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1
Voss, Oliver Paul. "AMPA receptor potentiators : mechanisms of neuroplasticity." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/25276.
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The AMPA receptor potentiator LY404187 is able to significantly increase the average length of neuritic processes in the neuroblastoma cell line SH-SY5Y only in the presence of s-AMPA, and this response is dependent on AMPA receptor activation. The compound also increases neurofilament protein levels as well as levels of the BDNF receptor Trk-B. The increase in neuritic length is blocked by addition of an antibody specific for BDNF indicating that this neurotrophin is required for the induction of neurite growth. The ability to induce morphological change in neuronal processes of the compound was then tested in a rodent model of lesions and sprouting. Unilateral ibotenic lesions of the entorhinal cortex in mice produce a progressive and substantial loss of synapses in the molecular layer of the dentate gyrus. Twice daily s.c. injections of LY404187 for 14 and 28 days post-lesion did not produce any significant change in synaptophysin immunoreactivity in the dentate gyrus. There was also no change in the volume of the lesion in the entorhinal cortex. In a secondary study the rate of neurogenesis in the dentate gyrus was also measured. Administration of LY404187 failed to induce a change in the number of BrdU +ve cells within the sub-granular zone of the dentate gyrus. Any long term structural of behavioural change caused by prolonged AMPA receptor potentiation is likely to be underpinned by changes in protein expression. The levels of key proteins involved in the intracellular response to AMPA receptor activation were measured by Western Blot and immunohistochemistry and levels of the neurotransmitters dopamine and serotonin were measured by HPLC. The effect of chronic administration to the AMPA receptor potentiator LY450108 on the rate of neurogenesis and the development of newly born neuron in the hippocampus was also investigated.
2
Krishnan, Ramya. "Characterization of Novel Small Molecule Potentiators of Oncolytic Virotherapy." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37551.
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The use of oncolytic viruses (OVs) to selectively destroy cancer cells is poised to make a major impact in the clinic and potentially revolutionize cancer therapy. Pre-clinical and clinical studies have shown that OV therapy is safe, well-tolerated and effective in a broad range of cancers. Still, resistance due to tumour heterogeneity highlights areas for improvement in OV based therapeutics. Combining OVs and small molecules is a promising strategy to selectively enhance OV-mediated anti-tumour effects. To this end, we have previously identified the synthetic compound Viral Sensitizer 1 (VSe1) that enhances the spread of oncolytic vesicular stomatitis virus (VSVΔ51) in resistant cancer cell lines up to 1000-fold, resulting in synergistic cell killing and improved efficacy in vitro and in vivo. The electrophilic nature of VSe1 prompted us to investigate the scaffold to identify active analogs with more favourable physiochemical properties and explore structure-activity relationships (SAR). In vitro assays and a rational approach in the design of VSe1 analogs allowed us to identify functional groups that can be modified without hampering activity. Lead compounds created in this study based on a pyrrole scaffold increase OV growth up to 2000-fold in vitro and demonstrate remarkable selectivity for cancer cells over normal tissue ex vivo and in vivo. Compared to the parental VSe1, these small molecules also possess enhanced stability with reduced electrophilicity and are well-tolerated in animals, leading to reduced tumour burden and prolonged survival in vivo when used in combination with VSVΔ51.
It was known from previous studies that VSe1 suppresses the type I interferon response generated by cancer cells to defend against viral infection. In this study, further investigation revealed that VSe1 and its analogs inhibit the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), resulting in dampened transcriptional expression and secretion of IFN-β and interferon stimulated genes, thereby increasing viral replication and spread. While these findings further elucidated the effect these compounds have on the innate antiviral response, the molecular mechanisms leading to NFκB inhibition remained unclear. We used the newly generated VSe1 analogs to perform ligand-based affinity capture studies leading to the identification of glutathione-s-transferases as interacting proteins, catalytically inhibited by VSe1 and to a lesser extent by its pyrrole analogs. Further inquiry revealed that VSe1 and its analogs cause an imbalance in cellular glutathione homeostasis and increase oxidative stress, which is associated with inhibition of the nuclear translocation of NFκB. However, further studies are required to assess whether these phenomena are directly or indirectly linked.
Overall, this study highlights a novel approach to improving OV therapy by using a previously uncharacterized class of compounds that ultimately alter the innate cellular antiviral response through inhibition of NFκB.
3
El-Sayes, Nader. "Small Molecule Potentiators of Oncolytic Virus Therapy Suppress the Innate Antiviral Response." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37115.
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Oncolytic Viruses (OVs) are often attenuated to increase their safety profile, however this can lead to reduced efficacy in heterogeneous malignancies and result in resistance to OV therapy. Our group utilizes small molecule enhancers of OV therapy termed viral sensitizers. These small molecules have been shown to enhance the replication and spread of oncolytic rhabdovirus VSVΔ51 in vitro and prolong survival in tumour-bearing mice. In this study, we evaluate the ef-fect of these viral sensitizers on the innate antiviral response in order to identify the mechanism of action responsible for their viral-sensitizing properties. Our previous data suggest that VSe1 and its structural analogues affect the type I IFN antiviral response and have the potential to af-fect cellular redox homeostasis. We hypothesized that VSe1 and its structural analogues potenti-ate VSV∆51 activity by inhibiting the type I IFN response via redox-mediated dysregulation. In this study, we demonstrate that the viral sensitizers inhibit the nuclear translocation and transcrip-tional activity of NFκB, which in turn dampens the expression of antiviral cytokines IFN-, TNFα and IL-6. We also provide evidence supporting the possibility that the NFκB inhibition may be a result of the formation of ROS intermediates by the viral sensitizers, which leads to re-duced nuclear translocation of NFκB subunits, thereby preventing NFκB-mediated cytokine production. Overall, this work contributes to the identification of the mechanism of action of our viral sensitizers and highlights the finding that oncolytic VSV infection can be enhanced through redox-mediated modulation of the innate antiviral response.
4
Kinting, Susanna [Verfasser], and Heinrich [Akademischer Betreuer] Leonhardt. "Functional rescue of mutant ABCA3 by correctors and potentiators / Susanna Kinting ; Betreuer: Heinrich Leonhardt." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1204005281/34.
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Quintela, Varela Hugo [Verfasser], and Dirk [Akademischer Betreuer] Trauner. "Biomimetic synthesis of (−)-PF-1018 and development of photoswitchable GABAA receptor potentiators / Hugo Quintela Varela ; Betreuer: Dirk Trauner." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1221960458/34.
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Mareux, Elodie. "Pharmacothérapie ciblée des déficits en ABCB11." Electronic Thesis or Diss., université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL083.
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ABCB11/BSEP (Bile Salt Export Pump) est exprimé à la membrane canaliculaire des hépatocytes. Sa fonction de transport d’acides biliaires dans la bile est essentielle à la sécrétion biliaire. Près de 400 variations du gène ABCB11 ont été identifiées et sont associées à des maladies hépatobiliaires rares, la plus sévère étant la cholestase intrahépatique progressive familiale de type 2 (PFIC2). L’efficacité des traitements médicaux est limitée. Par conséquent, une transplantation hépatique est indiquée avant l’âge adulte pour près de deux tiers des patients. Dans ce contexte, l’identification de thérapies alternatives est un enjeu capital.Cette thèse s’intéresse à la recherche de stratégies thérapeutiques personnalisées permettant de corriger les conséquences pathologiques de certaines variations d’ABCB11 identifiées chez des patients. Dans le cadre d’une stratégie de traitement par des molécules potentiatrices, nous avons étudié les variations A257V, G562D et T463I d’ABCB11 par modélisation moléculaire 3D. L’étude de l’expression et de la fonction de ces variants dans différents modèles cellulaires a confirmé que ces variations étaient responsables d’un défaut de fonction du transporteur Abcb11. L’ivacaftor (VX 770, Kalydeco®), approuvé cliniquement pour le traitement de la mucoviscidose, corrigeait le défaut d’activité de ces trois variants.Des effets similaires ont été observés avec les molécules GLPG1837, SBC040 et SBC219, connues comme potentiateurs de CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Dans une optique de thérapie combinatoire, nous avons également mis en évidence la capacité de ces potentiateurs à corriger le défaut de fonction des variants R1090C et R1090W, produits potentiels de la translecture du variant non-sens R1090X. Nous avons également évalué les molécules correctrices elexacaftor (VX-445) et tezacaftor (VX-661). Ces correcteurs, en monothérapie ou en combinaison, permettaient de restaurer l’adressage du variant R1128C, s’accompagnant d’une augmentation significative du transport de taurocholate. De façon intéressante, l’addition de molécules potentiatrices réduisait ces effets.L’ensemble de ces travaux constitue une preuve de concept que les défauts de certains variants d’ABCB11 peuvent être corrigés par ces molécules potentiatrices à haut potentiel thérapeutique. Ce type de traitements pourrait être envisagé pour les patients atteints de déficit en ABCB11 et permettrait ainsi d’augmenter la pharmacopée disponible pour traiter ce genre de pathologies et ainsi repousser voir palier à la transplantation hépatique pour les cas les plus sévèresABCB11/BSEP (Bile Salt Export Pump) is expressed at the canalicular membrane of hepatocytes. It ensures bile acids secretion into bile which is essential for biliary secretion. Nearly 400 variations of the ABCB11 gene have been identified and are associated with rare hepatobiliary diseases, the most severe being progressive familial intrahepatic cholestasis type 2 (PFIC2). The effectiveness of medical treatments is limited. Consequently, liver transplantation is required before adulthood for almost 2/3 of PFIC2 patients. In this context, the identification of alternative therapies is a major challenge.This thesis focuses on personalized therapeutic strategies to correct the pathological consequences of some ABCB11 variations identified in patients. The A257V, G562D and T463I variations of ABCB11 were studied by 3D molecular modelling. These variations were responsible for a defect in Abcb11 transport function. Ivacaftor (VX-770, Kalydeco®), a clinically approved cystic fibrosis treatment, corrects the activity defect of the three variants.Similar effects were observed with GLPG1837, SBC040 and SBC219, known as potentiators of CFTR (Cystic Fibrosis Transmembrane Conductance Regulator).From a combinatory therapy perspective, we also demonstrated the ability of these potentiators to correct the transport defect of the R1090C and R1090W variants, potential readthrough products of the R1090X nonsense variant. We also evaluated the ability of Elexacaftor (VX-445) and Tezacaftor (VX 661) correctors of CFTR. These correctors, alone or in combination, restored trafficking of the R1128C missense variant, leading to a significant increase in the transport function. Interestingly, the addition of potentiators abolishes this effect.Altogether, this thesis constitutes a proof of concept that molecules with high therapeutic potential can correct the molecular defects of ABCB11 variants. These treatments could increase the pharmacopoeia available for patients with ABCB11 deficiency and thus delay or even suppress the need for liver transplantation
7
Hanoteau, Aurélie. "Chemotherapy potentiates immune responses against murine tumors." Doctoral thesis, Universite Libre de Bruxelles, 2016. https://dipot.ulb.ac.be/dspace/bitstream/2013/231745/5/Thesis.pdf.
