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Younis, Usir, and Usir Younis. "Inhalational Delivery of a JAK3 Inhibitor for the Novel Treatment of Asthma and the Investigation of Pharmaceutical Salts in HFA Propellant Systems." Diss., The University of Arizona, 2018. http://hdl.handle.net/10150/626756.
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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.
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Torikoshi, Kazuo. "Protein inhibitor of activated STAT, PIASy regulates α-smooth muscle actin expression by interacting with E12 in mesangial cells." Kyoto University, 2013. http://hdl.handle.net/2433/174820.
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Martin, Nadine. "Rôle de la SUMO E3 ligase PIASy dans les mécanismes de contrôle de la prolifération cellulaire et de réponse aux dommages." Paris 6, 2007. http://www.theses.fr/2007PA066243.
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La modification des protéines par SUMO joue un rôle important dans de nombreuses fonctions cellulaires. Le travail présenté dans cette thèse vise à analyser l'action des protéines PIAS, régulateurs transcriptionnels et SUMO E3 ligases, et en particulier de PIASy dans le contexte de l'oncogenèse. Nous avons montré que PIASy induit la sénescence des fibroblastes primaires, en activant les voies Rb et p53, ou l'apoptose si la voie Rb est déficiente. Nous avons parallèlement identifié de nouveaux partenaires protéiques de PIASy. PIASy induit l'accumulation de FIP200 dans le noyau, ce qui inhibe l'action de FIP200 dans la voie mTOR. FIP200 coopère avec PIASy pour activer l'expression du gène p21. Par ailleurs, la modification de PARP-1 par SUMO stimulée par PIASy régule la réponse au choc thermique. Ainsi, ce travail a mis en évidence un rôle important de PIASy dans le contrôle de la prolifération cellulaire et la réponse aux dommages, et a précisé son action au niveau moléculaire
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Aubert-Jürgens, Ana. "STAT3 inhibitors for cancer treatment." [S.l.] : [s.n.], 2005. http://elib.tu-darmstadt.de/diss/000563.
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Ricard, Laure. "Les lymphocytes T folliculaires auxiliaires dans la sclérodermie systémique." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS340.
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La sclérodermie systémique (SSc) est une maladie auto-immune, caractérisée par une atteinte microvasculaire oblitérante, de la fibrose et des anomalies de l'immunité humorale. Les lymphocytes T folliculaires auxiliaires (Tfh) CD4+CXCR5+PD1+ coopèrent avec les lymphocytes B pour induire leur différenciation en plasmocytes sécréteurs d’immunoglobulines (Ig). Les Tfh circulants (cTfh) sont augmentés dans plusieurs maladies auto-immunes et les Tfh infiltrent la peau des patients SSc. Nous avons observé que les cTfh des patients SSc étaient augmentés en comparaison avec les témoins et notamment dans des formes sévères de SSc. Les cTfh des patients SSc ont un phénotype activé avec une forte expression de BCL-6 et des capacités augmentées à produire de l’IL-21. In vitro, les cTfh de patients SSc sont aussi plus efficaces pour stimuler la différenciation des plasmablastes (PB) ainsi que leur production d’Ig. Le blocage de l’IL-21 ainsi que le ruxolitinib, un inhibiteur de la voie JAK1/2 diminuaient la capacité des cTfh à stimuler la différenciation des PB et leur sécrétion d’Ig. Les mécanismes à l’origine de cette expansion aberrante des cTfh dans la SSc demeurent à définir. Les cellules dendritiques, les monocytes ainsi que des anomalies épigénétiques pourraient être à l’origine de cette expansion des cTfh. L’hématopoïèse clonale (HC) est définie par l’acquisition de mutations somatiques dans les cellules souches hématopoïétiques menant à des clones détectables. Nous avons observé une forte prévalence d’HC dans la SSc. Le gène le plus fréquemment muté était DNMT3A impliqué dans la régulation épigénétique. Le rôle de ces mutations dans la physiopathologie de la SSc reste à démontrerSystemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis, vascular microangiopathy and deregulated immune system with the presence of autoantibodies. Follicular helper T (Tfh) cells, defined as CD4+CXCR5+PD-1+ cooperate with B lymphocytes to induce the differentiation of plasmocytes secreting immunoglobulins (Ig). Circulating Tfh (cTfh) cells are increased in several autoimmune diseases, and Tfh cells can infiltrate the skin of SSc patients. We demonstrate that cTfh cell are increased in SSc patients compared with healthy controls (HC), which was more potent in severe forms of SSc. cTfh cells from SSc patients present an activated Tfh phenotype, with high expression of BCL-6 and increased capacity to produce IL-21 in comparison to HC. In vitro, cTfh cells from SSc patients had higher capacity to stimulate the differentiation of plasmablasts and their secretion of Ig through the IL-21 pathway. Blocking IL-21 or using the JAK1/2 inhibitor (ruxolitinib) reduced the Tfh cells’ capacity to stimulate the plasmablasts and decreased the Ig production. Mechanisms leading to this aberrant cTfh expansion remain to be established. Monocytes and dendritic cells could participate to this cTfh expansion. Epigenetics abnormalities could also contribute to cTfh activation in SSc. Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the acquisition of somatic mutations in hematopoietic stem cells leading to detectable clones. We observed a high prevalence of CHIP in SSc patients. The most common mutation occurred in DNMT3A gene involved in epigenetic regulation. The implication of these mutations in SSc pathophysiology remains to be demonstrated
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Ball, Sarah Lynnette. "Small molecule inhibitors, LLL12 and celecoxib, effectively inhibit STAT3 phosphorylation, decrease cellular viability and induce apoptosis in medulloblastoma and glioblastoma cell lines." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1298906960.
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Hill, Jacqueline M. "Transition state analogues as inhibitors of metallo-proteases." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260112.
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Fisher, Michael I. "Transition state analogue inhibitors of the aspartyl proteases." Thesis, University of Huddersfield, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363233.
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Ekegren, Jenny. "Design and Synthesis of Novel HIV-1 Protease Inhibitors Comprising a Tertiary Alcohol in the Transition-State Mimic." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6737.
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Ghafoory, Shima. "Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer." Thesis, Mälardalen University, Mälardalen University, School of Sustainable Development of Society and Technology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-7797.
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Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers.
Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells.
Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer.
Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors.
The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection.
At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project.
Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer
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Bhasin, Deepak. "Small Molecule Inhibitors asAnticancer Agents." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1305826098.
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Gonzalez, Palmén Lorena. "Homotrimeric dUTPases : Principles of Catalysis and Inhibitor Design." Doctoral thesis, Högskolan i Kalmar, Naturvetenskapliga institutionen, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-6119.
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The ubiquitous enzyme dUTPase hydrolyzes dUTP into dUMP and pyrophosphate, preventing DNA fragmentation and cell death due to accumulation of dUTP. Inhibitors of dUTPase could serve as drugs in the treatment of cancers and infectious diseases. This thesis presents five studies. A mutational study on the Escherichia coli dUTPase (S72A) provides new insights about the catalytic principles of the homotrimeric dUTPases. A model is presented in which transition state formation is associated with a rotation of the conserved Ser72 side chain. The model can explain the strict order of deamination and hydrolysis catalyzed by the bifunctional dCTP deaminase:dUTPases. The S72A/D90N double mutant is currently investigated. Preliminary data indicate that this form preserves the binding properties of the S72A mutant but is completely inactive, making it attractive for structural studies. In the remaining studies we compare the binding of substrate analogues to the human, the E. coli and the equine infectious anemia virus (EIAV) homotrimeric dUTPases. One study concerns 2´,3´-dideoxy-UTP (ddUTP) and shows that removal of the 3´-hydroxyl group increases KM, ten times with the cellular dUTPases and fifty times with the viral dUTPase, but does not affect kcat with any of these enzymes. Another study concerns the inhibitory effects of 3´-azido-2´,3´-dideoxy-UTP. This derivative binds to the bacterial dUTPase but not to the other forms making it a potential lead for the development of antibacterial dUTPase inhibitors. Yet another study investigates two uracil derivatives. Both compounds are found to inhibit the human, the bacterial but not the viral dUTPase. The inhibition is shown to be competitive.
