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In many tumors, persistently-active signal transducer and activator of transcription 3 (STAT3) imparts several oncogenic features such as survival, proliferation, angiogenesis, and immune escape. Therefore, STAT3 targeting in cancer and cancer-exposed dendritic cells (DCs) is important for cancer therapy. Our objective is developing delivery modalities of STAT3-targeting small interfering RNA (siRNA) using lipid-modified polyethylenimine (PEI) polyplexes and poly(D,L lactic-co-glycolic) acid (PLGA) nanoparticles (NPs), and evaluating the therapeutic outcomes in vitro and in vivo. Significant increase in siRNA condensation, protection, and cellular uptake by B16.F10 melanoma was seen by stearic-acid-modified PEI (PEI-StA) compared to unmodified PEI. Moreover, PEI-StA increased the STAT3 silencing potency of siRNA compared to PEI. STAT3 knockdown was accompanied with significant induction of interleukin-6 (IL-6) secretion and reduction of vascular endothelial growth factor (VEGF) production and cytotoxicity evidenced by increased Caspase 3 activity in vitro and in vivo, and significant inhibition in tumor growth. Analysis of tumor microenvironment showed CD3+ cells infiltration corresponding to STAT3 knockdown. The levels of CD4+ helper cells, CD8+ cytotoxic cells, and NKT cells significantly increased. DC infiltration and activation significantly increased in tumor mass following STAT3 knockdown as evidenced by high expression of CD86 and CD40. Moreover, IFN-, IL-12, and TNF- significantly increased following STAT3 knockdown by PEI-StA compared to PEI, suggesting Th1-type immunity. Allogenic capacity of DCs isolated from siRNA-treated mice was evidenced by the high T cell proliferation and IL-2 production in mixed lymphocytes reaction (MLR). Then, we explored STAT3 knockdown in DCs exposed to tumor derived factors (TDFs). We investigated encapsulation of siRNA complexes (PEI or PEI-StA) into PLGA NPs (PLGA-P and PLGA-PS). PLGA-P and PLGA-PS had an average diameter of ~ 370 nm and zeta potential of ~ -16 mV. Uptake and endosomal localization was confirmed. After TDFs exposure, DCs showed high STAT3 and low CD86 expression. STAT3 silencing by PLGA-P and PLGA-PS restored DC functionality as evidenced by upregulation of CD86, IL-12, and TNF- and MLR activity. PLGA significantly reduced PEI-associated toxicity. Therefore, STAT3 targeting in B16 cells by siRNA polyplexes of PEI and PEI-StA, or in DCs by PLGA-P and PLGA-PS provide potential strategies for cancer therapy. / Pharmaceutical Sciences

Universiẗat, Diss., 2005--Giessen.

Titel: Stat i världen – En studie av Islamiska staten i förhållande till begreppet stat  Kurs: Statsvetenskap C, Mittuniversitetet, vårterminen 2015 Författare: Zophie Lif Nyckelord: Statsvetenskap, Islamiska staten, stat, begreppsdefinition, kvalitativ metod Bakgrund Islamiska staten är en av flera islamistiska rörelser som på senare tid vunnit stora politiska och territoriella framgångar. Organisationen utmärker sig genom att göra anspråk på ett världsomspännande kalifat som ska omfatta hela världens muslimska befolkning.I västvärlden har Islamiska staten främst uppmärksammat för sitt extrema våld, medan de i Mellanöstern av många grupper uppfattas som möjliga makthavare. Syfte Uppsatsens syfte är undersöka om och i vilken utsträckning Islamiska staten teoretiskt kan definieras som en stat. Undersökningen har gjorts med utgångspunkt i frågeställningen "Om och i vilken utsträckning kan Islamiska staten i teoretisk mening definieras som en stat?" Metod Undersökningen är en kvalitativ textanalys, där Islamiska staten prövas mot begreppet stat såsom det definieras av Max Weber. Det empiriska materialet analyseras utifrån en kategorisering av Webers teori. Resultatet har visat att Islamiska staten delvis, om än inte fullt ut, uppfyller Webers kriterier för en stat. Slutsats Studien visar att Islamiska staten, helt eller delvis, uppfyller vissa av de preciserade kriterier som definierar en stat. Utifrån studiens resultat dras slutsatsen att Islamiska staten av idag inte i tillräcklig utsträckning uppfyller villkoren för en stat, men samtidigt att det inte går att utesluta att organisationen en dag kommer att uppnå det målet. / <p>2015-06-03</p>