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There is increasing evidence that the effect of chemotherapy on tumor rejection is not cell autonomous but relies on the immune system. Indeed, several reports have shown that human and murine tumors respond to chemotherapeutic agents more efficiently when the host immune system is intact. In particular, we have shown that cyclophosphamide treatment of DBA/2 mice bearing P815 mastocytoma induces rejection and long term protection in a CD4- and CD8-dependent manner. We used this tumor model, as it is poorly immunogenic, expresses tumor-associated P1A and tumor-specific P1E antigens, encoded by germline and mutated genes, respectively, and allows the identification of some tumor-specific CD8+ T cells.We have previously reported that tumor regression correlates with selective infiltration of CD8+ T cells specific for P1E/H-2Kd antigen in tumor bed upon cyclophosphamide treatment. Unexpectedly, the proportion of CD8+ T cells specific for the tumor-associated antigen P1A in the context of H-2Ld decreases concomitantly, indicating that cyclophosphamide alters the repertoire of CD8+ T cells recognizing tumor antigens. Using P1A KO mice, we found that preferential activation of CD8+ T cells to P1E is not solely due to thymic negative selection. The major role of “mutated” antigens in tumor resistance has been recently highlighted in humans and raises an interesting question about the immune mechanisms of tumor rejection. Additionally to its effect on the specific immune response, cyclophosphamide promotes tumor infiltration by effector memory (P1E/H-2Kd)+ CD8+ T cells which are characterized by higher expression of KLRG1 and Eomes. Our data point to a role of IL-15 and type 1 IFNs for their development, as increased levels of IL-15 and IRF7 were measured in tumor after cyclophosphamide. IFNAR1 blockade interferes with the tumor rejection in 50% of mice and decreases the (P1E/H-2Kd)+ CD8+ T cell infiltration induced by cyclophosphamide, suggesting a role of this cytokine in the expansion and/or recruitment of (P1E/H-2Kd)+ CD8+ T cells in vivo.Altogether, our results suggest that type 1 IFNs and IL-15 induced after cyclophosphamide promote the reprogramming of CD8+ T cells specific for the “mutated” P1E/H-2Kd antigen into effector memory lymphocytes.Option Biologie moléculaire du Doctorat en Sciences
info:eu-repo/semantics/nonPublished
8
Khalil, Dayekh. "Novel Combination Therapy: Monensin Potentiates Erlotinib-Induced Cytotoxicity." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/24405.
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Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced
in the past decade as a promising therapeutic option in Head and Neck Squamous Cell
Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result,
RTK inhibitors require a combination based therapeutic approach with other treatment
modalities. To uncover such a combination of agents, we performed a high throughput
Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC
cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the
cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of
Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous
protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis
suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol
biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.
9
Lortie, Karine. "The growth-arrest-specific protein gas7 potentiates neuronal differentiation." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/26701.
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The growth-arrest-specific gas7 protein is involved in neuronal development. Its role in neuronal differentiation and its potential neuroprotective activity were investigated in PC 12 and NT2 cells. gas7 overexpression in PC12 cells promoted neurite outgrowth and potentiated nerve growth factor-induced expression of the neuronal markers betaIII-tubulin, synaptotagmin, alpha7 subunit of the acethylcholine receptor, and dihydropyrimidinase related protein-3. This effect was exerted independently of cellular proliferation, as gas7 did not affect cell cycle progression. Endogenous gas7 expression was induced during neuronal differentiation of NT2 cells with retinoic acid, suggesting a role for gas7 in neuronal development. Finally, gas7 overexpression in PC 12 cells did not protect against toxicity triggered by oxygen-glucose deprivation, the calcium ionophore A23187 or sodium nitroprusside. The ability of gas7 to potentiate neuritogenesis and neuronal differentiation makes it a potential therapeutic target to promote re-establishment of neuronal connections in the injured or diseased brain, such as following stroke.
10
Seufert, Florian [Verfasser], and Ulrike [Gutachter] Holzgrabe. "Entwicklung von Inhibitoren des „macrophage infectivity potentiator“-Proteins / Florian Seufert. Gutachter: Ulrike Holzgrabe." Würzburg : Universität Würzburg, 2016. http://d-nb.info/1112041222/34.
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Ho, Wing-tak, and 何永德. "Glycyrrhizic acid potentiates dsRNA-induced nitric oxide generation inalveolar macrophages." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31971799.
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Ho, Wing-tak. "Glycyrrhizic acid potentiates dsRNA-induced nitric oxide generation in alveolar macrophages." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31971799.
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13
Nakamura, Takanori. "Disruption of multidrug and toxin extrusion MATE1 potentiates cisplatin-induced nephrotoxicity." Kyoto University, 2011. http://hdl.handle.net/2433/142112.
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Liang, Xiaoting, and 梁小婷. "Activation of NRG1-ERBB4 signaling potentiates mesenchymal stem cell-mediated myocardial repairs." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2015. http://hdl.handle.net/10722/208584.
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Mesenchymal stem cell (MSC) transplantation has achieved only modest success in the treatment of ischemic heart disease due to poor cell viability in the diseased microenvironment. Genetic manipulation on the MSCs holds promising prospects in enhancing cell tolerance against adverse environmental conditions.
Recent studies demonstrate that the activation of the NRG1 (neuregulin 1) - ERBB4 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) pathway can enhance pro-survival signaling, stimulate mature cardiomyocyte cell cycle re-entry and cell division. In this study, I aimed to determine whether activating NRG1-ERBB4 in MSCs can enhance their cardioprotective effects following myocardial infarction.
In chapter 3, I determined that MSC endogenously expresses NRG1, but not ERBB4. Considering the absence of ERBB4 in the MSCs might lead to mute response to its ligand NRG1, I exogenously manipulated ERBB4 into MSCs.
In chapter 4, MSCs, with or without ERBB4 overexpression were transplanted into mice following myocardial infarction. The transplantation of MSCs with ERBB4 expression considerably improved left ventricular ejection fraction and reduced infarctsize, compared to unmodified MSCs and direct NRG1 injection. ERBB4 overexpression induced greater MSC survival following infarction. The transduction of ERBB4 in MSCs increased cell mobility and apoptotic resistance via a PI3K/Akt pathway under hypoxic conditions in the presence of NRG1. The transplantation of MSCs with ERBB4 expression induced cardiomyocyte division and protected them against apoptosis during early phase of infarction.
In chapter 5, a novel autocrine loop regarding to NRG1-ERBB4-NRG1 signaling was identified. MSCs with ERBB4 overexpression in turn increased NRG1 synthesis and secretion. Conditioned medium of ERBB4-expressing MSCs containing elevated NRG1, promoted cardiomyocyte growth, division and anti-senescence, whereas neutralization of NRG1 blunted these effects. Injecting ERBB4-expressing MSCs restored NRG1 in the infarcted myocardium to a level comparable with that of the normal myocardium.
These findings collectively suggest overexpressing ERBB4 in MSCs enhances the effectiveness of MSCtherapy following myocardial in farction through potentiating MSC survival and revitalizing endogenous repair and regeneration. The combination of ERBB4 and MSC is more efficient than naïve MSC or solely recombinant NRG1 injection, emerging as potential target for developing novel strategy in treating myocardial diseases.published_or_final_version
Medicine
Doctoral
Doctor of Philosophy
15
Taylor, Marlon D., and Marlon D. Taylor. "Sulforaphane Potentiates Non-Melanoma Skin Cancer in UVB-Treated Nrf2 Knockout Mice." Thesis, The University of Arizona, 2016. http://hdl.handle.net/10150/622859.
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Sulforaphane is a natural product found in cruciferous vegetables which is known to have many chemopreventive properties including the induction of apoptosis and the inhibition of inflammation, cellular proliferation, and reactive oxygen species (ROS) formation. The reduction of ROS activity by sulforaphane is likely linked to the activation of NF-E2 related factor-2 (Nrf2), a transcription factor involved in cytoprotection against ROS and electrophilic stress. The skin is particularly vulnerable to oxidative stress caused by ultraviolet (UV) light, which is an established complete carcinogen. Sulforaphane has been shown to reduce both chemical and UVB-induced skin carcinogenesis in mouse models. Suppression of DMBA/TPA-induced skin tumorigenesis by sulforaphane has been shown to be dependent upon Nrf2 activity. Additional studies have shown that genetic activation of Nrf2 can protect keratinocytes against UVB-induced ROS. Nrf2 has also been implicated in regulating inflammatory responses after UVB exposure in the skin. However, the role of Nrf2 in the antitumorigenic activity of sulforaphane in the context of UVB-induced skin tumors is not well understood. We therefore performed murine experiments in order to clarify whether sulforaphane requires Nrf2 in order to block UVB-induced non-melanoma skin cancer. Consistent with the literature, we observed that wildtype (WT) mice topically treated with sulforaphane were less susceptible to UVB-induced tumor incidence and tumor burden compared to the vehicle control WT group. However, Nrf2 KO mice treated with sulforaphane presented with significantly greater UVB-induced tumor incidence and burden compared to the WT sulforaphane group, suggesting that sulforaphane may potentiate tumorigenesis in the context of UVB exposure if Nrf2 is absent. We therefore performed acute in vivo and in vitro experiments using topical sulforaphane (as per the tumor experiment) to investigate why Nrf2 KO mice developed more tumors than WT mice during UVB and sulforaphane treatment. Topical treatment of SKH-1 mice with sulforaphane did result in slight reduction of UV-induced epidermal hyperplasia in wildtype mice which was not present in Nrf2 KO mice (trends were not significant). Surprisingly, while wildtype mice developed significantly more epidermal inflammation in our acute treatment model than did the Nrf2 KO strain (as measured by skin fold thickness), inflammation was not significantly influenced by topical sulforaphane treatment in either strain of mice. However, cell culture studies using primary mouse keratinocytes indicate that sulforaphane’s ability to block UVB-induced ROS is lost in Nrf2 KO cells. Taken together, our ROS data may strengthen the hypothesis that sulforaphane increases the oxidative stress of cells during UVB treatment in the absence of Nrf2.
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Scarpa, Richard C. "Neurotensin potentiates the proliferative effects of growth factors in human embryonic lung fibroblasts /." Thesis, Connect to Dissertations & Theses @ Tufts University, 2004.
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Thesis (Ph.D.)--Tufts University, 2004.Adviser: David E. Cochrane. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 137-165). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
17
Vono, Maria. "The adjuvant MF59 induces ATP release from muscle that potentiates response to vaccination." Doctoral thesis, Università degli studi di Padova, 2014. http://hdl.handle.net/11577/3423482.
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Vaccines are the most effective agents to control infections [1]. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance the specific immune responses. Despite their effectiveness and their wide use, the mechanism of action of many adjuvants is poorly characterized [2]. Therefore, adjuvant research is crucial to better understand how they work and to exploit their full potential in vaccinology [3]. Release of endogenous danger signals has been linked to adjuvanticity, however the role of extracellular ATP during vaccination has never been explored. Extracellular ATP can work as "danger signal" and, as such is a strong modulator of immune responses [4-6]. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminium hydroxide, calcium phosphate (CaPi), incomplete Freund’s adjuvant (IFA) and the squalene-based oil in water emulsion MF59.