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Haque, Mohammad Rashedul. "Novel STAT3 small-molecule inhibitors as potential anticancer agents." Thesis, University of London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.535504.
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Mautsa, Nicodemus. "Structural and functional characterisation of the protein inhibitor of activated STAT3 (PIAS3)." Thesis, Rhodes University, 2011. http://hdl.handle.net/10962/d1004050.
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The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.
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Singh, Danny Ravinder. "Phosphorus containing transition state analogue inhibitors of the aspartyl proteases." Thesis, University of Huddersfield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368303.
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Chamberlain, Christopher Daniel. "Development and validation of assays used to evaluate STAT3 inhibitors." Thesis, King's College London (University of London), 2017. https://kclpure.kcl.ac.uk/portal/en/theses/development-and-validation-of-assays-used-to-evaluate-stat3-inhibitors(007f8426-c173-4802-955e-181bf9aec424).html.
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Transcription factors are important control proteins in cells that bind to their cognate DNA sequences in the promoter regions of genes, either up-regulating or down-regulating protein expression. In many cancer types, transcription factors are up-regulated and promote the expression of genes important in survival and metastasis. For this reason, transcription factors are good targets for novel anticancer agents. The STAT family of transcription factors (seven are now acknowledged) recognize and bind to a ~10 base pair sequence of DNA in the promoter region of a number of genes, enhancing the expression of oncogenic proteins such as Survivin, Cyclin D1, Bcl-2 and VEGF. There are currently no small-molecule STAT3 inhibitors in clinical use, so there is a need for the development of assays that can be used to screen molecules to identify lead compounds. The main focus of this project has been to develop an in vitro homogenous time resolved FRET (HTRF) assay that can be used in low-, medium- and high-throughput modes for the discovery of novel inhibitors. The project started with the cloning, production and purification of recombinant STAT3βTC, which is a homodimeric protein. This was challenging and time-consuming as initial solubility and stability issues were encountered. However, experimental conditions were eventually established that allowed useful quantities (i.e.10 mg batches) of purified and stable protein to be obtained. As part of the optimization process, the STAT3βTC was re-cloned into a HIS-Tag vector which facilitated purification using affinity (Ni2+) chromatography along with size exclusion chromatography to produce pure monomeric STAT3βTC. This could be dimerised to provide pure STAT3βTC homodimer. The pure protein was used to develop a HTRF assay by first labelling the STAT3βTC with Europium. Next, the cognate DNA recognition sequence in the form of an 18-mer duplex oligonucleotide was biotinylated and joined to the second fluorophore label (D2) via a streptavidin linkage. The strength of the FRET signal between these two components could then be used to measure the interaction between them. As part of a multi-well system, this could then be used to screen for small molecules capable of disrupting the protein/DNA complex. The assay was validated using unphosphorylated STAT3 that does not form the biologically-relevant homodimer, and non-biotinylated DNA, which would not form the active FRET pair. Further validation of the assay was carried out using known STAT3 inhibitors such as the peptidomimetics PYLKTK and YLPQTV, and the small-molecule inhibitors STA-21 and Stattic. It was then used to screen a 40-membered library of novel SH2-targeted molecules produced in-house, in which it successfully identified six “hit” molecules with low micro molar activity. These were further evaluated by establishing IC50 values in a number of cell lines including MDA-MB231, HELA, A4 and NCI-H1975. These studies revealed a correlation between the FRET assay results and the cytotoxicity of the molecules in the STAT3-dependent cell lines. The molecules were also studied in cellular experiments to establish their effect on STAT3-regulated genes such as Cyclin D1 and Survivin, in which a correlation was also observed. As a result, these molecules are now in further development. Finally, the assay has been modified for high-throughput use in a 384-well system, and will be used for robotic screening in the future.
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Etter, Jonathan Parker. "Development of Inhibitors in the IL-6/GP130/JAK/STAT Pathway as Therapeutic Agents." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376525461.
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Zhang, Yixi. "Targeting STAT3 in Ovarian Cancers: Reciprocal Activation of NF-kB by STAT3 Inhibition." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:27007758.
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The transcription factor STAT3 normally modulates cell proliferation with a rapid and transient downstream effect. However, in tumor cells, inappropriately activated STAT3 alters the gene expression profile and renders tumor cells unresponsive to cell death signals. In this study, we examine the biological and biochemical effects of some STAT3 inhibitors on ovarian and cervical cancer cells. Furthermore, we study the reciprocal relationship between STAT3 and NF-kB—another prosurvival transcription factor—in ovarian cancer cells.
Inappropriate activation of STAT3 occurs in many cancers and often results in resistance to conventional chemotherapies. In addition, overactive STAT3 signaling in tumor cells has been correlated with resistance to conventional chemotherapies. Therefore, for cancer patients, targeted inhibition of STAT3 potentially constitutes a powerful therapeutic tool. Initially, when ovarian cancer cells that depend on STAT3 for pathogenesis are treated with STAT3 inhibitors (i.e. nifuroxazide, ST3-01, etc), significantly reduced viability was observed. Surprisingly, results from quantitative RT-PCR analysis and reporter assays have identified an unexpected reciprocal relationship between STAT3 inhibition and NF-кB activation. Furthermore, reducing STAT3 expression by RNAi seemed to result in the upregulation of NF-кB genes including A20 and IL-8, which was consistent with the effects of STAT3 inhibitors. Moreover, the combination of reducing the levels of both STAT3 and the NF-кB subunit p65 was found to abrogate the upregulation of NF-кB target genes seen when STAT3 levels alone were reduced. This suggests that p65 expression is important for the activation of NF-кB by STAT3 inhibition. Subsequently, NF-кB nuclear translocation was examined in whole cell populations as well as in single cells. The results showed that no apparent p65 nuclear translocation was observed upon STAT3 inhibition, suggesting an alternative mechanism of NF-кB activation.
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Smith, Cressida Sally. "Design and synthesis of novel transition state isostere inhibitors of MurD." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418195.
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Wang, Xiaodong. "Design, Syntheses, and Bioactivities of Conformationally Locked Pin1 Ground State Inhibitors." Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/26625.
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Pin1 (protein interacting with NIMA 1) is a peptidyl-prolyl isomerase involved in mitosis. As a potential anti-cancer drug target, Pin1 interacts and regulates the activity of an increasing number of cell cycle enzymes by an unknown mechanism. These cell cycle enzymes include Cdc25, Cdc27, Cyclin D1, Myt1, Wee1, NIMA, Cdc2, Plk1 and c-Myc. Recent research has revealed that Pin1 is overexpressed in a variety of cancer cell lines and Pin1 inhibitors inhibit proliferation activity of several cancer cells overexpressing Pin1. The most potent Pin1 inhibitors identified so far are in the micromolar range and no pharmacophore has been identified.
In order to assist the understanding of the biological function of Pin1 using molecular probes, two amide isosteres of Ser-trans-Pro and Ser-cis-Pro dipeptides were designed and stereoselectively synthesized. The conformationally locked Ser–trans–Pro mimic, Boc-SerΨ[(E)CH=C]Pro–OH, was synthesized through the use of an Ireland-Claisen [3,3]-sigmatropic rearrangement in nine steps with 13% overall yield from a serine derivative. The Ser-cis-Pro mimic, Boc-SerΨ[(Z)CH=C]Pro–OH, was synthesized through the use of a Still-Wittig [2,3]-sigmatropic rearrangement in 11 steps with an overall yield of 20% from the same starting material.
Conformationally locked peptidomimetics, including two exactly matched peptidomimetics, Ac–Phe–Phe–pSer–Ψ(E)CH=C]Pro–Arg–NH2 and Ac–Phe–Phe–pSer–Ψ[(Z)CH=C]Pro–Arg–NH2, were synthesized from these Ser-Pro isosteres using Fmoc SPPS. A protocol for in vitro Pin1 inhibition assay was established for measuring the inhibition constant for these peptidomimetics. A conformationally locked cis peptidomimetic inhibits Pin1 with a Ki of 1.7 μM, 23-fold more potent than its trans counterpart, illustrating the preference of Pin1 for a cis amide bond in its PPIase domain. The A2780 ovarian cancer cell antiproliferation activity of these peptidomimetics parallels their respective Pin1 inhibition data. This research provides a start toward more drug-like Pin1 inhibitor design. Gly–trans–Pro isosteres were synthesized using the Ireland-Claisen route. The construction of a non-peptidic (Z)-alkene library for Pin1 inhibition was attempted using the Ser-cis-Pro mimic, Boc—SerΨ[(Z)CH=C]Pro–OH as the core.Ph. D.