Cisplatin is a widely used chemotherapeutic drug for the treatment of solid tumors. However, the drug accumulates in the cochlea, and damages inner ear structures, resulting in bilateral andpermanent hearing loss. Previous data from our laboratory indicate that activation of the transient receptor potential vanilloid 1 (TRPV1) receptor (by capsaicin) increases the NOX3 isoform of NADPH oxidase, leading to the generation of reactive oxygen species (ROS) in the cochlea, transient cochlear inflammation and transient hearing loss. We also demonstrated that the transient inflammation was produced by ROS-mediated activation of signal transducer and activator of transcription 1 (STAT1). Surprisingly, over time, this response desensitizes and capsaicin was subsequently able to protect against cisplatin ototoxicity. The goal of this study was to determine the mechanism of otoprotection against cisplatin ototoxicity following the administration of capsaicin. For this study we utilize both an immortalized organ of Corti outer hair cells and rat cochlea. Capsaicin (2.5 µM) increased both Ser727 p-STAT1 and Tyr705 p-STAT3 implicating its role in inflammation. Expression of cannabinoid receptors were observed in UB/OC-1 cells as well as rat outer hair cells (OHCs). However, inhibition of CB2 receptors (by AM630) reduced capsaicin-mediated Tyr705 p-STAT3, but had little effect on Ser727 STAT1. Capsaicin protected UB/OC-1 cells against cisplatin-induced apoptosis. This protection was reversed by CB2 antagonist but potentiated by TRPV1 inhibition. Significant cell death was observed following treatment of UB/OC-1 cells with AM630 alone, underscoring the importance of CB2 receptors in survival of these cells. CB2 agonist, JWH, significantly increased the protective signal, STAT3. Furthermore, capsaicin-mediated protection was reversed by the inhibition of STAT3, implicating STAT3 in otoprotection. In animal studies, oral administration of capsaicin (0.5% solution) induced transient inflammation but led to a long term recovery. Animals pre-treated with oral capsaicin were protected against cisplatin-induced hearing loss as compared to vehicle-treated animals, suggesting protection against hearing loss. Capsaicin increased the expression of both CB1 and CB2 receptors in the organ of Corti, which might confer the long term protective actions of this agent against hearing loss. In rats pretreated with AM630, the protective action of capsaicin was abolished. We conclude that otoprotection mediated by capsaicin is produced by activation of CB receptors in the cochlea which are coupled to both STAT1 and STAT3 activation. However, our data support the conclusion that activation of STAT3 confers the otoprotective action of capsaicin. In contrast, activation of STAT1 by capsaicin could contribute to the transient inflammatory response previously observed in vivo. The net protective action of capsaicin could result from an increase in the STAT3/STAT1 ratio of cells in the cochlea, which antagonizes the ability of cisplatin lower this ratio and promote cell death.