Experiments were performed ex vivo in excised mice muscles (tibialis anterior and quadriceps) and in vivo in live mice injected with the reporter system luciferase-luciferin that reports on ATP changes. We found that intramuscular injection in general is always associated to a weak transient release of ATP. In contrast, a greatly enhanced ATP release was found upon injection of MF59 but not by all other adjuvants tested.
Therefore, we wanted to dissect whether and how ATP release would contribute to the activity of MF59. The strong adjuvanticity of MF59 [7-8] has been ascribed to its capability to induce an immunocompetent environment in the muscle, characterized by a rapid and transient influx of a large number of immune cells participating in antigen uptake and transport to draining lymph nodes [9-11]. We found that the local injection of apyrase, an ATP-hydrolyzing enzyme, reduced the immune cells recruitment induced by MF59 but not by alum or IFA. These findings indicated that the ability of MF59 to induce migration of different immune cells into the injected muscle is partly due to induced ATP release. Moreover, co-injection of apyrase and MF59 at the muscle injection site reduces the number of antigen positive cells in the draining lymph nodes in a cell type-specific manner. Indeed, co-injection of apyrase negatively impacts the number of antigen positive B cells induced by MF59, suggesting that B cells could be a key component in ATP-mediated signaling during vaccination. Strong innate immune responses lead to enhanced adaptive immune responses [12]. Accordingly, we compared the impact of MF59-induced ATP release on T cells responses and antibody titers. Groups of mice were immunized with an experimental trivalent influenza vaccine (TIV) either as plain antigens or together with MF59 with or without apyrase. Apyrase strongly inhibited influenza specific T cell responses, total IgG and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link for the first time extracellular ATP to an enhanced response to vaccination.I vaccini rappresentano senza dubbio l’arma più efficace per combattere e tenere sotto controllo le infezioni [1]. In aggiunta agli antigeni del patogeno, i vaccini contengono adiuvanti utilizzati per potenziare le risposte immunitarie specifiche verso determinati antigeni. Nonostante la loro efficacia e il loro largo uso, il meccanismo di azione di molti adiuvanti è ancora scarsamente caratterizzato [2]. Pertanto, far luce sui meccanismi d’azione degli adiuvanti vaccinali è fondamentale per sviluppare prodotti nuovi, più efficienti e sicuri, e poter così sfruttare appieno il potenziale della vaccinologia [3]. Dopo la vaccinazione, è stato osservato al sito di iniezione il rilascio locale di molecole endogene con la capacità di segnalare “danno” al sistema immunitario, note come allarmine. Per esempio, un rilascio locale di acido urico e DNA è stato osservato nel modello murino dopo vaccinazione con alum, il più diffuso tra gli adiuvanti approvati per uso sull’uomo. Tuttavia, finora non è mai stato esplorato un potenziale ruolo dell’ATP durante la vaccinazione. L’ATP, tra le sue tante funzioni, quando rilasciato nell’ambiente extracellulare in concentrazioni opportune può fungere da allarmina e, come tale è un forte modulatore delle risposte immunitarie [4-6]. Pertanto, in questo lavoro abbiamo indagato se un rilascio di ATP è coinvolto nel meccanismo d’azione di quattro comuni adiuvanti vaccinali: idrossido di alluminio (alum), calcio fosfato (CaPi), adiuvante incompleto di Freund (IFA) e MF59. Sono stati condotti esperimenti ex vivo su muscoli murini isolati (tibiale anteriore e quadricipite) e in vivo in topi immunizzati intramuscolo con l’adiuvante da testare e il sistema reporter luciferina-luciferasi in grado di segnalare il livello di ATP al sito d’iniezione. Abbiamo osservato che l'iniezione intramuscolare è sempre associata a un debole e transitorio rilascio di ATP. Il rilascio basale di ATP è notevolmente potenziato dall’iniezione di MF59 ma non dagli altri adiuvanti testati. Pertanto, abbiamo esplorato se e come il rapido e transitorio rilascio di ATP indotto da MF59 al sito d’iniezione potesse contribuire al suo meccanismo d’azione. Il forte potere adiuvante di MF59 [7, 8] è stato attribuito alla sua capacità di istituire un ambiente immunocompetente al sito di iniezione nel muscolo, caratterizzato da un rapido e transitorio afflusso di un gran numero di cellule immunitarie che captano e assorbono l’antigene e lo trasportano ai linfonodi drenanti [9-11]. Abbiamo qui dimostrato, che la co-iniezione di apirasi, un enzima in grado di idrolizzare l’ATP, riduce fortemente l’afflusso di cellule immunitarie indotto da MF59 ma non quello indotto da alum o IFA. Questi risultati indicano che l’abilità di MF59 di indurre un forte afflusso di cellule immunitarie al sito di iniezione è in parte dovuta alla sua intrinseca capacità di rilasciare ATP. Inoltre, abbiamo osservato che la co-iniezione di apirasi e MF59 riduce il numero di cellule antigene-positive che dal muscolo raggiungono i linfonodi drenanti. Tale riduzione si è rivelata tipo cellulare-specifica, infatti il trattamento con apirasi impatta negativamente il numero di cellule B antigene-positive indotto da MF59 nei linfonodi drenanti, suggerendo che le cellule B potrebbero essere un elemento chiave nei “pathways” mediati da ATP durante la vaccinazione. Efficienti risposte immunitarie di tipo innato si traducono spesso in forti risposte adattative [12]. Pertanto, abbiamo analizzato un eventuale ruolo dell’ATP rilasciato da MF59 sull’attivazione delle cellule T e la produzione di titoli anticorpali antigene-specifici. Di conseguenza, gruppi di topi sono stati immunizzati con un vaccino influenzale trivalente, iniettato come tale o adiuvato con MF59 con o senza apirasi. L’apirasi ha fortemente ridotto la proliferazione delle cellule T vaccino-specifiche e i relativi titoli anticorpali. Questi dati dimostrano che un locale e transitorio rilascio di ATP a livello del sito d’iniezione è necessario per lo sviluppo di risposte immunitarie innate e adattative indotte da MF59 e associano per la prima volta un rilascio extracellulare di ATP a un potenziamento delle risposte immunitarie indotte dalla vaccinazione.
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Bechtel, Cale. "Back squat potentiates both vertical and horizontal jump performance in collegiate ice hockey players." Thesis, California State University, Long Beach, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10638622.
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Back squats (BSQ) have been shown to effectively potentiate lower body power in a subsequent performance activity. There is a plurality of post activation potentiation (PAP) studies in which the BSQ and vertical jump (VJ) are used. To date, there is little information regarding BSQ and horizontal jump (HJ) performance. Nine collegiate ice hockey players from the California State University, Long Beach ice hockey team volunteered for the study. Participants performed five testing sessions separated by 96 hours. The first testing session was a one repetition maximum (1RM) BSQ to assign the athletes specific intensity. The intensity chosen was 87% of the athletes’ 1RM, which means they should complete five repetitions (87%) for the potentiated testing sessions. The four testing sessions were randomized consisting of a back squat followed by horizontal jump (BSQ-HJ), back squat followed by vertical jump (BSQ-VJ), horizontal jump only (CT-HJ) and vertical jump only (CT-VJ). During the potentiated conditions participants had a rest interval of 5 minutes between the BSQ and VJ or HJ. Alpha-level was set a priori at 0.05. The results indicate that both vertical (p = 0.017) and horizontal (p = 0.003) jump were significantly increased (VJ = +5.51cm, HJ = +11.55cm). The present study helps indicate that muscular power performance can be improved in VJ and HJ using the PAP training phenomenon in collegiate ice hockey players.
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Miller, Laurence L. "A competitive NMDA receptor antagonist potentiates the effects of morphine on spatial and discrimination learning /." Electronic version (PDF), 2005. http://dl.uncw.edu/etd/2005/millerl/laurencemiller.pdf.
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Kuk, Raafat. "Lestaurtinib (CEP-701) Potentiates the Anticonvulsant Effect of Phenobarbital against Kainic Acid-induced Status Epilepticus." Thesis, The University of Arizona, 2018. http://hdl.handle.net/10150/626859.
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Chan, Hoi-ching, and 陳凱靜. "Heat shock protein 90 inhibitor 17-AAG potentiates anticancer activityof bortezomib in NK cell malignancies." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46632025.
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Zhang, Wei. "LOSS OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 1 (MRP1/ABCC1) POTENTIATES DOXORUBICIN-INDUCED CARDIOTOXICITY IN MICE." UKnowledge, 2015. http://uknowledge.uky.edu/toxicology_etds/12.
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Doxorubicin (DOX) is a broad-spectrum and effective chemotherapeutic agent, but its use in oncologic practice is limited by dose-dependent cumulative cardiotoxicity. DOX-induced cardiotoxicity is in large part due to its ability to cause oxidative stress. Multidrug resistance associated protein 1 (MRP1/ABCC1) is a member of the ATP-binding cassette (ABC) transporter superfamily. By effluxing a wide variety of endogenous and exogenous substrates, Mrp1 plays important physiological roles in multiple tissues and also protects normal tissues against toxicants. However, the role of MRP1 in heart is largely unknown.
The role of Mrp1 in DOX-induced cardiotoxicity was investigated in Mrp1 null (Mrp1-/-) and their C57BL (WT) littermates. Chronic DOX caused body weight loss and hemotoxicity, and these adverse effects were significantly exacerbated in Mrp1-/- vs WT mice. Importantly, loss of Mrp1 potentiated DOX-induced cardiotoxicity, presenting as worsened cardiac function and more cellular apoptosis in DOX treated Mrp1-/- mice. Mrp1 also protected neonatal mouse cardiomyocytes (CM) and cardiac fibroblasts (CF) culture against DOX cytotoxicity in vitro. This was demonstrated by the decreased cell survival, more apoptosis and more DNA damage in DOX treated Mrp1-/- vs WT cells.
In addition, the effects of deletion of Mrp1 was studied on glutathione (GSH)/glutathione disulfide (GSSG) homeostasis, glutathione conjugate of 4-hydroxy-2-nonenal (GS-HNE) accumulation, protein oxidative damage and expression of antioxidant enzymes. Loss of Mrp1 led to significantly higher GSH and GSSG basal levels in heart. Following DOX treatment, Mrp1-/- CM and CF showed increased GSH and GSSG levels vs WT cells. Meanwhile, DOX increased expression of the GSH synthesis enzymes in Mrp1-/- but not WT cells. Thus, increased GSH synthesis may contribute to the further increase in the GSH pool in DOX-treated Mrp1-/- cells. DOX induced comparable increases of GS-HNE concentration in WT and Mrp1-/- mice hearts. Finally, expression of extracellular superoxide dismutase (ECSOD/SOD3) was significantly lower in Mrp1-/- vs. WT CM treated with either saline or DOX.
In summary, this study is the first to document a protective role of Mrp1 in DOX-induced cardiotoxicity. It gives critical information regarding the potential adverse sequelae of introduction of MRP1 inhibitors as adjuncts to clinical chemotherapy of multidrug resistant tumors.
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Kahya, Hasan Faisal Hussein. "Removal of acetylation by pneumococcal esterases potentiates neuraminidase activity for mucin utilisation, colonisation and virulence." Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/38750.