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Couto, Jason. "Biologic Activity of the Novel Small Molecule STAT3 Inhibitor Against Canine Osteosarcoma Cell Lines." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1373986927.
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Ziegler, Inna. "Posttranslationale Modifikationen der IL-6-Typ-Zytokin-Rezeptoren gp130 und LIFR und ihr Einfluss auf die Assoziation mit Detergenz-resistenten Membranmikrodomänen (DRM)." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:100-opus-3124.
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Misale, Antonio. "Synthesis of angucycline-based small molecules as potential STAT3 : STAT3 protein-protein interaction inhibitors for cancer therapy." Thesis, University of London, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555844.
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Inhibition of the ST AT3: ST AT3 protein-protein interaction is an attractive approach for cancer therapy as it can lead to suppression of tumour cell growth and induce apoptosis. The racemic ochromycinone (STA21) is one of the few known small-molecule STAT3:STAT3 inhibitors. Our synthetic efforts focused on synthesis of the natural product YM-181741, which possesses at least three points for chemical variation to prepare compound libraries as potential STAT3:STAT3 inhibitors. Synthesis of the angucycline molecule was achieved employing an approach based on a Gold (lII)-catalysed intramolecular [4+2] benzannulation reaction. A facile and highly efficient route for the preparation of racemic form of the natural product was developed that offers a high degree of flexibility for modification of the scaffold at different stages of its synthesis. The enantioselective synthesis of (S)- YM181741 was successfully carried out through the (R)-diyne building block via an enantioselective copper-catalysed 1,4-conjugate addition reaction on a system bearing a v-coordinating group, in order to install the chirality on the diyne moiety. The optimised reaction conditions afforded the Michael adduct in good yield and high enantiomeric excess (up to 96% ee). Further chemical elaboration of the (±)- YM-181741 natural product was investigated in order to explore the necessary chemical diversity to assess a preliminary model of interaction between the SH2 domain of ST AT3 and the angucycline scaffold. The biological evaluation of the focused library allowed the identification of angucycline derivatives possessing high binding affinity for the SH2 region and the ability to inhibit STAT3 transcriptional activity.
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Wang, Xinning [Verfasser]. "Tetrazole-containing STAT5 Inhibitors Derived from Furazan-based Phosphate Mimetics / Xinning Wang." Berlin : Freie Universität Berlin, 2020. http://d-nb.info/121464130X/34.
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Laver, Travis. "Mechanism of inteferon-beta-mediated inhibition of IL-8 gene expression." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/laver.pdf.
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Miller, David James. "Phosphinic acids as inhibitors of D-Ala-D-Ala adding enzyme." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242865.
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Xu, Guoyan. "Pin1 Inhibitors: Towards Understanding the Enzymatic Mechanism." Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/37823.
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An important role of Pin1 is to catalyze the cis-trans isomerization of pSer/Thr-Pro bonds; as such, it plays an important role in many cellular events through the effects of conformational change on the function of its biological substrates, including Cdc25, c-Jun, and p53. The expression of Pin1 correlates with cyclin D1 levels, which contributes to cancer cell transformation. Overexpression of Pin1 promotes tumor growth, while its inhibition causes tumor cell apoptosis. Because Pin1 is overexpressed in many human cancer tissues, including breast, prostate, and lung cancer tissues, it plays an important role in oncogenesis, making its study vital for the development of anti-cancer agents.
Many inhibitors have been discovered for Pin1, including 1) several classes of designed inhibitors such as alkene isosteres, non-peptidic, small molecular Pin1 inhibitors, and indanyl ketones, and 2) several natural products such as juglone, pepticinnamin E analogues, PiB and its derivatives obtained from a library screen. These Pin1 inhibitors show promise in the development of novel diagnostic and therapeutic anticancer drugs due to their ability to block cell cycle progression. In order to develop potent Pin1 inhibitors, the concept of transition-state analogues was used for the design of three classes of compounds: ketoamide, ketone, and reduced amide analogues.
Specifically, a convergent synthesis of α-ketoamide inhibitors of Pin1 was developed. An α-hydroxyorthothioester derivative of Ser was reacted directly with an aminyl synthon. The reaction was catalyzed by HgO and HgCl2 to form an α-hydroxyamide. Hydrolysis and coupling were combined in one step in 80% yield. Two diastereomers of a phospho-Ser-Pro α-ketoamide analogue were synthesized. The resulting IC50 values of 100 µM and 200 µM were surprisingly weak for the Pin1 peptidyl-prolyl isomerase.
Diastereomeric ketones were synthesized by coupling cyclohexenyl lithium to the serine Weinreb amide, via the Michael addition of a carboxylate synthon. The IC50 values of the two ketone diastereomers were determined to be 260 μM and 61 μM, respectively.
Five reduced amide inhibitors for Pin1 were synthesized through a selective reduction using borane. The most potent inhibitor was found to be Fmocâ pSerâ Ψ[CH2N]-Proâ tryptamine, which had an IC50 value of 6.3 µM. This represents a 4.5-fold better inhibition for Pin1 than a comparable cis-amide alkene isostere. The co-crystal structure of Acâ pSerâ Ψ[CH2N]-Proâ tryptamine bound to Pin1 was determined to 1.76 à resolution.
Towards understanding the two proposed mechanisms of Pin1 catalysis, nucleophilic-additition mechanism and twisted-amide mechanism, three classes of Pin1 inhibitors (ketoamide, ketone, and reduced amide analogues) involving a total of nine compounds were synthesized and evaluated. The weak inhibitory activities of ketoamide and ketone analogues do not support the nucleophilic-addition mechanism, while the twisted-amide mechanism of Pin1 catalysis is promising based on the reduced amide inhibitors with good potencies.Ph. D.
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Schust, Jochen. "Neue Ansätze zur Identifizierung niedermolekularer Inhibitoren der STAT3-Aktivierung und -Homodimerisierung." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=982197438.
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Al-Lami, Naeemah. "Synthesis of nitrogen-containing bicyclic sesquiterpenes as potential transition state inhibitors of aristolochene synthase." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/53674/.
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Aristolochene synthase from Penicillium roqueforti(PR-AS) is sesquiterpene synthase that catalyses the Mg2+-dependent conversion of farnesyl diphosphate FDP to (+)-aristolochene. Through the use of site directed mutagenesis, fluorinated FDPs and an aza-analogue of the eudesmane cation, the reaction was previously shown to involve germacrene A and eudesmane cation as intermediates. The subsequent series of rearrangements that transform the eudesmane cation to (+)-aristolochene have not been investigated previously. To probe the carbocationic nature of these 1,2-hydride and methyl shifts, new aza-analogues were designed to mimic the geometric and electrostatic properties of postulated carbocation intermediates in the catalytic mechanism of PR-AS. Here is described the synthesis of both enantiomers of 10-aza-eremophilane in enantiomerically pure from the common precursor (4S)-limonene oxide and their analysis as inhibitors of PR-AS. The synthesis of (7R,4S,5S)-10-aza-eremophilane cation was accomplished in 8 steps, starting from a known keto ester that in turn was obtained by degradation of (-)-limonene oxide. An identical synthetic protocol was repeated from (4R)-limonene oxide to give the enantiomer of 10-aza-eremophilane cation. Inhibition studies with compound (7R,4S,5S)-10-aza-eremophilane indicated that this ammonium salt acted as a moderate competitive inhibitor of PR-AS (Ki = 38 μM), and showed that eremophilane cation is likely a true intermediate on the pathway from FDP to aristolochene during PR-AS catalysis. The inhibition potency of 10-aza-eremophilane was increased by the addition of diphosphate PPi (Ki = 2.9 μM). This synergetic kinetic effect suggests that the possible involvement of PPi as a stabilizing anion for the eremophilane carbocation in PR-AS biosynthesis. Inhibition studies of the enantiomer of (7R,4S,5S)-10-aza-eremophilane cation, (7S,4R,5R)-10-aza-eremophilane cation, which has incorrect stereocenteres, with PR-AS indicated that this ammonium salt was a poor inhibitor of PR-AS (Ki = 1.03 mM). The data obtained for this compound highlight the chiral environment of the active site of PR-AS, and more importantly supports the postulate that terpene synthases form a product-like contour at their active site that steers the carbocationic cascade catalyzed by PR-AS toward the production of a single enantiomer. iv In the second part of the present work, progress was made towards the stereoselective synthesis of 5-aza-eudesmane cation. This teriary amine is a structural mimic of the 5-eudesmane carbocation, another putative intermediate in the reaction cascade catalysed by PR-AS. However, this tertiary amine was not obtained with desired stereochemistry, nevertheless, two diastereoisomers of the desired compound were obtained.