Cisplatin is a commonly used chemotherapeutic agent for multiple solid tumors. However, cisplatin-induced neurotoxicity, nephrotoxicity and hearing loss hamper its use in clinical setting. Although, neurotoxicity and nephrotoxicity can be prevented, there is no cure for cisplatin-induced hearing loss. Cisplatin-induced hearing loss results from damage to outer hair cells (OHCs) in basal turn of the cochlea, to spiral ganglion neurons (SGN), stria vascularis (SV) and fibrocytes of spiral ligament (SL). At the cellular level, cisplatin produces profound increases in reactive oxygen species (ROS) that stimulate cell signaling pathways leading to cochlear inflammation, apoptosis and permanent hearing loss. Thus, potential otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatin chemotherapeutic efficacy. In this study, I characterized the otoprotective actions of the green tea extract, epigallocatechin 3-gallate (EGCG), which possesses anti-oxidant, anti-inflammatory and anti-tumor properties. Oral administration of EGCG to male Wistar rats reduced cisplatin-induced hearing loss, assessed by auditory brainstem responses. These changes were associated with a reduction in cisplatin-induced loss of OHCs primarily in the basal region of the cochlea, along with reduced oxidative stress, inflammatory and apoptotic markers. In addition, EGCG protected against cisplatin-induced decrease in inner hair cell (IHCs) ribbon synapses, labeled with CtBP2. EGCG also protected against cisplatin-induced loss of Na+/K+ ATPase α1 immunoreactivity in the stria vascularis and spiral ligament. In vitro studies using University Bristol/Organ of Corti-1 (UB/OC-1) cells showed that EGCG reduced cisplatin-induced ROS generation and the activation of ERK and STAT1, while it preserved the activity of STAT3 and levels of Bcl-xL. Moreover, EGCG suppressed oxidative stress, inflammatory and apoptotic markers in cisplatin-treated UB/OC-1 cells. Co-administration of EGCG did not alter cisplatin-induced apoptosis of human-derived head and neck cancer cells, ovarian cancer cells or colon cancer cells. In studies using a xenograft model of head and neck cancer in severe combined immunodeficient (SCID) mice, I showed that EGCG did not interfere with cisplatin chemotherapeutic efficacy. These data suggest that EGCG is a potential otoprotective agent for treating cisplatin-induced hearing loss without compromising its chemotherapeutic efficacy.

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

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.

Epilepsy, a disease characterized by recurring spontaneous seizures, affects over 65 million people, 2% of the world’s population. Over 30% of patients are refractory to all current medical therapy, and for those that can be treated, many suffer from severe drug side-effects. Understanding the molecular basis of epilepsy is vital to the advancement of better therapeutic options and an eventual cure. Upregulation of brain-derived neurotrophic factor (BDNF) is highly associated with epileptogenesis in human patients, as well as animal models. Our laboratory discovered that BDNF induces the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway in neurons and that inhibition attenuates spontaneous seizures in a temporal lobe epilepsy model. The mechanism behind JAK/STAT signaling in neurons and its relationship to epilepsy still remains to be elucidated and is the subject of my thesis. Surprisingly, even though BDNF is such a major signaling molecule, its full genomic impact has never been assessed. We conducted a high-density RNA-sequencing analysis of the BDNF transcriptome in cortical neurons and probed such regulation with selective JAK inhibitors. Results suggest that 68% of BDNF-induced changes in gene expression implicated in epilepsy are regulated by JAK/STAT signaling. Eighty percent of BDNF-induced changes coding for proteins involved in synaptic neurotransmission (receptor subunits and ion channels) involve JAK/STATs. Additionally, these datasets include genes that have never been associated with BDNF regulation (such as Dopamine Receptor D5 and Galanin Receptor 1). Most interestingly, the datasets reveal that BDNF-induced JAK/STAT signaling in neurons is non-canonical, as STAT3 phosphorylation at tyrosine 705 is not required for action. To directly examine STAT3’s role in epileptogenesis, we studied the transcriptome of transgenic mice that express lower levels of STAT3 specifically in neurons. Using the intrahippocampal kainic-acid (KA) model of epilepsy, our datasets suggest that STAT3 knockdown in vivo, and selectively in neurons, protects mice from KA-induced dysregulation of the sphingolipid metabolism pathway that is associated with the trafficking, sorting, and stability of membrane-bound proteins, including neurotransmitter receptors and ion channels. Finally, we discuss a model for JAK/STAT signaling in neurons that includes structural aspects of an intracellular BDNF receptor (p75NTR) associated with JAK2. / 2021-06-14T00:00:00Z