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The genome of pneumococcal strains contains 4 putative esterase genes (SPD_0534, estA; SPD_0932; SPD_1239; and SPD_1506, axe). Esterases have been reported to be important for bacterial physiology and virulence in other microorganisms but their role in S. pneumoniae is unknown. We hypothesised that esterases potentiate neuraminidase activity by removing acetylation in sialic acid. This hypothesis was tested using isogenic mutants and recombinant esterases in microbiological, biochemical and In vivo assays. The results showed that pneumococcal esterases are specific for short acyl chains, all gene contributed to overall esterase activity but SPD_0534 (EstA) was found to be responsible for main esterase activity. Both Axe and EstA could use acetylated xylan and Bovine Sub-maxillary Mucin (BSM), a highly acetylated substrate, but only EstA was active against tributyrin (triglyceride). Incubation of BSM with either Axe or EstA led to the acetate release in a time and concentration dependent manner, and pretreatment of BSM with either EstA or Axe increased sialic acid release by subsequent exposure to neuraminidase. qRT-PCR results showed that the expression level of estA and axe increased when exposed to BSM and in respiratory tissues. Mutation of either estA alone or in combination with nanA (codes for neuraminidase A), or the replacement of serine 121 to alanine in EstA, reduced the pneumococcal ability to utilise BSM as sole carbon source, sialic acid (Sia) release, colonisation and virulence in a mouse model of pneumococcal infection.
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Knoch, Megan E. "Short Term Exposure to Light Potentiates Phase Shifting to Nonphotic Stimuli in the Syrian Hamster." Kent State University / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=kent1117224927.
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Arendt, Kristina Anna Maria [Verfasser], and Rudolf [Akademischer Betreuer] Hatz. "An in vivo inflammatory loop potentiates KRAS blockade / Kristina Anna Maria Arendt ; Betreuer: Rudolf Hatz." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1213658829/34.
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Rowe, R. K., G. I. Ellis, J. L. Harrison, A. D. Bachstetter, G. F. Corder, Eldik L. J. Van, B. K. Taylor, F. Marti, and J. Lifshitz. "Diffuse traumatic brain injury induces prolonged immune dysregulation and potentiates hyperalgesia following a peripheral immune challenge." SAGE Publications, 2016. http://hdl.handle.net/10150/614986.
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Background: Nociceptive and neuropathic pain occurs as part of the disease process after traumatic brain injury (TBI) in humans. Central and peripheral inflammation, a major secondary injury process initiated by the traumatic brain injury event, has been implicated in the potentiation of peripheral nociceptive pain. We hypothesized that the inflammatory response to diffuse traumatic brain injury potentiates persistent pain through prolonged immune dysregulation. Results: To test this, adult, male C57BL/6 mice were subjected to midline fluid percussion brain injury or to sham procedure. One cohort of mice was analyzed for inflammation-related cytokine levels in cortical biopsies and serum along an acute time course. In a second cohort, peripheral inflammation was induced seven days after surgery/injury with an intraplantar injection of carrageenan. This was followed by measurement of mechanical hyperalgesia, glial fibrillary acidic protein and Iba1 immunohistochemical analysis of neuroinflammation in the brain, and flow cytometric analysis of T-cell differentiation in mucosal lymph. Traumatic brain injury increased interleukin-6 and chemokine ligand 1 levels in the cortex and serum that peaked within 1-9 h and then resolved. Intraplantar carrageenan produced mechanical hyperalgesia that was potentiated by traumatic brain injury. Further, mucosal T cells from brain-injured mice showed a distinct deficiency in the ability to differentiate into inflammation-suppressing regulatory T cells (Tregs). Conclusions: We conclude that traumatic brain injury increased the inflammatory pain associated with cutaneous inflammation by contributing to systemic immune dysregulation. Regulatory T cells are immune suppressors and failure of T cells to differentiate into regulatory T cells leads to unregulated cytokine production which may contribute to the potentiation of peripheral pain through the excitation of peripheral sensory neurons. In addition, regulatory T cells are identified as a potential target for therapeutic rebalancing of peripheral immune homeostasis to improve functional outcome and decrease the incidence of peripheral inflammatory pain following traumatic brain injury.
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Hassanieh, Sarah. "CO-ADMINISTRATION OF SILDENAFIL POTENTIATES DOXORUBICIN-INDUCED APOPTOSIS IN PROSTATE CANCER: THE ROLE OF NF-kappaB." VCU Scholars Compass, 2008. http://scholarscompass.vcu.edu/etd/1655.
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Our recent studies have shown that that erectile dysfunction (ED) drugs including Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) enhance killing of several types of cancer cells by anticancer drug, Doxorubicin (DOX). We observed increased cell death by apoptosis in response to the combined treatment with ED drugs and DOX. However, the mechanism of such enhancement of cell death by combined treatment of ED drugs and DOX is not fully understood. Nuclear factor-κB (NF-κB) is an oxidant-sensitive transcription factor that plays a critical role in the immediate-early activation of a multitude of genes that have been documented to play critical role in programmed cell death (apoptosis). NF-κB activation has been shown to block apoptosis and its inhibition improves existing anti-oncogenic therapy such as chemotherapy. In the present study, we tested the hypothesis whether combined treatment of prostate cancer cells, PC3, with Sildenafil plus DOX would attenuate the activation of NFκB by inhibiting translocation of the p65 and p50 subunits to the nucleus and by phosphorylation of cytosolic IκB In addition, we investigated the effect of DOX and DOX plus Sildenafil on the expression of BCL family of proteins which play critical role in apoptosis. We treated PC3 cells with 1.5 μM DOX with or without 10 µM Sildenafil for 6 hours and 72 hours. The nuclear translocation of p65 and p50 and expression of BCL family of proteins was determined by western blot analysis. Our results show that combined treatment of DOX and Sildenafil significantly reduced the nuclear translocation of p65 and p50 as compared with DOX alone (P < 0.05). This correlated with the significant reduction in the expression of Bcl-2, BclxL and phosphorylation of BAD. These data provide an important mechanism by which Sildenafil treatment augments the apoptotic potential of DOX in PC3 cancer cells.
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Leipert, Jenny, Franziska Kässner, Susanne Schuster, Norman Händel, Antje Körner, Wieland Kiess, and Antje Garten. "Resveratrol Potentiates Growth Inhibitory Effects of Rapamycin in PTEN-deficient Lipoma Cells by Suppressing p70S6 Kinase Activity." Taylor & Francis, 2016. https://ul.qucosa.de/id/qucosa%3A38595.
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Patients with phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome and germline mutations in PTEN frequently develop lipomatosis, for which there is no standard treatment. Rapamycin was shown to reduce the growth of lipoma cells with heterozygous PTEN deficiency in vitro, but concomitantly induced an upregulation of AKT phosphorylation. Since it was shown that resveratrol stabilizes PTEN, we asked whether co-incubation with resveratrol could suppress the rapamycin-induced AKT phosphorylation in PTEN-deficient lipoma cells.
Resveratrol incubation resulted in decreased lipoma cell viability by inducing G1-phase cell cycle arrest and apoptosis. PTEN expression and AKT phosphorylation were not significantly changed, whereas p70S6 kinase (p70S6K) phosphorylation was reduced in PTEN-deficient lipoma cells after resveratrol incubation. Rapamycin/resveratrol co-incubation significantly decreased viability further at lower doses of resveratrol and resulted in decreased p70S6K phosphorylation compared to rapamycin incubation alone, suggesting that resveratrol potentiated the growth inhibitory effects of rapamycin by reducing p70S6K activation. Both viability and p70S6K phosphorylation of primary PTEN wild-type preadipocytes were less affected compared to PTEN-deficient lipoma cells by equimolar concentrations of resveratrol. These results support the concept of combining chemopreventive natural compounds with mammalian target of rapamycin (mTOR) inhibitors to increase the efficacy of chemotherapeutic drugs for patients suffering from overgrowth syndromes.
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Mandrusiak, Lisa. "Transglutaminase potentiates proteasome dysfunction induced by polyglutamine-expanded androgen receptor : a potential pathogenic mechanism for spinobulbar muscular atrophy." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79042.
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Research into CAG-triplet repeat expansion mutations responsible for human neurodegenerative disease is a rapidly expanding genetic field. The CAG repeat (encoding glutamine) expansion is thought to confer a novel, toxic gain of function to the protein that resulting in neuron-specific cell death. The mechanism of cytotoxicity in these diseases is unknown, but often centers on the shared pathological characteristic of protein aggregate formation. Recently, the calcium-dependent enzyme transglutaminase has been implicated in aggregate formation in several of these disorders. Cellular processes such as proteolysis, calcium homeostasis, gene transcription, and mitochondrial function are likely affected by polyglutamine-expanded proteins and contribute to pathogenesis.Expansion of the CAG repeat in the androgen receptor leads to the X-linked disorder spinobulbar muscular atrophy. To examine underlying disease mechanisms we investigated the link between polyglutamine-expanded androgen receptor and transglutaminase. We found N-terminal androgen receptor fragments to be a substrate for transglutaminase, and cells expressing polyglutamine-expanded androgen receptor exhibited ligand-depended proteasome dysfunction. This effect was prevented by the presence of cystamine, a transglutaminase inhibitor, suggesting involvement of a transglutaminase-catalyzed reaction in disease pathogenesis and providing a potential basis for the development of therapies for CAG-repeat expansion disorders.
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Pascal, Maud. "Innate Lymphoid Cells under Neuronal Control in the Small Intestine : vasoactive Intestinal Peptide potentiates ILC2 and ILC3 functions." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS318.