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Maughan, Michael A. T. "Cyclic imine sugars : towards the synthesis of transition-state mimics as potential glycosyltransferase inhibitors." Thesis, Durham University, 2003. http://etheses.dur.ac.uk/3694/.
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This thesis describes the development of methodology for the synthesis of cyclic imine-sugars, and their use in the synthesis of aza-sugars as potential sugar-processing enzyme inhibitors. Our goals include the synthesis of cyclic imines of L-rhamnose, D-glucose, and L-idose stereochemistry, and the introduction of functionality via nucleophilic addition reactions. Work on the synthesis of cyclic imines commenced with the use of the simple model systems piperidme and 2-methylpiperidine. N-Chlorination of these systems was performed and the products converted into their cyclic imine derivatives through elimination of HCl. Nucleophilic addition reactions to these systems were attempted, but the low stability and reactivity of the imines led to the isolation of only one adduct. The synthesis of novel cyclic pyrrolidine imine-sugars of L-rhamnose stereochemistry was performed by a Staudinger aza-Wittig reaction. The aza-Wittig reaction of a known L-rhamnose derived azido-sugar gave a novel cyclic L-rhamnopyrrolidine aldimine A novel synthesis of this azido-sugar was also devised. Successful nucleophilic additions to the cyclic L-rhamnopyrrolidine aldimine were performed with a range of Grignard reagents giving novel protected aza-sugars in good yields and with excellent diastereoselectivities. A novel cyclic L-rhamnopyrrolidme ketimine was also synthesised via a Staudinger aza-Wittig from a novel azido-sugar, although time constraints prevented screening this system with nucleophiles. The synthesis of novel cyclic piperidine imine-sugars of D-glucose, and L-idose stereochemistry was performed, both via N-chlorination/elimination of the protected parent aza-sugars, and via the Staudinger aza-Wittig reaction of novel azido-sugars. The elimination of HCl from six-membered iV-chloro aza-sugars of D-glucose and L- idose stereochemistry was investigated, and methodology developed in the case of D- glucose system for the regioselective elimination of HCl to give either the aldimme or the ketimine derivative. Comparison of elimination reactions of the N-chloro aza- sugars of D-glucose, and L-idose stereochemistry allowed rationalisation of the observed regiocontrol. Screening of the cyclic imines of D-glucose and L-idose stereochemistry with nucleophiles, followed by deprotection of the adducts, allowed the synthesis of novelaza-sugars via late-stage introduction of functionality. Low yields were obtained for these additions, but diastereocontrol was generally good, and could be rationalised by accepted stereoelectronic and steric approach control factors. The formation of cyclic piperidine imines of L-idose, and D-glucose stereochemistry was also performed via the Staudinger aza-Wittig reaction. These systems were found to be identical to those synthesised by the N-chlorination/elimination protocol. We also performed the first synthesis of the enantiomer of the natural product (+)- adenophorine thereby allowing assignment of the absolute configuration of the natural product (+)-adenophorine. The key synthetic step in this synthesis was stereoselective reduction of a novel intermediate cyclic piperidine ketimine-sugar of L-idose stereochemistry.
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Feng, You. "Kinetic Mechanism and Inhibitory Study of Protein Arginine Methyltransferase 1." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/chemistry_diss/68.
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Protein arginine methyltransferase 1 (PRMT1) is a key posttranslational modification enzyme that catalyzes the methylation of specific arginine residues in histone and nonhistone protein substrates, regulating diverse cellular processes such as transcriptional initiation, RNA splicing, DNA repair, and signal transduction. Recently the essential roles of PRMT1 in cancer and cardiovascular complications have intrigued much attention. Developing effective PRMT inhibitors therefore is of significant therapeutic value. The research on PRMT inhibitor development however is greatly hindered by poor understanding of the biochemical basis of protein arginine methylation and lack of effective assays for PRMT1 inhibitor screening.
Herein, we report our effort in the kinetic mechanism study as well as the fluorescent probe and inhibitor development for PRMT1. New fluorescent reporters were designed and applied to perform single-step analysis of substrate binding and methylation of PRMT1. Using these reporters, we performed transient-state fluorescence measurements to dissect the rate constants along the PRMT1 catalytic coordinate. The data give evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, we identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. On the other hand, we discovered a type of naphthyl-sulfo (NS) compounds that block PRMT1- mediated arginine methylation at micromolar potency through a unique mechanism: they directly target the substrates but not PRMT enzymes for the observed inhibition. We also found that suramin, an anti-parasite and anti-cancer drug bearing similar functional groups, effectively inhibited PRMT1 mediated methylation. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation. Addionally, to dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. While certain acetylations present a repressive impact on PRMT-1 mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases activity of PRMT1 but increases that of PRMT5. Furthermore, hyperacetylation increases the content of ordered secondary structures of H4 tail. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation, and unravel that complex intercommunications exist between different posttranslational marks in cis.
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Csatary, Erika Elizabeth. "Asymmetric Multicomponent Aza-Diels-Alder Reaction for Construction of Multicyclic Heterocycles and Development of XZH-5 Derivatives as Inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3)." Miami University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=miami1435110305.
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Altundas, Abdullah Bilal. "Synthesis of XZH-5 Derivatives as Inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) and Synthesis of π-Extended Tetraphenylporphyrins." Miami University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=miami1473201129.
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Yu, Wenying. "Computational, Synthetic, Biochemical and Biological Studies and Characterization on STAT3 Inhibitors for Potential Anticancer Therapy." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1373328058.
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Turner, Kimberly Ann. "Deliberate Memory in Three-Year-Old Children: Interrelations among Task Approaches, Working Memory, and Inhibitory Control." NCSU, 2008. http://www.lib.ncsu.edu/theses/available/etd-03242008-181800/.
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Preschool children are capable of displaying strategies in memory tasks and demonstrating an early understanding of memorization (e. g., Wellman, 1988; Baker-Ward, Ornstein, & Holden, 1984). Questions remain, however, about the origins of strategic behavior in early childhood. A great deal of recent attention has been devoted to the interrelations among working memory and measures of executive functioning/inhibitory control in elementary-school children (e.g., Schneider, Schumann-Hengsteler, & Sodian, 2005). The goal of this investigation was to extend this work to preschool children in order to examine possible influences on the emergence of deliberate remembering. Specifically, interrelations among working memory, inhibitory control, and deliberate task approaches were examined in 168 three-year-olds who participated in a large-scale, broadly-focused investigation of development, the Durham Child Health and Development Study. Although predicted relations among multiple domains of cognitive functioning were not observed, important findings did emerge. Previous results examining the use of deliberate task approaches were replicated in a more diverse and younger sample. Support for the presence of deliberate remembering in young preschoolers was found in a significant positive relation between language ability and the extent of deliberate task approaches. Finally, an unexpected relation between deliberate task approaches and subsequent recall performance was found; this result is discussed in relation to Utilization Deficiency. Implications for understanding some of the contributors to the emergence of deliberate remembering are presented, and directions for future research are discussed.
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Shumate, Howard W. "Repeated Alcohol Use and Sober-State Reactive Aggression: The Mediating and Moderating Role of Sober-State Executive Cognitive Functioning." Thesis, Virginia Tech, 2005. http://hdl.handle.net/10919/33397.
