regulator of the JAK1 dependent STAT3 phosphorylation. Furthermore, the level of Cav1 interaction with JAK1 depended on mechanical stress. We could show that Cav1-JAK1 interaction was mediated by the caveolin scaffolding domain (CSD), abolishing JAK1 kinase activity, hence, interfering with STAT3 activation upon IFN-α stimulation. Altogether our results show that caveolae are mechanosignaling organelles that disassemble under mechanical stress, releasing non-caveolar Cav1, which binds to the JAK1 kinase and inhibits its catalytic activity, preventing thereby JAK-STAT signal transduction.

No description available.

Le facteur d'activation plaquettaire (PAF) est un puissant médiateur pro-inflammatoire impliqué dans des processus physiologiques et pathologiques.Le PAF exerce ses effets suite à la liaison à son récepteur, le récepteur du PAF (PAFR), qui est un récepteur à sept domaines transmembranaires et couplé aux protéines G (RCPG). La signalisation du PAFR est en partie médiée par les protéines G et implique principalement des sous-unités G[indice inférieur [alpha]i] et G[indice inférieur [alpha]q] dans plusieurs types cellulaires.Le PAFR peut également activer des effecteurs variés : des canaux ioniques, des phospholipases (PLA[indice inférieur 2], PLC, PLD), ainsi que plusieurs kinases (PKC, PI3K et MAPK). Nous avons récemment démontré que le PAFR peut activer de façon indépendante des protéines G la voie des Janus kinase (JAK) et des Signal Transducers and Activators of Transcription (STAT). JAK2, TYK2 et STAT1, 2, 3 et 5 sont ainsi activés dans la lignée cellulaire myéloïde humaine MonoMac1. Les SOCS sont une famille de protéines qui régulent négativement la signalisation des cytokines et qui ont récemment été démontrés comme impliqués dans la signalisation de certains RCPG dont le CXCR4 (récepteur de chimiokine 4 de la famille des C-X-C) et l'AT1 (récepteur de l'angiotensine II). Une stimulation au PAF induit de manière transcriptionnelle l'accumulation de l'ARNm de la protéine suppressors of cytokine signalling 3 (SOCS3) dans les monocytes humains et la lignée cellulaire MonoMac1, mais ne l'induit pas dans les cellules endothéliales de veines de cordons ombilicaux humain (HUVEC). En plus d'une augmentation de l'ARNm de SOCS3, une augmentation de la protéine SOCS3 est également observée suivant une stimulation au PAF. Premièrement, nous voulions déterminer l'importance de SOCS3 dans la signalisation du PAFR à l'aide de différentes lignées cellulaires (HEK293, COS7, MonoMac1) et diverses techniques de biologie cellulaire. Ensuite, nous désirions évaluer l'impact des différents domaines de SOCS3 dans ces fonctions par des approches de biologie moléculaire. Finalement, nous avons évalué le/s rôle/s de SOCS3 sur différents aspects fonctionnels pro-inflammatoires du PAF (Voies signalisation, adhésion cellulaires, etc.). Nous démontrons dans la présente thèse que SOCS3 module la signalisation du PAFR en plus de présenter certains aspects moléculaires entourant la relation entre le PAFR, TYK2 et SOCS3. Les présents travaux démontrent que SOCS3 peut être recruté de façon transitoire à la seconde boucle intracellulaire et la queue cytoplasmique du PAFR. Son domaine kinase inhibitory region (KIR) semble être requis pour le recrutement induit au PAFR, alors que son domaine SOCS box semble être impliqué dans le recrutement basal. Suivant une stimulation au PAF, SOCS3 est phosphorylé sur un/des résidus tyrosine. Cette modification est sous le contrôle essentiel de TYK2, alors que son mutant K930I (kinase inactive) ne le fait pas. SOCS3 joue un rôle très important dans la modulation des voies de signalisation du PAFR ainsi que sur certains effets biologiques de celui-ci. Ces actions se révèlent être également très spécifiques : SOCS3 ne module pas la voie de G[indice inférieur [alpha]q] (IP3 ), mais module la migration et également l'adhésion cellulaire induite par le PAF. SOCS3 ne module pas la phosphorylation des STAT 1, 3 et 5 induite par le PAF, mais module négativement la voie de TYK2 (activation du promoteur du PAFR) et la phosphorylation des STAT 1, 3 et 5 induite par l'OSM. SOCS3 ne module pas la voie des JNK MAPK, prolonge la voie des ERK MAPK et module négativement l'activation précoce de la voie de p38 MAPK. Enfin, SOCS3 joue un rôle de modulation négative dans la transcription induite par le PAF des promoteurs du PAFR, de l'IL-6 et de l'IL-8. En conclusion, cette thèse propose de nouveaux mécanismes par lesquels SOCS3 module certains aspects de la signalisation et effets biologiques du PAF et de son PAFR. Comme le PAF est impliqué dans plusieurs phénomènes à caractères inflammatoires, le travail présenté ici a porté son attention sur plusieurs aspects pro-inflammatoires du PAF plutôt que sur une pathologie en particulier, ce qui permettra de mieux comprendre divers aspects liant le PAFR, la kinase TYK2 et SOCS3.

INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas. / BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.

Titel: JAK Medlemsbank och räntan – Ett räntefritt alternativ. Nivå: Kandidatnivå. Författare: Anna Amborn och Linda Elgestad. Handledare: Maria Fregidou-Malama. Datum: 2012-06-28. Syfte: Studien syftar till att undersöka och analysera JAK Medlemsbanks syn på det ekonomiska systemet i allmänhet och räntan i synnerhet, samt undersöka om det går att låna pengar utan ränta. Vi belyser också att det finns dolda räntekostnader i det vi konsumerar.Metod: Metoden som använts är en kvalitativ metod och data har främst samlats in genom tre ostrukturerade intervjuer med anställda och engagerade personer inom JAK Medlemsbank. Material till teoriavsnittet har samlats in från databaser, artiklar och böcker. Resultat & slutsats: Studien visar att JAK Medlemsbank är vad den utger sig för att vara, alltså en räntefri bank. Dock är detta beroende av att ränta definieras som en arbetsfri inkomst dvs. en inkomst som inte täcker några verkliga kostnader. Studien visar att ett lån i JAK Medlemsbank ger en relativt hög månadskostnad vilket också gör att det krävs en hög månadsinkomst för att låna ett större belopp. Förslag till fortsattforskning: Det räntefria alternativet är ett relativt outforskat område vilket ger stora möjligheter till fortsatt forskning. Ett av dessa outforskade områden är räntans andel av priset på en vara. För att utforska detta område anser vi att det krävs mer tid då många företag verkat motvilliga att lämna ut sådan information. Uppsatsens bidrag: Studien har bidragit till att samla in information om JAK Medlemsbank, deras ideologi och verksamhet. Den bidrar också till att redogöra för att det finns ett räntefritt alternativ till de konventionella bankerna samt beskriver hur detta alternativ fungerar. Studien ger således en övergripande bild av JAK’s verksamhet samt belyser de nackdelar som räntan medför. Nyckelord: Räntefri, JAK Medlemsbank, ränta, avgift, lånekostnad, kooperativ. / Title: JAK Members bank and the interest rate – An interest-free alternative. Level: Final assignment for Bachelor Degree in Business Administration. Author: Anna Amborn och Linda Elgestad. Supervisor: Maria Fregidou-Malama. Date: 2012-06-06. Aim: The study aims to investigate and analyze the JAK Members bank view of the economic system in general and the interest rate in particular, and examine if it is possible to borrow money without interest. We also highlight that there is a hidden interest rate in what we consume. Method: The method used is a qualitative method and the data was mainly gathered through three unstructured interviews with employees and involved people in the JAK Members bank. Materials for the theory section were collected from databases, articles and books. Result & Conclusions: The study suggests that JAK Members bank is what they claim to be, that is an interest-free bank. However, this depends on that the interest rate is defined as a work-free income i.e. an income that does not cover real costs. The study also shows that a loan in JAK Members bank will give a relatively high monthly fee which leads to a pretty high required income from the borrower. Suggestions for future research: The interest-free option is a relatively unexplored area which provides significant opportunities for further research. One of those unexplored areas is the interest share of the price of goods. To explore this area, we believe that it takes time because many companies seemed reluctant to disclose such information. Contribution of the thesis: The study has helped to gather information about JAK Members bank, their ideology and activities. It also helps to explain that there is an interest-free alternative to the conventional banks, and describes how this option works. The study thus provides an overall picture of the JAK Bank´s activities and highlights the disadvantages that interest rate brings. Keywords: Interest-free, JAK Members bank, interest rate, fees, loan costs, cooperatives.