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L’intestin est une vaste surface de l’organisme constamment exposée aux substances ingérées et aux nombreux micro-organismes qui colonisent sa muqueuse. Afin d’assurer le maintien de son intégrité, plusieurs dispositifs de reconnaissance et de défense impliquent cellules immunitaires et neurones, faisant de lui un organe de choix pour l’étude des interactions neuroimmunes. La compréhension des éléments assurant un fonctionnement coordonné des Systèmes immunitaire (SI) et Nerveux (SN) dans l’intestin reste cependant partielle. Ce travail a porté sur l’étude des mécanismes régissant le fonctionnement intégré des SN et SI de l’intestin suite à une prise alimentaire. Nous démontrons que la libération de Peptide Intestinal Vasoactif (VIP) suite à une prise alimentaire impacte la fonction de cellules clés dans l’organisation de l’immunité intestinale, les cellules lymphoïde innées (ILC). Pour la première fois, on démontre qu’un neuropeptide modifie de manière anticipée la biologie des ILC2 et des ILC3 pour potentialiser l’effet de cytokines inductrices caractéristiques des immunités de type 2 ou de type 3, permettant une activation rapide et conséquente des ILC. Ce travail complexifie le réseau de régulation du fonctionnement des ILC et dévoile un nouveau rôle du VIP dans le maintien de l’homéostasie intestinale via sa capacité à anticiper et potentialiser les réponses immune de manière intégrée. La compréhension de ce nouveau mécanisme d’interactions neuroimmunes dans l’intestin ouvre la voie vers le développement de nouvelles stratégies thérapeutiques basées sur les propriétés du VIP pour prévenir et traiter des maladies infectieuses et inflammatoires de l’intestinThe intestine represents an extremely wide interface constantly exposed to substances that we ingest and to numerous micro-organisms that colonize its mucosae. Several mechanisms of recognition and defense involving both immune cells and neurons exist to ensure protection of the gut, setting the gut as a paradigm for neuroimmune interactions. However, how the nervous and immune systems coordinate and synchronize their action in the gut remain unclear. In this thesis, I aimed to elucidate the mechanisms underlying one type of neuroimmune communication occurring in the gut, during a physiological process: feeding. In this context, I demonstrated that the food-induced release of the Vasoactive Intestinal Peptide (VIP) impacts the function of the recently discovered “gatekeepers” of the gut immune system, Innate Lymphoid Cells (ILCs). For the first time, I showed that a neuropeptide induces an anticipatory priming of both ILC2 and ILC3, which could potentiate the effect of the canonical type 2 and type 3 inducer cytokines to lead a rapid and strong activation of ILCs. This work provides new insights in the highly complex regulatory network of ILCs and uncovers a new role for VIP in maintaining gut homeostasis through its ability to prime and eventually boost immune responses in an integrated and context dependent manner. The understanding of the neuroimmune interplay involving VIP in the small intestine opens the path toward the development of new therapeutic strategies based on VIP properties to treat infectious and inflammatory diseases of the gastrointestinal tract
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Yaseen, Alae Abod. "THE NATURAL POLYPHENOL RESVERATROL POTENTIATES THE LETHALITY OF HDAC INHIBITORS IN ACUTE MYELOGENOUS LEUKEMIA CELLS THROUGH MULTIPLE MECHANISMS." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2519.
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This study examined the mechanisms underlying the interactions between the natural polyphenol Resveratrol and HDAC inhibitors in both U937 myelomonocytic leukemia cell line and blood samples from AML patients and normal cord blood. Simultaneous exposure to Resveratrol and HDAC inhibitors (Vorinostat-SAHA or Panobinostat-LBH589) resulted in potentiating the lethality caused by any single agent of the combination, this interaction found to be synergistic at multiple concentrations. Exposing U937 cells to minimal toxic doses of Resveratrol and HDACIs results in release of mitochondrial pro-apoptotic proteins AIF and cytochrome c, pro-apoptotic caspase activation especially caspase-8, and induction of DNA damage. These events were associated with increase deacetylation of NF-κB and reactive oxygen species generation, as well as G0-G1 cell cycle arrest. Genetic knockdown of SIRT1 (a deacetylator of NF-κB that is upregulated by Resveratrol) resulted in significant increase in NF-κB acetylation and activity. However, SIRT1 knock down failed to protect U937 cells against combination-induced cell death, implying the possibility of the involvement of other mechanisms in inducing cell death rather than NF-κB deactivation only. Co-incubation of the antioxidant vi MnTBAP significantly reduced Resveratrol/HDACIs induced cell death, and resulted in a marked decrease in caspase-8, caspase-3, and PARP activation. Finally, the combined treatment of Resveratrol/HDACIs induce cell cycle changes possibly through Resveratrol action of blocking cell cycle in S phase exposing more cells to HDACIs lethality. Collectively, these finding indicate that the combined regimen of Resveratrol and HDAC inhibitors promote lethality in U937 cells and primary AML cells by a variety of mechanisms. The approved use of both agents in clinical setting make future clinical studies for development of this drug regimen a potential option in the battle with leukemia.
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Shulak, Laura. "Vorinostat potentiates vesicular stomatitis virus oncolysis in prostate cancer cells by modulating autophagy in an NF-kB dependent manner." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121210.
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Vesicular stomatitis virus (VSV) replication and oncolytic potential is reversibly stimulated in combination with epigenetic modulators such as the histone deacetylase inhibitor (HDI) Vorinostat (SAHA). Based on this reversible effect of Vorinostat on viral oncolysis, we reasoned that critical host genes involved in oncolysis may be reversibly regulated in prostate cancer PC3 cells following removal of Vorinostat. A transcriptome analysis in PC3 cells identified a subset of NF- κB target genes that were reversibly regulated by Vorinostat and involved in regulation of inflammatory and stress-responses. Consistent with the induction of NF- κB target genes, Vorinostat-mediated enhancement of VSV oncolysis correlated with hyper-acetylation of the NF- κB transcription factor subunit RELA/p65; furthermore, VSV replication and cell killing were suppressed when NF- κB signaling was inhibited using pharmacological or genetic approaches. Additional bioinformatics analysis revealed that stimulation of NF- κB signaling also resulted in the increased expression of several autophagy-related genes. Inhibition of autophagy by 3-methyladenine led to an increase in expression of IFNβ-stimulated genes, and both 3-methyladenine treatment and genetic ablation of autophagy led to a decrease in VSV replication and oncolysis. Together, these data demonstrate that Vorinostat stimulates NF- κB activity in a reversible manner via modulation of RelA/p65 signaling, leading to induction of autophagy and enhancement of VSV replication and oncolysis. These studies thus positively correlate NF- κB signaling and autophagy with VSV replication and oncolysis.La réplication et l'activité oncolytique du virus de la stomatite vésiculaire (VSV) sont stimulées d'une manière réversible lorsque le VSV est combiné avec des modulateurs épigénétiques tels que le vorinostat (SAHA), un inhibiteur de l'histone déacétylase (IDH). En se basant sur cette observation, nous avons émis l'hypothèse que les gènes importants de l'hôte impliqués dans l'oncolyse peuvent-être eux aussi réversiblement régules dans les cellules du cancer de la prostate PC3 après le retrait du vorinostat. En effet, une analyse du transcriptome des cellules PC3 a identifié un set de gènes impliqués dans la voie NF-κB, plus précisément dans la réponse inflammatoire et aux réponses au stress, qui est régulé par le vorinostat. Conformément à l'induction des gènes cibles de la voie NF-κB, l'amélioration de l'effet oncolytique du VSV par le vorinostat corrèle avec une hyper-acétylation du NF-κB RELA/p65; En outre, la réplication du VSV et la mort des cellules ont été supprimées lorsque la voie signalétique NF-κB a été inhibée par des approches pharmacologiques et géniques. Nous avons également observé que l'expression de plusieurs gènes impliqués dans l'autophagie a été augmentée par la stimulation de la voie NF-κB. De plus, une analyse supplémentaire bioinformatique a révélé que l'expression de plusieurs gènes impliqués dans l'autophagie était également augmentée par la stimulation de NF-κB. En addition, l'inhibition de l'autophagie par le 3-méthyladénine a entrainé une amplification de l'expression des gènes stimulés par l'IFN-β. En outre, le traitement avec le 3-méthyladénine et l'inhibition génique de l'autophagie a résulté en une diminution de la réplication du VSV et de l'oncolyse. Ensemble, ces résultats démontrent que le vorinostat stimule l'activité du NF-κB de manière réversible via la modulation de RelA/p65, conduisant à l'induction de l'autophagie et à l'amélioration de la réplication du VSV et l'oncolyse. Ainsi, cette étude met en évidence le lien entre l'activation de l'axe NF-kB- autophagie et la réplication du VSV et son effet oncolytique.
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Braun, Floriane Claudia Maria [Verfasser]. "In vivo silencing of A20 via TLR9-mediated targeted siRNA delivery potentiates anti-tumor immune response / Floriane Claudia Maria Braun." Greifswald : Universitätsbibliothek Greifswald, 2016. http://d-nb.info/1088766536/34.
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Huang, Yu Hong. "Sustained release of prostaglandin E1 potentiates the impaired therapeutic angiogenesis by basic fibroblast growth factor in diabetic murine hindlimb ischemia." Kyoto University, 2009. http://hdl.handle.net/2433/126420.
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Usui, Ryota. "GPR40 activation initiates store-operated Ca²⁺ entry and potentiates insulin secretion via the IP3R1/STIM1/Orai1 pathway in pancreatic β-cells." Kyoto University, 2020. http://hdl.handle.net/2433/253196.
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King, Steven Bradley. "Chronic Stress Potentiates The Response To Intra-Bed Nucleus Of The Stria Terminalis (bnst) Pituitary Adenylate Cyclase Activating Peptide (pacap) Infusion." ScholarWorks @ UVM, 2016. http://scholarworks.uvm.edu/graddis/461.
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Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in brain structures involved in emotion. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. In order to determine whether chronic stress would potentiate the behavioral and/or endocrine response to subthreshold BNST PACAP infusion, rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, stressed and control rats were bilaterally infused into the BNST with 0.5 µg PACAP. Startle response to intra-BNST PACAP infusion was assessed post-infusion in Experiment 1. In Experiments 2 and 3, blood was sampled via a tail nick 30 min following PACAP infusion to assess the corticosterone response to PACAP following CVS. We found an increase in startle amplitude and an increase in plasma corticosterone levels 30 minutes following BNST PACAP infusion only in rats that had been previously exposed to CVS. These results were likely mediated via PAC1 receptors, as equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels. These results suggest that repeated exposure to stressors sensitizes the neural circuits underlying the behavioral and endocrine responses to BNST PACAP infusion and BNST PACAP/PAC1 receptor signaling likely plays a critical role in mediating stress responses.
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Juli, Christina [Verfasser], and Ulrike [Akademischer Betreuer] Holzgrabe. "Synthese und Charakterisierung von potenziellen Inhibitoren des „Macrophage infectivity potentiator“ (Mip) Proteins von Legionella pneumophila - Ein neuer Ansatz in der Legionellose-Therapie / Christina Juli. Betreuer: Ulrike Holzgrabe." Würzburg : Universitätsbibliothek der Universität Würzburg, 2012. http://d-nb.info/102621095X/34.
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Youssefian, Leena. "THE SMALL MOLECULE BCL-2 INHIBITOR HA14-1 POTENTIATES THE LETHALITY OF A REGIMEN COMBINING MEK1/2 AND CHK1 INHIBITORS IN MULTIPLE MYELOMA CELLS." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1799.
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Previously, we have found that the co-administration of MEK1/2 inhibitors and Chk1 inhibitors synergistically induce multiple myeloma cell apoptosis through upregulation of the BH3-only pro-apoptotic protein Bim. However, these apoptotic events were largely blocked by the characteristic over-expression of Bcl-2 of Bcl-xL in multiple myeloma cells. HA14-1, a small molecule Bcl-2 inhibitor, may therefore circumvent this resistance to apoptosis by blocking Bcl-2 and Bcl-xL anti-apoptotic protein actions. In our project, we hypothesize that the co-administration of HA14-1 with MEK/Chk1 inhibitors will enhance apoptosis in multiple myeloma (MM) cells. To test this hypothesis, we exposed MM cells U266 and RPMI8226, or those cells with Bcl-2 over-expressing stable clones to minimally toxic concentrations of MEK1/2 inhibitor (PD184352) with Chk1 inhibitor (CEP3891) for 24 hours, followed by the Bcl-2 inhibitor (HA14-1). To date, our data indicates that co-administration of HA14-1 with the PD184352/CEP3891 regimen significantly enhances apoptotic death in U266/Bcl-2 multiple myeloma cells compared with the PD184352/CEP3891 regimen. Future studies are designed to elucidate mechanisms underlying Bcl-2 and Bcl-xL anti-apoptotic protein interactions with the Bak and Bim apoptotic proteins, focusing release of Bak and Bim from Bcl-2/Bcl-xL, and subsequent Bax/Bak activation.