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This study examined the cumulative, more insidious, impact of repeated drinking on sober-state aggression based on research that has pointed to the negative neural effects of chronic alcohol consumption, especially on frontal lobe functioning. In particular, it examined the relationship between repeated alcohol use and sober-state reactive aggression as it is mediated or moderated by sober-state executive cognitive functioning (ECF), thus expanding upon research that has examined the relationship between acute alcohol intoxication and consequent aggression while under the influence (Giancola, 2000b). It was hypothesized that ECF would mediate the relationship between repeated alcohol use and sober-state reactive aggression in college students in that a history of alcohol use would lower sober-state ECF which in turn would increase sober-state impulsive aggression in individuals. It was further hypothesized with a moderational model that high levels of ECF would offset the more insidious effects of repeated alcohol use on subsequent sober-state aggressive acts. Moreover, those effects would remain after controlling for potential confounds of violence exposure, gender, and intelligence.
Eighty college students, aged 18-23 years, from Virginia Tech were recruited to participate in this study. A self-report measure for aggression, neuropsychological tests for ECF, and a lifetime drinking interview schedule were used to assess the relationship between cumulative alcohol use, sober ECF, and sober aggression. A combination of bivariate and hierarchical regression analyses was used to analyze the data.
The hypotheses of this study were not supported. Instead, the results supported a positive relationship between prior exposure to violence and later escalation of alcohol use and perpetrated violence. Additionally, these results support the presence of a â binge drinkingâ pattern within the sample.Master of Science
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Dimberg, Lina. "Apoptosis Regulation in Multiple Myeloma." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7099.
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Frase, Hilary. "TOWARDS DEVELOPING SPECIFIC INHIBITORS OF THE ATP-DEPENDENT LON PROTEASE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1175637588.
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Khatchaturyan, Levon [Verfasser], Udo [Akademischer Betreuer] Markert, Uta-Christina [Akademischer Betreuer] Hipler, and Ulrike [Akademischer Betreuer] Kämmerer. "Die Rolle von PIAS (Protein Inhibitors of Activated STATs) in der Regulation von STAT (Signal Transducer and Activator of Transcription) : vermittelten Funktionen trophoblastärer Zellen / Levon Khachaturyan. Gutachter: Udo Markert ; Uta-Christina Hipler ; Ulrike Kämmerer." Jena : Thüringer Universitäts- und Landesbibliothek Jena, 2012. http://d-nb.info/1020402113/34.
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DAKA, PHILIAS. "ENAMINE-METAL LEWIS ACID BIFUNCTIONAL CATALYSTS FOR ASYMMETRIC ALDOL REACTIONS. DESIGN AND SYNTHESIS OF STAT3 INHIBITORS." Miami University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=miami1374852476.
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Tse, Joyce. "Inhibition of STAT3 decreases OSM induced EDA-FN expression in human lung fibroblasts." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/33839.
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Fibrosis is excessive deposition of connective tissue components that results in the destruction of normal tissue architecture and compromises organ function. When fibrosis occurs in the major organs such as the lung, for example in idiopathic pulmonary fibrosis (IPF), it inevitably leads to organ failure and premature death of the afflicted individual. The development of fibrosis follows a similar pathway to normal wound healing, although there is chronic progression of the disease without resolution, suggesting the fine control of cellular functions that occur during wound healing is disturbed. Determining where this control is lost is paramount to preventing and treating this condition. Fibroblasts are the main cell type responsible for extracellular matrix (ECM) production. The transcription factor signal-transducer-and-activator-of-transcription-3 (STAT-3) regulates genes involved in cell differentiation and wound healing. It has been shown that fibroblasts isolated from normal and IPF lungs differ in STAT3 dependent interleukin-6 (IL-6)/glycoprotein 130 (gp130) cell signaling and proliferation. Therefore, we aimed to evaluate whether STAT3 inhibition could decrease expression of ECM proteins, including collagen and extra-domain A fibronectin (EDA-FN) in human lung fibroblasts. We also sought to examine the effect of knocking down STAT3 function on fibroblast proliferation. Cells were exposed to Oncostatin-M (OSM) or IL-6, and collagen-1 and EDA-FN protein expression was analyzed by western blotting, while cell proliferation was assessed by bromo-deoxyuridine (BrdU) incorporation. STAT3 function was inhibited in two ways: Firstly, inhibition with a small molecule inhibitor, STA-21, blocks STAT3 dimerization and nuclear translocation and secondly, inhibition of STAT3 gene transcription by short interfering RNA (siRNA). Both methods inhibited OSM induced EDA-FN expression and proliferation in human fetal lung (HFL) fibroblasts. However, STAT3 had negligible effects in adult lung fibroblasts. We attempted to resolve the disparate effects by inhibiting another downstream signaling pathway, the extracellular receptor kinase (ERK)-1/2, which is also activated by gp130. In conclusion, OSM induced EDA-FN expression and cell proliferation in HFL fibroblasts are dependent upon STAT3 activation. In contrast, STAT3 has minimal involvement in adult cells. The mechanisms underlying these disparate effects remain to be elucidated. Interestingly, inhibiting either STAT3 or ERK1/2 inhibited OSM induced proliferation in HFL fibroblasts.
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Moreno, Fortuño David. "Prions i Agregons com a Inhibidors de Start: una Via a l’Envelliment Cel·lular." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/404674.
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Saccharomyces cerevisiae és un model escaient per estudiar el procés d’envelliment a nivell cel.lular gràcies al seu mecanisme de divisió asimètrica. Fent una analogia amb l’envelliment en organismes superiors, on les cèl.lules somàtiques són pròpies de l’individu i envelleixen amb ell, mentre que les cèl.lules de la línia germinal són capaces de formar un nou individu i són virtualment immortals en la població, en S. cerevisiae podem diferenciar dos tipus de cèl.lules: les cèl.lules mare, que envelleixen i poden produir unes 20-30 cèl.lules filles, i les cèl.lules filles, que són capaces d’esdevenir mares i tornar a fer 20-30 cèl.lules filles més independentment de l’edat de la seva mare en el moment de néixer, excepte si la mare ja és molt vella. Per tant, en estudiar l’envelliment de les cèl.lules mare del llevat de gemmació podem aprendre molts dels mecanismes que condueixen l’envelliment cel.lular, especialment a nivell molecular.
Entre les característiques que adquireixen les cèl.lules mare de llevat durant el procés d’envelliment, l’acumulació de dipòsits proteics insolubles, proteïnes carbonilades i agregats proteotòxics sembla jugar un paper molt important. Donada la seva preponderància també en organismes superiors, juntament amb les afectacions mitocondrials, ens interessava entendre la relació entre la presència d’aquests dipòsits proteotòxics i la maquinària de cicle cel.lular, especialment durant G1 sobre la Xarxa de Start. Sabem que les xaperones tenen un paper clau en Start per a executar l’entrada en el cicle cel.lular i, en ser segrestades per els agregats proteotòxics en cèl.lules envellides podrien ser causa directa de senescència replicativa.