The Janus kinase (JAK) pathway is an integral part of signaling through a variety of ligands and receptors in mammals. The extensive reutilization and pleiotropy of this pathway in vertebrate development is conserved in other animals as well. In Drosophila melanogaster, JAK signaling is involved in embryonic pattern formation, sex determination, larval blood cell development, wing venation, planar polarity in the eye, and formation of other adult structures. Here we describe several roles for JAK signaling in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired, is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. A primary defect of chambers with reduced JAK activity is fusion of successive chambers. These chambers exhibit an expansion of the polar cell population and concomitant loss of interfollicular stalk cells. Mosaic analysis of both JAK pathway transducers, hopscotch and stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Another role of JAK signaling is in oocyte localization. In chambers mosaic for loss of hop activity, oocyte mislocalization results. Proper localization occurs only when the posterior follicle cells are wild type for hop.

Submitted by Augusto Santana Nascimento (nascimento@aluno.ibb.unesp.br) on 2017-01-19T12:49:36Z No. of bitstreams: 1 Dissertacao Augusto.docx: 2881032 bytes, checksum: c87b3b303b6f8075ca2bde19b2863849 (MD5) / Rejected by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: A versão final da dissertação/tese deve ser submetida no formato PDF (Portable Document Format). O arquivo PDF não deve estar protegido e a dissertação/tese deve estar em um único arquivo, inclusive os apêndices e anexos, se houver. Por favor, corrija o formato do arquivo e realize uma nova submissão. Agradecemos a compreensão. on 2017-01-20T11:53:55Z (GMT) / Submitted by Augusto Santana Nascimento (nascimento@aluno.ibb.unesp.br) on 2017-01-23T17:19:59Z No. of bitstreams: 1 Dissertacao Augusto.pdf: 2309354 bytes, checksum: a8372ec1b075dc9d75fd293868f6988a (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-01-25T11:40:26Z (GMT) No. of bitstreams: 1 nascimento_as_me_bot.pdf: 2309354 bytes, checksum: a8372ec1b075dc9d75fd293868f6988a (MD5) / Made available in DSpace on 2017-01-25T11:40:26Z (GMT). No. of bitstreams: 1 nascimento_as_me_bot.pdf: 2309354 bytes, checksum: a8372ec1b075dc9d75fd293868f6988a (MD5) Previous issue date: 2016-11-18 / Embora algum progresso tenha sido alcançado nos últimos anos, ainda são necessários estudos capazes de desvendar os mecanismos moleculares envolvidos com o fenótipo de resistência a múltiplas drogas (MDR) em células tumorais. Com esta finalidade, estabelecemos o perfil quinômico (através do microarranjo de peptídeos, PepChip) das linhagens MCF7 e MCF7Res, fenótipo parental e resistente respectivamente, de células de câncer de mama. Os resultados obtidos pelo microarranjo de peptídeos e posteriormente, validados por western blotting, apontaram o envolvimento da via de sinalização Jak-Stat e isoformas de PKC no processo de resistência das células de câncer de mama. Além disso, mostramos envolvimento de p42/44-mapk, Ras e um aumento na expressão de MMP-9. Estes resultados mostram o potencial agressivo destas células resistentes, visto que estas vias estão envolvidas em mecanismos responsáveis pela proliferação e invasão celular. Como as proteínas Jak1 e Jak2 mostraram-se envolvidas, decidimos avaliar níveis de fosforilação de Stats 1, 2, 3, 5 e 6 e mostramos que todas estavam up-fosforiladas nas células resistentes. Baseado nestes resultados, decidimos avaliar através de um ensaio funcional, o papel de Jak2 no fenótipo resistente e, desta forma, avaliamos a viabilidade das células MCF7Res em pré-tratamento com 2 concentrações subtóxicas do inibidor de Jak2 (5µM e 10µM) e nossos resultados claramente mostraram que, inibindo Jak2, as células MCF7Res ficam mais sensíveis a daunorrubicina, aumentando a taxa de morte celular frente à resposta ao quimioterápico. Baseado nos resultados obtidos pelo fosfoproteoma concluímos que o fenótipo MDR envolve metabolismo específico em células tumorais de mama, onde isoformas de PKCs e sinalização Jak-Stat exercem função de destaque. Assim, estes dados apontam o potencial uso de inibidores de Jak2 como estratégia para o tratamento de pacientes não responsivos a terapias convencionais.