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Bruneau, Alix. "Régulation de l'expression membranaire du transporteur de phospholipides biliaires ABCB4 : effet de mutations Functional Defect of Variants in the Adenosine Triphosphate–Binding Sites of ABCB4 and Their Rescue by the Cystic Fibrosis Transmembrane Conductance Regulator Potentiator, Ivacaftor (VX-770) Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS048.
Full textAbstract:
ABCB4 est exprimé à la membrane canaliculaire des hépatocytes où il sécrète un composant majeur de la bile : la phosphatidylcholine. Plus de 500 mutations d’ABCB4 sont associées à des maladies biliaires. La pathologie la plus sévère est la PFIC3, qui se développe tôt dans l’enfance et progresse rapidement vers l’insuffisance hépatique. La transplantation hépatique reste la seule thérapie efficace. Le développement d’alternatives représente donc un enjeu majeur. Cette thèse s’intéresse à l’effet de cinq mutations décrites chez des patients et situées dans les sites de liaison à l’ATP d’ABCB4. En combinant la modélisation 3D avec des études in vitro, nous avons montré que ces mutations sont responsables d’un défaut d’activité d’ABCB4. Nous avons mis en évidence que le VX-770, un médicament approuvé en clinique pour traiter les patients atteints de mucoviscidose, restaure l’activité des cinq mutants. Ces travaux ouvrent des perspectives de repositionnement du VX-770 pour le traitement ciblé de patients atteints de pathologies biliaires liées à ABCB4. La seconde partie de cette thèse consiste à étudier le rôle de l’interaction du domaine N-terminal d’ABCB4 avec la kinase MRCKalpha. L’inhibition ou l’extinction de cette kinase montrent que MRCKalpha joue un rôle dans la régulation de l’expression membranaire d’ABCB4 en contrôlant son internalisation depuis la membrane plasmique. En inhibant la protéine MLC2, effecteur de MRCKa, nous avons ensuite montré que l’effet de MRCKalpha sur l’expression d’ABCB4 passe par MLC2, qui est un partenaire du domaine linker d’ABCB4. Notre travail montre un rôle commun de ces deux partenaires dans la régulation de l’internalisation d’ABCB4ABCB4 is exclusively expressed at the canalicular membrane of hepatocytes where its function is to translocate phosphatidylcholine (PC) into bile. Variations in ABCB4 gene sequence are associated with several chronic and progressive liver diseases. The most severe is PFIC3 which develops early in childhood and most often requires liver transplantation. Less severe diseases are the intrahepatic cholestasis of pregnancy and the low phospholipid- associated cholelithiasis syndrome which occur in young adults. Up to now, about 500 disease-causing ABCB4 variants have been reported. A challenge is to find pharmacological treatments for the severe forms of the diseases. We have studied the effect of five disease-causing variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Using three-dimension structural modeling and in vitro studies, we showed that the five mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. PC secretion activity of the mutants was rescued by the clinically approved CFTR potentiator ivacaftor (VX-770). These results pave the way for personalized therapy in ABCB4-related diseases.The second part of my project was aimed at investigating the potential role of two ABCB4 partners, the kinase MRCKalpha and its effector the myosin light chain II (MLCII) in the expression and function of ABCB4. We found that downregulation of both partners didn’t affect the canalicular localization of ABCB4 but led to a reduction of its endocytosis. Our results open new insights into the mechanisms underlying the regulation of ABCB4 expression and function
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Leuillier, Matthieu. "Rôle de l'activité phosphatase de l'époxyde hydrolase soluble dans la régulation de l'homéostasie métabolique et cardiovasculaire. In vivo inactivation of the phosphatase activity of soluble epoxide hydrolase potentiates brown adispose thermogenesis and protects against cardiovascular damage and remodeling Discovery of the first in vivo active inhibitors of the soluble epoxide hydrolase phosphatase domain Altered bioavailability of epoxyeicosatrienoic acids is associated with conduit artery endothelial dysfunction in type 2 diabetic patients." Thesis, Normandie, 2019. http://www.theses.fr/2019NORMR150.
Full textAbstract:
Près de 40 ans après sa découverte initiale en 1972, il a été montré en 2003 que l'époxyde hydrolase soluble (sEH), codée par le gène EPHX2, est une protéine bifonctionnelle qui présente non seulement une activité époxyde hydrolase au niveau de sa partie C-terminale mais également une activité lipidophosphatase sur son domaine N-terminal. En effet, au niveau de sa partie C-terminale, l’activité hydrolase métabolise des époxydes d'acides gras polyinsaturés. Notamment, elle transforme les acides époxyeicosatriénoïques, facteurs vasodilatateurs et anti-inflammatoires biologiquement actifs générés par les cytochromes P450, en acides dihydroxyeicosatriénoïques qui sont des composés biologiquement moins actifs. Cette activité, est aujourd’hui la cible d’une nouvelle classe pharmacologique d’inhibiteurs. Contrairement à la fonction biologique de l’activité hydrolase, celle de l’activité phosphatase de la sEH reste, à ce jour, peu connue. Bien qu’à l’origine, il ait été montré que cette activité participait à la stabilisation de l’activité hydrolase ou à la dimérisation de l’enzyme, certaines données récentes révèlent que l’activité phosphatase de la sEH métabolise d'autres médiateurs lipidiques importants, comme les acides lysophosphatidiques intracellulaires, qui sont impliqués dans un large éventail de fonctions biologiques telles que le tonus vasculaire et l'inflammation, en monoacylglycérols. De plus, des études in vitro ont également suggéré que les deux activités de la sEH possèdent un rôle complémentaire dans la régulation du taux de cholestérol ainsi que dans l’homéostasie vasculaire. Même si les souris recombinantes qui n'expriment pas le gène EPHX2 existent depuis un certain temps, elles ne permettent pas d'étudier spécifiquement l’activité phosphatase car les deux activités de l’enzyme sont éliminées. Toutefois, des études portant sur les différences entre les effets de la délétion génétique de la sEH et ceux de l'inhibition pharmacologique de son activité hydrolase indiquent que l'activité phosphatase de la sEH possède probablement un rôle physiologique. Dans notre étude, afin de pouvoir étudier le rôle de l’activité phosphatase de la sEH et en raison de l’absence d’inhibiteur de cette activité utilisable in vivo, des rats transgéniques originaux exprimant une sEH sans activité phosphatase ont été générés grâce à la méthode CRISPR/Cas9. Un phénotypage métabolique et cardiovasculaire approfondi a été effectué sur ces animaux. Les résultats de cette étude ont mis en évidence que les rat Knock-In (KI) pour la sEH phosphatase présentent une diminution de leur poids corporel et de leur masse grasse comparativement à des rats sauvages du même âge. De plus, leur sensibilité à l’insuline est augmentée. Ce profil métabolique bénéfique est expliqué d’une part par une diminution de la consommation alimentaire et, d’autre part, par une augmentation de l'oxydation des graisses, potentialisant la thermogenèse dans le tissu adipeux brun, et de la dépense énergétique. Par ailleurs, lorsque les rats KI sont nourris avec un régime riche en graisses saturées, la prise de poids reste inférieure à celle des rats sauvages. De plus, ils ne développent pas d’insulino-résistance ou de stéatose hépatique. D’autre part, au niveau cardiaque, les rats KI présentent une activité mitochondriale basale plus élevée associée à une contractilité ventriculaire gauche accrue. Par ailleurs, les animaux KI sont protégés contre les lésions cardiaques d’ischémie-reperfusion et contre le développement de l'hypertension artérielle pulmonaire. Notre étude révèle ainsi que l’activité phosphatase de la sEH est un acteur clé du métabolisme lipidique et énergétique contribuant ainsi, comme l’activité hydrolase, à la régulation de l'homéostasie cardiométaboliqueNearly 40 years after its initial discovery in 1972, soluble epoxide hydrolase (sEH), encoded by the EPHX2 gene, was shown in 2003 to be a bifunctional protein that exhibits not only an epoxide hydrolase activity on its C-terminal domain but also a lipid phosphatase activity on its N-terminal domain. Indeed, the hydrolase activity metabolizes epoxides of polyunsaturated fatty acids. In particular, sEH converts the vasodilator and anti-inflammatory epoxyeicosatrienoic acids converts, generated by cytochromes P450, into dihydroxyeicosatrienoic acids, which are less biologically active. This activity is now the target of a new class of pharmacologicla inhibitors. Unlike the biological function of the hydrolase activity, the biological function of sEH phosphatase activity remains, this time, unknown. Although shown originally to contribute to the stabilization of hydrolase activity or dimerization of the protein, some recent data indicate that the sEH phosphatase metabolizes also important lipid mediators, such as intracellular lysophosphatidic acids, involved in a wide range of biological functions such as vascular tone and inflammation, into monoacylglycerols. In addition, in vitro studies also suggested that the two activities of sEH have a complementary role in cholesterol regulation and vascular homeostasis. Although recombinant mice that do not express the EPHX2 gene have been around for some time, they do not allow to specifically study the phosphatase activity because both activities are eliminated. However, studies examining the differences between the effects of the genetic deletion of sEH and those of the pharmacological inhibition of its hydrolase activity indicate that the phosphatase activity of sEH probably has also a distinct physiological role. In our study, to assess the role of sEH phosphatase activity in absence of an inhibitor of this activity usable in vivo, original transgenic rats expressing sEH without phosphatase activity were generated using the CRISPR/Cas9 method. A thorough metabolic and cardiovascular phenotyping was performed on these animals. The results of this study showed that Knock-In (KI) rats for the sEH phosphatase have a decrease in body weight and fat mass compared to wild type rats of the same age. In addition, their sensitivity to insulin is increased. This beneficial metabolic profile is explained on one hand by a decrease in food consumption and, on the other hand, by an increase in fat oxidation, potentiating thermogenesis in brown adipose tissue enhancing energy expenditure. In addition, when KI rats were fed a high fat diet, weight gain remains lower than that of the wild type rats. In addition, they do not develop insulin resistance or hepatic steatosis. Finally, at the cardiac level, KI rats have higher basal mitochondrial activity associated with increased left ventricular contractility. In addition, KI animals are protected against cardiac ischemia-reperfusion lesions and the development of pulmonary arterial hypertension. Our study thus reveals that the phosphatase activity of sEH is a key player in lipid and energy metabolism, thus contributing, like the sEH hydrolase activity, to the regulation of cardiometabolic homeostasis
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Oliveira, Cristiana Paula Martins da Silva. "Photodynamic therapy intheinactivation of bacteriophages with porphyrin and potentiators in wastewater." Master's thesis, 2019. http://hdl.handle.net/10773/29245.