Per tal de comprovar aquesta hipòtesi, en primer lloc hem constatat que els agregats proteotòxics endarrereixen la progressió durant G1, aboleixen la coordinació entre velocitat de creixement i la mida crítica de Start, i redueixen fortament la longevitat de les cèl.lules del llevat. D’altra banda, mitjançant diferents aproximacions que permeten l’estudi de l’envelliment de les cèl.lules mare del llevat (algunes de les quals desenvolupades en aquest treball), hem observat que en els últims cicles de cèl.lules envellides es produeix un clar retard en la progressió durant G1, així com que la majoria de cèl.lules moren estant en aquesta fase del cicle cel.lular, quan en les cèl.lules mare joves aquesta fase és la més curta. Especialment rellevant en el nostre estudi, hem demostrat que el grau de disponibilitat de xaperones disminueix clarament en cèl.lules velles. Finalment, veiem que la superexpressió de l’activador del cicle cel.lular Cln3 allarga la longevitat de les cèl.lules, fins i tot si aquesta activació es fa sobre cèl.lules parcialment envellides prèviament com si ho fem sobre mutants d’algunes xaperones clau per a l’activació del cicle cel.lular. En resum, com a resultat de l’acumulació d’agregats proteotòxics en cèl.lules envellides, la pèrdua d’activitat xaperona arribaria a comprometre l’entrada en el cicle cel.lular i, així, podria explicar la pèrdua de capacitat proliferativa que té lloc durant el procés d’envelliment cel.lular.Saccharomyces cerevisiae is a suitable model to study the aging process at the cellular level thanks to its mechanism of asymmetric division. Analogous to aging in higher organisms, which contain somatic cells that age with the individual and germline cells that are able to form a new individual and are virtually immortal in the population, in S. cerevisiae we can distinguish two types of cells: mother cells, which age and can produce 20 to 30 daughter cells, and daughter cells which are capable of becoming mothers and produce about 20-30 more daughter cells regardless of the age of the mother at birth, unless the mother is already very old). Therefore, studying mother cell aging in budding yeast can uncover many of the mechanisms that drive cell aging, especially at the molecular level. Among the features that mother cells acquire during aging, the accumulation of insoluble protein deposits, carbonylated proteins and proteotoxic aggregates is thought to play a very important role. As this seems to play a prominent role in higher organisms, along with mitochondrial affectations, we were interested in understanding the relationship between the presence of these proteotoxic deposits and the machinery of cell cycle, especially during G1 on the Start network. We know that chaperones play a key role at Start to trigger entry into the cell cycle and, by being hijacked by proteotoxic aggregates in aged cells, they could be a direct cause of replicative senescence. To test this hypothesis, we first assessed that proteotoxic aggregates delay G1 progression, abolish the coordination between growth rate and critical size at Start, and strongly reduce the longevity of yeast cells. On the other hand, using different approaches that allow to study aged mother cells of yeast (some of which developed in this work), we have found that there is a clear delay in G1 progression in the last cycles of aged cells, most cells dying within this cell cycle phase, when in young mother cells this is the shortest phase of the cell cycle. Especially relevant to our work, we have shown that availability of chaperones is remarkably lower in aged mother cells. Finally, we have seen that overexpression of the cell cycle activator Cln3 is able to extend cell longevity, even if this overexpression is triggered in partially aged cells or in cells lacking some key chaperones for the activation of the cell cycle. In summary, as a consequence of the accumulation of proteotoxic aggregates in aged cells, loss of chaperone activity would eventually compromise cell cycle entry and, therefore, would explain why cells lose their proliferative capacity during the aging process.
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Brown, Megan. "Characterization of STAT3 Expression, Signaling and Inhibition in Feline Oral Squamous Cell Carcinoma." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429621442.
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Goodall, Scott. "Probing the structure of acetylcholinesterase inhibitors in their binding site using solid state nuclear magnetic resonance." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270619.
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Hernández, Ribes Gracia. "ESTUDIO DE LA RUTA CELULAR JAK2/STAT3 COMO POTENCIAL INHIBIDOR EN EL MODELO DE FIBROSIS PULMONAR." Doctoral thesis, Universitat Politècnica de València, 2016. http://hdl.handle.net/10251/64087.
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[EN] Idiopathic pulmonary fibrosis (IPF) is the pulmonary disease with higher incidence and worse prognosis. Recent evidence suggests that cucurbitaceae, selective inhibitors of the JAK2/STAT3 pathway, may improve the pathogenesis of IPF, as anti-inflammatory and antioxidative properties have been confirmed in other diseases. However the role in IPF is unknown.
Two pharmacological models were investigated in the present study. In the preventive model, Wistar rats were instilled intratracheally with a single dose of bleomycin (BLM)(3.75 U/kg; n=12) to induce lung injury. CuI (20mg/kg/day; n=6) or CuI vehicle (control and IPF group) was administered intraperitoneally daily for 21 days. The therapeutic model was exactly the same starting CuI administration on day 7 until day 21. Animal evolution together with the ventilation-perfusion ratio was controlled through CT/SPECT imaging. Cellular count and characterization was performed in broncoalveolar lavage (BAL) together with the total protein content and the IL-6 and IL-13 concentration in BAL and lung tissue. Hematoxilin-eosin and masson trichrome stains allowed the study of the lung's tissue histology. TGF-ß1, CTGF, COL1A and ET-1 gene and protein expression were both measured by real time PCR and WB as pulmonary vascular remodeling markers. P-STAT3, P-SMAD3 and P-JAK2 proteins were determined by protein and immunohistochemistry quantification. Finally, JAK2 and STAT3 expression and distribution was studied in lung tissue of fibrotic patients with pulmonary hypertension. CT/SPECT quantification showed reduction of the fibrotic areas in the CuI treated group by reestablishing the air space in the lungs back to day 0 levels for the preventive and the therapeutic model. Furthermore, ventilation/perfusion correlation was restored in the therapeutic model after administrating CuI during 14 days. Hematoxilin-eosine stains demonstrated how the group treated pharmacologically presented an improvement in the lung tissue architecture, reversing vascular remodeling and right heart hypertrophy. Masson trichrome stain revealed a reduction of the collagen deposits. Ashcroft score, used to determine the severity of pulmonary fibrosis, was measured and diminished significantly in the CuI treated group. The results showed a gene and protein overexpression of TGF-ß1, CTGF, COL1A and ET-1 in BLM relative to Control rats. This was counteracted with CuI treatment which reduced the expression back to control. In terms of immunohistochemistry, the results demonstrated a decrease in the COL1A deposits in the treated group versus the absence of treatment group. Protein and immunohistochemistry analysis of JAK2, STAT3 and SMAD3 demonstrated an overexpression in bleomycin rats while the protein expression was inhibited in the CuI treated group. In accordance to the results obtained, the immunohistochemistry analysis of the lung parenchyma in patients with pulmonary hypertension related to pulmonary fibrosis showed and overexpression of the phosphorylated forms of JAK2 and STAT3, lacking its expression in healthy lung tissue. The preventive and therapeutic administration of JAK2/STAT3 inhibitor can be a potential treatment for pulmonary fibrosis, as it improves the parameters related to the disease.[ES] La fibrosis pulmonar idiopática (FPI) es la enfermedad pulmonar con mayor incidencia y peor pronóstico. Estudios recientes sugieren que la familia de las cucurbitaceae, inhibidores selectivos de la ruta JAK2/STAT3, pueden mejorar la patogénesis de la enfermedad, al haberse confirmado sus propiedades antinflamatorias y antioxidantes en otras enfermedades. Sin embargo se desconoce su papel en FPI. En el presente trabajo se estudiaron dos modelos farmacológicos. En el modelo preventivo las ratas Wistar fueron instiladas con una dosis única de bleomicina intratraqueal (BLM)(3.75 U/kg; n=12) para inducir las lesión pulmonar. Durante 21 días se administró CuI (20mg/kg/día; n=6) o vehículo de CuI (control y grupo FPI) por vía intraperitoneal. El modelo curativo se diferencia del preventivo en el comienzo de la administración farmacológica a los 7 días de inducir la enfermedad. El seguimiento de la evolución animal y el ratio de ventilación-perfusión se realizó mediante las técnicas de imagen TC/SPECT. Se realizó el recuento y caracterización de las células totales extravasadas en lavado broncoalveolar (LBA) así como el contenido de proteína total y la concentración de IL- 6 e IL-13 en LBA y tejido pulmonar. Las tinciones de hematoxilina-eosina y masson tricrómico permitieron el estudio de la histología del tejido pulmonar. Se determinó la expresión génica y proteica de TGF-ß1, CTGF, COL1A y ET-1 mediante las técnicas de real time PCR y WB como marcadores de remodelado vascular. Las proteínas P-STAT3, P-SMAD3 y P-JAK2, fueron determinadas mediante cuantificación proteica e inmunohistoquimia. Por último se estudió la expresión y distribución de JAK2 y STAT3 en tejido de pacientes con fibrosis pulmonar e hipertensión pulmonar. La cuantificación de las imágenes TC/SPECT mostraron una reducción de las áreas fibróticas en el grupo tratado con CuI. El tratamiento farmacológico permitió el restablecimiento del espacio aéreo pulmonar hasta valores control en ambos modelos estudiados. El grupo con tratamiento farmacológico restauró el ratio de ventilación/perfusión tras administrar CuI durante 14 días. Las tinciones de hematoxilina eosina revelaron como el grupo animal tratado farmacológicamente presenta una mejora de la histología pulmonar, revirtiendo el remodelado vascular y la hipertrofia del ventrículo derecho. La tinción de masson tricrómico mostró una disminución de los depósitos de colágeno. Se determinó el valor de Ashcroft, evaluador del grado de fibrosis pulmonar, que descendió significativamente en el grupo tratado con CuI. Los resultados presentan una sobrexpresión génica y proteica de TGF-ß1, CTGF, COL1A y ET-1 en los grupos de bleomicina frente a las ratas control. Dicha condición fue revertida mediante el tratamiento con CuI que restableció los valores a niveles control. Los análisis proteicos e inmunohistoquíimicos de JAK2, STAT3 y SMAD3 revelaron una sobreexpresión en las ratas con bleomicina mientras que la expresión proteica fue inhibida en el grupo tratado con CuI. En consonancia con los resultados obtenidos, el análisis inmunohistoquímico del parénquima pulmonar de pacientes con FPI e HP asociada muestran una sobreeexpresión de las formas fosforiladas de JAK2 y STAT3 frente a la ausencia de expresión en tejido pulmonar sano. La administración curativa y preventiva de un inhibidor de la ruta JAK2/STAT3 puede ser un potencial tratamiento para la fibrosis pulmonar, ya que mejora parámetros indicativos de la patología.