Made available in DSpace on 2014-06-12T15:07:01Z (GMT). No. of bitstreams: 2 arquivo3001_1.pdf: 1265385 bytes, checksum: 195917f7eadaa711ca792e1630574a84 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco / Um dos determinantes envolvidos no complexo ciclo de transmissão da dengue é o nível de susceptibilidade do Aedes aegypti ao vírus dengue (DENV), ou seja, a competência vetorial, que varia entre populações de mosquitos. Identificar moléculas envolvidas na interação mosquito-vírus pode auxiliar no conhecimento dos mecanismos envolvidos na competência vetorial, até então pouco elucidados. Estudos recentes mostraram a participação de certos mecanismos na interação mosquito-DENV, porém, pouco se sabe do real papel destes na modulação da competência vetorial em mosquitos de campo ou até da relação entre eles. Mediante isso, objetivamos analisar a expressão de três moléculas representantes de diferentes mecanismos de defesa antiviral no Ae. aegypti, em resposta à infecção com vírus dengue sorotipo 2 (DENV-2), sendo elas REL1, HOP e Dicer-2, em populações de campo e de laboratório do mosquito. Para isso, as diferentes linhagens foram artificialmente infectadas com DENV-2, e tecidos variados foram coletados em diversos momentos após infecção. Tanto a quantificação viral quanto a expressão das moléculas selecionadas nas amostras foram realizadas por PCR em tempo real quantitativo (qRT-PCR). Os resultados mostraram que tanto o padrão de infecção viral quanto a expressão das moléculas variaram entre as populações de A. aegypti nos diferentes momentos após infecção com DENV-2. Os resultados aqui obtidos poderão ser bastante relevantes na pesquisa da interação vetor-vírus e poderão auxiliar no desenvolvimento de novas estratégias de controle da dengue, como na pesquisa com mosquitos transgênicos

INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas. / BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.

Asthma is a significant lung disease involving chronic inflammation and remodeling of the airways, resulting in reduced quality of life for those who suffer from the condition. Current therapeutic guidelines suggest the use of inhaled corticosteroids for long-term anti-inflammatory relief to manage moderate to severe chronic asthma; however, inhaled corticosteroids fail to provide prophylactic or reversal treatment of damaged airways incurred by chronic asthma as well as exhibiting adverse side effects (skeletal complications, diabetes, and weight gain).Therefore, there is a need for a new type of drug therapy to address these gaps in the treatment of chronic asthma. There is growing interest aimed towards the inhibition of the Janus Kinase and Signal Transducer and Activator of Transcription (JAK-STAT) pathway for the treatment of asthma. Despite the promising opportunity to investigate this new pathway towards this clinical application, no published work is available using an established and characterized JAK 1/3 inhibitor for the treatment of chronic asthma delivered via inhalation. This work investigated tofacitinib citrate, a selective JAK 3 inhibitor, and its potential to be delivered locally to the lungs for the treatment of chronic asthma. Several preformulation studies were conducted to determine the basic physical and chemical properties of the compound and its free base, tofacitinib, for proper inhalational formulation development. The drug was delivered to BALB/c mice challenged with house dust mite (HDM) allergen via nebulization utilizing a nose-only chamber. After a three week dosing schedule, mice treated with tofacitinib citrate exhibited an increase in monocyte cell numbers with a simultaneous decrease in eosinophil cell count, gathered from BAL fluid. Further, the experimental groups treated with tofacitinib citrate had a decrease in total protein concentrations in comparison to the experimental groups that were only challenged with HDM or were both exposed to HDM and vehicle. These findings demonstrated that the proper formulation was developed for nebulized delivery of tofacitinib citrate, and that the compound was capable of reducing total protein concentrations and eosinophil cell recruitment, both recognized as biomarkers for an asthmatic response. Although significant work is still needed to be done, these data hold promise for the potential of a locally delivered JAK 3 inhibitor as a treatment for chronic asthma. Further, the solubility of tofacitinib citrate and five other pharmaceutical salts were determined in HFA 134a, HFA 227, and DFP with varying cosolvent content (0-20% v/v ethanol). The experimental solubilities of the free acid and base compounds were larger than the solubilities of their respective salts in all three systems for tofacitinib, albuterol, and salicylic acid. Warfarin, phenytoin, and ciprofloxacin had similar solubilities with their respective salt forms. Solubilities also increased with increasing cosolvent concentration for all compounds investigated. The model propellant, DFP, provided a slightly stronger correlation of solubility values with HFA 134a in comparison to HFA 227. The observed solubility values were also compared to calculated values obtained from the ideal solubility model, where it was determined that the observed solubility was indeed also dependent on its surrounding solvent interactions and not solely on its ideal solubility (melting point). While some physical changes were observed for the pharmaceutical salts in HFA 134a and 227, more quantitative studies are needed for a larger database of compounds to better understand the factors that contribute to the solubility of pharmaceutical salts (and their correlation to DFP), in HFA-based systems. This information could potentially contribute to a predictive model, saving time and money during the process of pMDI formulation development.