Full textAbstract:
Pathogenic viruses are frequently introduced into marine and estuarine waters through the discharge of treated and untreated sewage, since current treatments are unable to provide virus-free wastewater (WW) effluents, affecting the receiving waters quality and, consequently, human health. The removal of harmful constituents by the conventional treatments comprises a combination of chemical, physical and biological methods. Usually, WW from urban areas is secondarily, rarely tertiary, treated. Although the secondary effluent contains high concentrations of microorganisms, the effect of water dilution makes it acceptable in terms of quality indicators. In tertiary treatment, chlorination is the most common method used to ensure microbiological safety in tertiarily treated effluents. However, its massive utilization, both in free and combined chlorine forms, may lead to the formation of chemical disinfection by-products though the reaction with organic matter present in the effluents, being those chemicals toxic to aquatic organisms, representing potential health hazards. Unfortunately, these conventional methods are limited and may not be adequate to reach the quality levels specified by the guidelines. Photodynamic therapy (PDT) with porphyrins may be a promising approach for the inactivation of pathogens as they are effective in inactivating microorganisms without the formation of potentially toxic products. Some studies have reported an enhancer effect on antimicrobial photodynamic therapy (aPDT) by the combined used of some photosensitizer (PS) with potassium iodide (KI) and hydrogen peroxide (H2O2). The main objective of this study was to evaluate the aPDT efficacy of a PS based on a low-cost formulation constituted by five cationic porphyrins (Form) and its potentiation effect by KI and H2O2 in the inactivation of a T4-like bacteriophage in WW. The experiments were done in phosphate buffered saline and in filtered and non-filtered contaminated wastewater. The aPDT assays in filtered WW (0.45 μm pore-size) were performed with different concentrations of Form (1.0 to 10 μM). In a second phase was evaluated the effect of KI (100 mM) in the photodynamic action of Form (1.0 to 10 μM). The results of these experiments demonstrated that Form is efficient in filtered WW treatment and that the efficacy of bacteriophage photoinactivation is correlated with the concentration of the used PS. When combined with KI, the Form is clearly less effective to inactivate the bacteriophage. To evaluate if the organic matter present in water influences the efficiency of PS, the WW was filtered using three different pore-sized membranes (0.45, 0.30 and 0.22 μm). The results demonstrated that the increase of organic matter promote a significant decrease in the efficiency of Form. In order to evaluate if the efficiency of aPDT to inactivate bacteriophages is maintained when the treatments are performed in non-filtrated WW, the effect of Form alone (10 μM) and combined with H2O2 (2, 5 and 9%) in non-filtered WW was evaluated. The Form alone proved to be an efficient PS to photoinactivate the bacteriophage in non-filtered WW, but the presence of H2O2 enhanced the photodynamic effect. The FORM can be an effective alternative to control viruses in WW, particularly if combined with H2O2.Os vírus patogénicos são frequentemente introduzidos nas águas marinhas e estuarinas através da descarga de esgoto tratado e não tratado, uma vez que os tratamentos atuais não inativam os vírus presentes nas águas residuais (WW), afetando a qualidade das águas recetoras e, consequentemente, a saúde humana. Nos tratamentos convencionais, a remoção de constituintes nocivos consiste no uso de métodos químicos, físicos e biológicos. Geralmente, a WW de áreas urbanas é tratada secundariamente e não terciariamente. Embora o efluente secundário contenha altas concentrações de microrganismos, o efeito da diluição na água torna-o aceitável em termos de indicadores de qualidade. A cloração é o método mais comum usado para garantir a segurança microbiológica em efluentes tratados terciariamente. No entanto, a sua utilização maciça, tanto na forma de cloro livre como combinada, pode levar à formação de subprodutos químicos como resultado da reação com a matéria orgânica presente nos efluentes, sendo esses produtos químicos tóxicos para os organismos aquáticos, apresentando riscos para a saúde. Os métodos convencionais são limitados e podem não ser adequados para manter os níveis de qualidade especificados nas diretrizes. As porfirinas quando usadas como fotossensibilizadores (PS) na terapia fotodinâmica (PDT) podem ser desinfetantes promissores para a inativação de microrganismos patógenicos, pois são eficazes na inativação de microrganismos sem formação de produtos tóxicos. Alguns estudos mostraram efeito potenciador de alguns PS usados em terapia fotodinâmica antimicrobiana (aPDT) quando estes são usados em combinação com iodeto de potássio (KI) e peróxido de hidrogénio (H2O2). O principal objetivo deste estudo foi avaliar a eficácia da aPDT de um PS baseado numa formulação de baixo custo constituída por cinco porfirinas catiónicas (Form) e o seu efeito potenciador por KI e H2O2 na inativação de um bacteriófago tipo T4. As experiências foram realizadas em solução salina tamponada com fosfato e em água residual contaminada filtrada e não filtrada. Os ensaios de aPDT em WW filtrada (tamanho do poro de 0,45 μm) foram realizados com diferentes concentrações de Form (1,0 a 10 μM). Numa segunda fase foi avaliado o efeito do KI (100 mM) na ação fotodinâmica da FORM (1,0 a 10 μM). Os resultados dessas experiências demonstraram que a Form é eficiente no tratamento de WW filtrada e que a eficácia da fotoinativação de bacteriófagos está correlacionada com a concentração do PS usado. Quando combinada com o KI, a Form é claramente menos eficaz na inativação do bacteriófago. Para avaliar se a matéria orgânica presente na água influencia a eficiência do PS, a WW foi filtrada usando três membranas com tamanho de poros diferentes (0,45, 0,30 e 0,22 μm). Os resultados mostraram que o aumento da matéria orgânica promove uma diminuição significativa na eficiência da Form. Para avaliar se a eficiência da aPDT para inativar bacteriófagos é mantida quando os tratamentos são realizados em WW não filtrada, o efeito da Form sozinha (10 μM) e combinado com H2O2 (2, 5 e 9%) em WW não filtrada foi avaliado. A Form por si só provou ser um PS eficiente para fotoinativar o bacteriófago em WW não filtrada, mas a presença de H2O2 aumentou significativamente o efeito fotodinâmico. A Form pode ser uma alternativa eficaz para controlar vírus na WW, principalmente se combinada com H2O2.
This work was supported by funding FEDER through COMPETE – Programa Operacional Factores de Competitividade, and by National funding through Fundação para a Ciência e Tecnologia (FCT) and Marine Studies (CESAM).
Mestrado em Biologia Molecular e Celular
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Braz, Márcia Alexandra Dias. "Photoinactivation of MRSA on skin: effect of potassium iodide and iodopovidone as potentiators of a porphyrinic formulation." Master's thesis, 2019. http://hdl.handle.net/10773/28032.
Full textAbstract:
Staphylococcus aureus, Gram-positive bacterium, is one of the leading causes
of a wide range of serious clinical infections, such as skin and soft tissue
infections. To treat these infections, antibiotics are used as the first treatment
option. However, S. aureus, namely methicillin-resistant S. aureus (MRSA), can
acquire resistance to the conventionally used antibiotics. This fact is associated
with high mortality, morbidity and high financial costs associated with the
treatment. Therefore, new approaches for the control of infections by this
bacterium are needed. Antimicrobial photodynamic therapy (aPDT), a multitarget
therapy, has emerged as a promising alternative treatment for localized
infections in response to the growing problem of antibiotic resistance. This
therapy requires the use of three key elements: photosensitizer (PS), light
(usually in the visible range) and oxygen. In the presence of these elements
reactive oxygen species (ROS) responsible for oxidative damage and microbial
cell death, are generated. Despite several in vitro studies reporting the
effectiveness of this therapy in inactivating a broad spectrum of
microorganisms, including S. aureus, there are few in vivo and ex vivo studies.
In this sense and given the importance of the composition of test matrices in
aPDT, in order to transpose this therapy for clinical application, it is necessary
to test the PSs in clinically relevant matrices. Thus, the aim of this work was to
evaluate the effectiveness of various aPDT approaches to control S. aureus
infections on skin. The use of aPDT adjuvants is important in order to increase
the efficiency of aPDT, reducing at the same time the amount of PS and/or the
treatment time and therefore the costs involved. In this work, a porphyrin
formulation (FORM), based on a non-separated mixture of 5 cationic porphyrins
and with recognized efficiency as an alternative to the highly efficient PSs
included in the formulation was used, in order to reduce the costs and
synthesis time relatively to the use of the separated porphyrins. FORM has
already been shown to be effective in aPDT of various bacteria, including S.
aureus. Potassium iodide (KI), recognized for increasing the efficiency of some
PSs in a broad spectrum of microorganisms, including S. aureus, through the
formed iodine species, was also used as a potentiator of aPDT. Iodopovidone
(PVP-I), a commonly used antimicrobial agent as disinfectant and antiseptic,
being a water-soluble complex of iodine bound to polyvinylpyrrolidone, was
also tested as a potentiator of aPDT. In a first phase, the aPDT protocol was
developed in phosphate buffered saline (PBS, in vitro). Thereafter, the efficacy
of FORM, as PS, alone and in combination with KI or PVP-I to photoinactivate
MRSA on skin was evaluated. For this, porcine skin (considered a good test
model for human skin) was artificially contaminated with MRSA and treated
with FORM, FORM + KI or FORM + PVP-I, under white light. The results
showed that FORM was effective to inactivate MRSA in PBS, where total
inactivation was achieved for all tested concentrations (0.5, 1.0 and 5.0 μM).