[CAT] La fibrosi pulmonar idiopàtica (FPI) és la enfermetat pulmonar amb major incidència i pitjor pronostic. Estudis recents suggereixen que la família de les cucurbitàcies, Inhibidors selectius de la ruta JAK2 / STAT3, poden millorar la patogènesi de la malaltia, en haver-se confirmat les seues propietats antiinflamatòries i antioxidants En altres patologies. No obstant això es desconeix el seu paper en FPI. En el present treball es van estudiar 2 models farmacològics. En el model preventiu les rates Wistar foren instilades amb una dosi única de bleomicina intratraqueal (BLM) (3,75 U / kg; n = 12) per a induir les lesions pulmonars. Durant 21 dies es va administrar CuI (20 mg / kg / dia, n = 6) o Vehicle de CuI (control i grup FPI) per vía intraperitoneal. El model curatiu es diferència del preventiu en el començament de l'administració farmacológica als 7 dies d'induir l'enfermetat. El seguiment de l'evoluciò dels animals i el ratio de Ventilació-perfusió es va realitzar mitjançant tècniques d'imatge TC/SPECT. Es realitzà el recompte i caracterització de les cèl·lules totals extravasades en el llavat broncoalveolar (LBA). Així com el contingut de proteina total i la concentració d'IL-6 i IL-13 en LBA i teixit pulmonar. Les tincions d'hematoxilina-eosina i tricròmic de Masson van permetre l'estudi de la histologia del teixit pulmonar. Es va determinar l'expressió gènica i proteica de TGF-ß1, CTGF, COL1A i Et-1 mitjançant tècniques de PCR en temps real i WB com marcadors de remodelat vascular. Les proteïnes P-STAT3, P-SMAD3 i P-JAK2, varen ser determinades mitjançant quantificació proteica i inmunohistoquimia. Per últim se estudià l'expressió i distribució de JAK2 i STAT3 en teixit de pacients amb fibrosi pulmonar e hipertensió pulmonar. La quantificació de les imatges TC/SPECT mostraren una reducció de les àrees fibrótiques en el grup tractat amb CuI. El tractament farmacològic permet el restabliment de l'espai aeri pulmonar fins valors de control en els dos models estudiats. El grup amb tractament farmacològic va restaurar el ratio de ventilació/perfusió tras administrar Cul durant 14 dies. Les tincions d'hematoxilina eosina van revelar com el grup animal tractat farmacològicament presenta una Millora de la histologia pulmonar, revertint el remodelat vascular i la hipertròfia del ventricle dret. La tinció de tricròmic de Masson va mostrar una disminució dels dipòsits de col·lagen. Es va determinar el valor d'Ashcroft, avaluador del grau de fibrosi pulmonar, que va baixar significativament a el grup tractat amb CuI. Els resultats presenten una sobreexpressió gènica i proteica de TGF-ß1, CTGF, COL1A i Et-1 en els grups de bleomicina en comparacióa les rates control. Aquesta condició va ser revertida mitjançant el tractament amb CuI que va restablir els valors fins a nivel dels control. A nivell immunohistoquímic els resultats mostren una disminució dels dipòsits de COL1A en el grup tractat comparativament al grup sense tractament. Els anàlisi proteics i inmunohistoquíimics de JAK2, STAT3 i SMAD3 van revelar una sobreexpressió en les rates amb bleomicina mentre que l'expressió proteica va ser inhibida en el grup tractat amb CuI. D'acord amb els resultats obtinguts, l'anàlisi immunohistoquímic del parènquima pulmonar de pacients amb FPI i HP associada mostren sobreeexpresió de les formes fosforilades de JAK2 i STAT3 davant l'absència d'expressió en teixit pulmonar sa. La administració curativa i preventiva d'un inhibidor de la ruta JAK2 / STAT3 pot ser un potencial tractament per la fibrosi pulmonar, ja que millora paràmetres indicatius de la patologia.
Hernández Ribes, G. (2016). ESTUDIO DE LA RUTA CELULAR JAK2/STAT3 COMO POTENCIAL INHIBIDOR EN EL MODELO DE FIBROSIS PULMONAR [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/64087
TESIS
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Ah, Koon Laurent. "Etude du rôle modulateur de STAT1 sur les réponses cellulaires induites par deux stratégies anticancéreuses : -Chimiothérapie par les agents alkylants : -Biothérapies utilisant un oligonucléotide leurre inhibiteur de STAT3." Paris 13, 2012. http://scbd-sto.univ-paris13.fr/secure/edgalilee_th_2012_ah_koon.pdf.
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Les membres de la famille des facteurs STAT ont des rôles modulateurs très différents. Dans cette famille, STAT1 et STAT3 sont très similaires, peuvent réguler les mêmes gènes cibles mais sont caractérisés par des rôles opposés. STAT1 est considéré comme un anti-oncogène tandis que STAT3 serait plutôt un oncogène. Le facteur de transcription STAT1 est impliqué dans plusieurs voies de signalisations visant à protéger la cellule. STAT1 est un facteur antiprolifératif et proapoptotique. Le rôle modulateur de STAT1 de l’action de certains agents génotoxiques a été démontré et semble dépendre à la fois de l’agent et du type cellulaire étudiés. STAT3 est souvent activé dans les tumeurs et est localisé dans le noyau. Ses fonctions pro-prolifératives et anti-apoptotiques en font une cible de choix pour des traitements antitumoraux. Dans un premier temps, nous avons montré que STAT1 régulait l’expression de MLH1, protéine essentielle du système Mismatch Repair (MMR), et de c-Abl. L’absence de STAT1 était associée à la persistance des lésions de l’ADN, un arrêt du cycle cellulaire en phase G 2 /M et à une sensibilité accrue à l’agent alkylant MNNG. En outre, STAT1 entrait dans la composition d’un complexe actif STAT1/MLH1/c-Abl/p53 qui pourrait moduler le devenir cellulaire après exposition au MNNG. Dans un deuxième temps, nous avons montré que l’inhibition de STAT3 par un oligonucléotide leurre (ODN) contenant une séquence GAS piégeait les dimères de STAT3 activés dans le cytoplasme en empêchant la liaison aux aryophérines et induisait la mort cellulaire via STAT1. Cependant, notre ODN peut aussi lier STAT1 et nécessite donc une optimisation pour accroître sa spécificité. L’association d’une stratégie « ODN leurre » à une chimiothérapie classique pourrait permettre de potentialiser l’action des anticancéreux et de diminuer leurs effets secondairesSTAT family members have various modulating roles. Within this family, STAT1 and STAT3 are very similar and can regulate the same target genes but do have opposite effects. Whereas STAT1 is considered as an anti-oncogene, STAT3 is rather an oncongene. The transcription factor STAT1 modulates several pathways involved in cellular protection. STAT1 has antiproliferative and proapoptotic effects. A role for STAT1 as a modulator of the effect of genotoxic agents has been demonstrated and seems to depend on both the type of genotoxic agent and of the cell line studied. STAT3 is often activated in several types of cancers and is located in the nucleus. Anticancer strategies targeting the pro-proliferative and antiapoptotic STAT3 are promising. We have first shown that STAT1 regulated the expression of MLH1, an essential factor of the mismatch repair (MMR) system, and of c-Abl. STAT1 deficiency was associated with DNA lesions persistency, sustained G 2 /M cell cycle arrest and increased sensibility to the alkylating agent MNNG. Also, STAT1 entered into an active complex including STAT1/MLH1/c-Abl/p53 which could modulates the cell fate following MNNG exposure. Second, we have shown that inhibition of STAT3 with a decoy oligonucleotide (ODN) containing a GAS sequence could trap activated STAT3 dimers in the cytoplasm. This ODN impaired binding of STAT3 to karyopherin and induced a STAT1-dependent cell death. However, this ODN can also bind to STAT1 and hence it needs to be optimized in order to improve STAT3-specificity. The combination of a decoy-ODN strategy with a conventional chemotherapy should allow to synergize the action of anticancer drugs and to decrease their side effects
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Startzman, Ashley N. "Inhibition of stat3 protein as an approach to sensitizing ovarian cancer cells to cisplatin." Honors in the Major Thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1143.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Medicine
Molecular Biology and Microbiology
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Wong, Ee Lin [Verfasser]. "The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation / Ee Lin Wong." Berlin : Freie Universität Berlin, 2019. http://d-nb.info/1201346711/34.
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Camerino, Eugene. "Trifluoromethyl ketones: Potential insecticides towards Anopheles gambiae." Thesis, Virginia Tech, 2013. http://hdl.handle.net/10919/54015.
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Malaria continues to cause significant mortality in sub-Saharan Africa and elsewhere, and existing vector control measures are being threatened by growing resistance to pyrethroid insecticides. With the goal of developing new human-safe, resistance-breaking insecticides we have explored several classes of acetylcholinesterase inhibitors. In vitro assay studies have shown that trifluoromethyl ketones (TFK\'s) are potent inhibitors of An. gambiae AChE (AgAChE), that inhibit the enzyme by making a covalent adduct with the catalytic serine of the enzyme. However research in the Carlier group has shown that trifluoromethyl ketones bearing benzene and pyrazole cores have shown very little toxicity to An. gambiae, perhaps due to hydration and rapid clearance. Focus was directed towards synthesis of oximes, oxime ethers, and hydrazones as potential prodrugs to prevent immediate hydration and reach the central nervous system. The synthesis of various oximes, oxime ethers, and hydrazones has been shown to give cimpounds toxic to Anopheles gambiae within 3- to 4- fold of the toxicity of propoxur. However, thus far we have not been able to link the toxicity of these compounds to a cholinergic mechanism. Pre-incubation studies suggest that significant hydrolysis of these compounds to TFKs does not occur or 22 h at pH 7.7 or 5.5.
Future work will be directed towards TFKs that have better pharmacokinetic properties. Work will also be directed at synthesis of oxime and hydrazone TFK isosteres to determine the mechanism of action of these compounds.
Master of Science
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Wooldridge, Lydia Katherine. "Supplementing Bovine Embryo Culture Media to Improve the Production and Quality of In Vitro Produced Bovine Embryos." Diss., Virginia Tech, 2020. http://hdl.handle.net/10919/105143.
Full textAbstract:
Initial studies in this work explored the role of interleukin-6 (IL6) and leukemia inhibitory factor (LIF) in preimplantation bovine embryos. Neither cytokine affected the total percentage of embryos which developed to the blastocyst stage in vitro. However, supplementation of IL6 increased blastocyst inner cell mass (ICM) cell number without affecting trophectoderm (TE) cell number. Additionally, we found that IL6 activated signal transducer and activator of transcription 3 (STAT3) specifically within ICM cells. LIF, however, did not affect ICM cell number or activate STAT3 in ICM cells, and was not pursued further. This increase in ICM cell number by IL6 was largely comprised of hypoblast (GATA6+:NANOG-) cells, and most IL6-responsive cells in day 9 blastocysts were hypoblast cells (as measured by STAT3 activation). However, some epiblast (NANOG+) cells were also IL6-responsive, and IL6 appeared to initially slow epiblast differentiation. Finally, IL6-treated blastocysts also had increased transcripts of hypoblast/primitive endoderm (PE) markers. These results indicate that IL6 may improve pregnancy retention of IVP embryos by improving yolk sac development, but further work is needed to confirm this theory.
Activation of STAT3 by IL6 could be blocked with a chemical Janus kinase 2 (JAK2) inhibitor (AZD1480). JAK2 inhibition from day 5 to 8 resulted in blastocyst ICMs with fewer than 10% the normal cell number, regardless of IL6 supplementation. This indicates that STAT3 is critical for bovine ICM development. Further analysis revealed that inhibition of JAK2/STAT did not prevent ICM formation but disrupted its maintenance.
Additionally, we assessed the suitability of zinc sulfate and a bovine embryonic stem cell culture media (TeSR) for improving bovine embryo development in vitro. Zinc sulfate increased day 8 blastocyst total and ICM cell number. Therefore, zinc sulfate appears to improve blastocyst quality. The TeSR medium improved embryo development beyond day 8. In normal synthetic oviduct fluid, blastocysts degenerated after day 8, while blastocysts moved to TeSR had greatly increased cell numbers, and even exhibited PE migration out from the ICM, a phenomenon that has not been reported in vitro. This indicates that extended blastocyst culture is possible with TeSR media.Doctor of Philosophy
Bovine embryos have been produced in vitro for the purpose of being transferred to recipient cattle to produce a calf since the 1980s. This practice allows cattle breeders to increase the number of offspring from their best females each year, and also allows for more rapid progress in generational genetic improvement. However, only approximately 10% of bovine oocytes survive and produce a calf. This poor efficiency of bovine in vitro embryo production negatively impacts the procedure's widespread use. A significant portion of these embryo losses are likely a result of inadequate in vitro culture conditions, particularly of the embryo culture media, the fluid in which embryos are grown. This media is often called "synthetic oviduct fluid," or SOF, because it is designed to mimic the fluid present in the cow's oviduct, where the embryo would normally reside. However, SOF is much simpler in nature than actual cow oviduct fluid, and this leads to reduced embryonic survival of in vitro produced embryos. Unfortunately, we know very little of what molecules control and promote bovine embryo development. Therefore, one major goal of bovine embryo research is to identify these factors and add them to SOF. The goal of this work was to examine the ability of three molecules, interleukin-6 (IL6), leukemia inhibitory factor (LIF), and zinc sulfate, to increase the number and quality of blastocysts produced through in vitro culture techniques. Additionally, I tested the replacement of SOF with a complex cell culture media, known as TeSR. This medium is more complex than SOF, and therefore should better promote embryo development. This work revealed that IL6, but not LIF, improves in vitro produced (IVP) bovine blastocyst quality. Unfortunately, neither IL6 nor LIF affected the percentage of embryos which survived to the blastocyst stage. However, IL6, but not LIF, increased the number of cells in the inner cell mass (ICM) of the blastocysts. ICM cells are the portion of the embryo which will produce the future calf. IVP bovine embryos are known to have fewer cells than normal, in vivo derived, blastocysts, and this issue is believed to cause some embryonic death after embryo transfer. Therefore, treatment with IL6 may increase the percentage of embryos which will survive after transfer and produce a calf. We also found the addition of zinc sulfate to SOF to benefit embryo quality. None of the concentrations of zinc significantly improved the percentage of embryos which survived to the blastocyst stage, but 2 µM zinc did increase ICM cell number. Like IL6, this may improve embryo survival after transfer. The use of the TeSR media as a replacement for SOF had some benefits. Unfortunately, this media is unusable for producing embryos for transfer to recipients, as we discovered early embryos could not survive in the media. However, blastocyst-stage embryos thrived in it, and could be cultured in vitro for a longer period of time as a result. Therefore, this media will be a useful tool for studying bovine embryo development in vitro, however it is unlikely to benefit calf production. In summary, this work provides evidence that zinc sulfate and IL6 are beneficial additions to SOF. However, future work is needed to determine if embryos produced with these factors are more able to produce a calf. Additionally, we discovered that TeSR is a superior extended blastocyst culture medium.