For the combinations FORM + KI and FORM + PVP-I, total inactivation of
MRSA and considerable reduction in irradiation time compared to FORM alone
was observed using the lowest tested PS concentration (0.5 μM). On skin, ex
vivo, a reduction in MRSA survival of 3.1 Log10 colony forming units (CFU) mL-1
with 50 μM FORM, was observed. Although the FORM + KI and FORM + PVP-I
combinations enhanced the efficacy of aPDT in inactivating MRSA in PBS, this
effect was not observed in ex vivo assays. Overall, aPDT using FORM as PS,
even without adjuvants, is a promising therapy for the inactivation of MRSA on
skin.Staphylococcus aureus é uma bactéria de Gram-positivo que pode causar vários tipos de infeções, incluindo doenças graves, como infeções de pele e tecidos moles. Os antibióticos são usados como primeira opção no tratamento destas infeções. Algumas estirpes de S. aureus, nomeadamente S. aureus resistente à meticilina (MRSA), podem adquirir resistência aos antibióticos mais frequentemente utilizados. Tal facto está associado a alta mortalidade, morbidade e altos custos financeiros associados ao seu tratamento. Assim, é necessário desenvolver novas abordagens para controlar infeções causadas por esta bactéria. A terapia fotodinâmica antimicrobiana (aPDT), uma terapia multi-alvo, surgiu como um tratamento alternativo promissor para infeções localizadas em resposta ao crescente problema da resistência aos antibióticos. Esta terapia requer o uso de três elementos-chave: fotossensibilizador (PS), luz (geralmente na gama do visível) e oxigénio. Na presença destes elementos, são geradas espécies reativas de oxigénio (ROS) que causam danos oxidativos nos microrganismos e consequentemente a sua morte. Apesar dos vários estudos in vitro que relatam a eficácia desta terapia na inativação de um amplo espectro de microrganismos, incluindo S. aureus, poucos são os estudos in vivo e ex vivo. Neste sentido, e dada a importância da composição das matrizes teste em aPDT, com vista à transposição desta terapia para aplicação clínica, é necessário testar os PSs em matrizes clinicamente relevantes. Assim, o objetivo deste trabalho foi avaliar a eficácia de vários protocolos de aPDT no controlo de infeções causadas por S. aureus na pele. De forma a aumentar a eficácia da aPDT, o uso de adjuvantes pode ser usado reduzindo também a quantidade de PS e/ou o tempo de tratamento e, portanto, os custos envolvidos no tratamento. Neste trabalho foi usada uma formulação porfirínica (FORM), baseada numa mistura não separada de 5 porfirinas catiónicas e reconhecida como uma alternativa ao uso dos PSs individuais incluídos nesta mistura, permitindo reduzir os custos e o tempo de síntese relativamente ao uso de porfirinas separadas. A FORM já mostrou ser eficaz na aPDT de várias bactérias, incluindo S. aureus. O iodeto de potássio (KI), reconhecido por aumentar a eficiência de alguns PSs para um amplo espectro de microorganismos, incluindo S. aureus, através da formação de espécies de iodo foi usado como potenciador da aPDT. A iodopovidona (PVPI), agente antimicrobiano vulgarmente usado como desinfetante e antisséptico, sendo um complexo solúvel em água de iodo ligado à polivinilpirrolidona, foi também testado como potenciador da aPDT. Numa primeira fase, o protocolo de aPDT foi desenvolvido em solução salina tamponada com fosfato (PBS, in vitro). Posteriormente, foi avaliada a eficácia da FORM, como PS, sozinha e em combinação com KI ou PVP-I para fotoinativar MRSA na pele. Para isso, pele suína (considerada um bom modelo de teste para a pele humana) foi artificialmente contaminada com MRSA e tratada com FORM, FORM + KI ou FORM + PVP-I, sob luz branca. Os resultados mostraram que a FORM foi eficaz para inativar MRSA em PBS, tendo sido observada inativação total para todas as concentrações testadas (0,5; 1,0 e 5,0 μM). Para as combinações FORM + KI e FORM + PVP-I, foi observada inativação total de MRSA e considerável redução no tempo de irradiação em comparação com a FORM sozinha, para a menor concentração de PS testada (0,5 μM). Na pele, ex vivo, foi observada uma redução na sobrevivência de MRSA de 3.1 Log10 unidades formadoras de colónias (UFC) mL-1 com 50 μM de FORM. Embora as combinações FORM + KI e FORM + PVP-I aumentassem a eficácia da aPDT na inativação de MRSA em PBS, esse efeito não foi observado nos ensaios ex vivo. No geral, a aPDT usando a FORM como PS, mesmo sem adjuvantes, é uma terapia promissora para a inativação de MRSA na pele.
Mestrado em Microbiologia
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Seufert, Florian. "Entwicklung von Inhibitoren des „macrophage infectivity potentiator“-Proteins." Doctoral thesis, 2016. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-134820.
Full textAbstract:
Die Melioidose und die Legionärskrankheit werden von den beiden Erregern Burkholderia pseudomallei bzw. Legionella pneumophila verursacht. Eine hohe Mortalitätsrate trotz langwieriger Behandlungen sowie die zunehmende Resistenz vieler Bakterien gegenüber den eingesetzten Antibiotika verdeutlichen die Notwendigkeit alternativer Behandlungsmethoden. Als neues Angriffsziel gilt das bereits in vielen Pathogenen gefundene „macrophage infectivity potentiator“-Protein, kurz Mip, das als Virulenzfaktor die Infektion forciert. Bei Legionella pneumophilia ist LpMip dafür verantwortlich, dass das Bakterium in die Lunge eindringen kann. Dabei überwindet der Erreger mit Hilfe des Mips die Epithelzellschicht und die extrazelluläre Matrix. Für BpMip ist der Sachverhalt der Invasion noch Gegenstand aktueller Forschung. Beide Mips zeigen eine hohe Sequenzhomologie zu humanem FKBP12 (FK506-bindende Proteine) und gehören deshalb zur Superfamilie der Peptidyl-Prolyl-cis/trans-Isomerasen (PPIasen), die die Fähigkeit besitzen, die cis/trans-Isomerisierung von Peptidbindungen der Aminosäure Prolin zu katalysieren. Die bereits bekannten FKBP12- und Mip-Inhibitoren Rapamycin und FK506 sind aufgrund ihrer immunsuppressiven Wirkung nicht zur Behandlung der beiden Krankheiten einsetzbar. Im Vorfeld dieser Arbeit konnte durch Synthese des literaturbekannten nicht-immunsuppressiven FKBP12-Inhibitors eine Leitstruktur gewonnen werden, die sowohl die PPIase-Aktivität von LpMip als auch von BpMip inhibiert. Zunächst konnten in dieser Arbeit durch Optimierung des Synthesewegs die Inhibitoren enantiomerenrein hergestellt werden. Ebenso wurde verifiziert, dass das S-Enantiomer das aktivere Konfigurationsisomer ist. Daneben wurde durch Synthese der Verbindung 8a/S-8a die anti-PPIase-Aktivität und die Löslichkeit im PBS-Puffer verbessert sowie die Zytotoxizität im Vergleich zu S-1a gesenkt Diese Verbindung zeigte jedoch eine schlechte Aktivität im Infektionsassay. In weiteren Kooperationen mit dem Biozentrum Würzburg und dem Dstl wurden die Inhibitoren ebenfalls erfolgreich an den Mips von Chlamydia trachomatis, Neisseria gonorrhoeae, Francisella tularensis undYersinia pestis getestet. In dieser Arbeit wurden erstmals Mip-Inhibitoren an Burkholderien in einer In-vivo-Studie untersucht. Die Wirksamkeit der Inhibitoren im Tiermodell soll in Folgestudien bewiesen werden. Damit ist eine aussichtsreiche Basis für zukünftige alternative Behandlungsmethoden der gram-negativer Bakterien gelegtDevelopment of macrophage infectivity potentiator inhibitors
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wang and 王秀勻. "Rapamycin potentiates cAMP-induced differentiation in NG108-15 cells." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/69739597164108939347.
Full textAbstract:
碩士國防醫學院
生物化學研究所
97
NG108-15 cell line fused from N18TG-2 and C6-BU-1 is a good model for investgating neuronal development and differentiation. We found that cell proliferation would be inhibited by treating DMEM with only 1% serum. It has been already known that inhibiting cell proliferation and elevating concentration of cytosolic cyclic adenosine 3',5'-monophosphate (cAMP) would let NG108-15 cell differentiation through activating transcription factor, cAMP response element binding protein (CREB). There are three features when NG108-15 cell differentiate. (1) Neurite outgrowth and Formation of varicosity. (2) Activity of voltage sensitive calcium channel, VSCC, elevates. (3) Neuronal marker protein, Microtubule Associated Protein 2 (MAP2), formate. Rapamycin is an inhibitor of Mammalian Target of Rapamycin Complex 1 (mTORC1). mTOR is a protein kinase that control cell cycle from G1 to S phase, promote proliferation and inhibit autophagy. Therefore, rapamycin would arrest cell cycle in G0/G1 and promote autophagy. We found that treating dibutyryl cAMP (dbcAMP) and rapamycin at the same time would promote NG108-15 cell differentiation earlier than only treating dbcAMP. It would have higher neurite and varicosity number, VSCC activity and MAP2 content. Silencing mTOR would mimic the effect of rapamycin in NG108-15 cells. Furthermore, potentiating differentiation caused by cotreating dbcAMP and rapamycin would be inhibited by adding autophagic inhibitor or silencing Beclin1. Rapamycin could also induce differentiation in NG108-15 cells. Rapamycin would not change phosphorylatic level of ERK and CREB. Besides, dbcAMP would not induce autophagy. In sum, differentiation of NG108-15 cells would be potentiated through inducing autophagy by treating rapamycin or silencing mTOR.
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Juli, Christina. "Synthese und Charakterisierung von potenziellen Inhibitoren des „Macrophage infectivity potentiator“ (Mip) Proteins von Legionella pneumophila - Ein neuer Ansatz in der Legionellose-Therapie." Doctoral thesis, 2012. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-72950.
Full textAbstract:
Die vorliegende Arbeit beschäftigt sich mit der Synthese und Charakterisierung potenzieller Inhibitoren des Oberflächenproteins Mip von Legionella pneumophila. Der gramnegative Mikroorganismus ist der ursächliche Erreger der Legionellose. Die Erkrankung kann in zwei verschiedenen Formen auftreten, dem Pontiac-Fieber, einer Grippe-ähnlichen Atemwegserkrankung, und der Legionärskrankheit, einer schweren Lungenentzündung mit einer Mortalitätsrate von bis zu 30 %. Natürliche und künstlich geschaffene Süßwassersysteme bilden den biologischen Lebensraum der Bakterien. Aus dieser Umgebung werden sie über technische Vektoren wie Duschen oder Klimaanlagen durch Inhalation kontaminierter Aerosole auf den Menschen übertragen, wo sie alveoläre Makrophagen besiedeln und eine pulmonale Infektion hervorrufen. Um die Zellen in der Lunge zu erreichen, müssen die Mikroorganismen jedoch zuerst die alveoläre Barriere, bestehend aus einer Epithelzellschicht und extrazellulärer Matrix, überwinden. Dafür ist das Oberflächenprotein Mip, der Hauptvirulenzfaktor, verantwortlich. Mip ist ein Homodimer, das sich aus einer C- und einer N-Domäne zusammensetzt. Während der N-Terminus für die Dimerisierung des Proteins verantwortlich ist, weist der C-Terminus die typische Faltung einer Peptidyl-Prolyl-cis/trans-Isomerase (PPIase) auf. Der Vergleich der Aminosäuresequenz der Mip-C-Domäne mit der Domäne verschiedener humaner PPIasen zeigte eine besonders große Homologie zu FKBP12, welches zur Familie der FK506-bindenden Proteine gehört und eine wichtige Rolle innerhalb des menschlichen Immunsystems spielt. Bemerkenswerterweise hemmen bekannte immunsuppressive FKBP12-Inhibitoren wie FK506 und Rapamycin neben der humanen PPIase ebenfalls das bakterielle Mip. Außerdem wurde beobachtet, dass die C-terminale Mip-PPIase an Kollagen IV, den Hauptbestandteil in der menschlichen Lunge, bindet und somit für die Transmigration der Legionellen in die Lunge verantwortlich ist. Mip-Inhibitoren sollten demnach eine Legionellen-Infektion verhindern können. Zur Verifizierung der Hypothese sollten daher im Rahmen dieser Arbeit neue Leitstrukturen für Mip-PPIase-Inhibitoren entwickelt werden. Mit Hilfe von Molecular-Modelling-Untersuchungen basierend auf der NMR-Struktur 2VCD wurde als eine mögliche Leitstruktur N,N-Dimethylphenylsulfonsäureamid identifiziert. Deshalb sollten diese Verbindung sowie Analoga hergestellt werden. Obwohl die Immunsuppressiva FK506 und Rapamycin Mip-Inhibitoren darstellen, können sie auf Grund ihrer immunsuppressiven Eigenschaften nicht in der Legionellosetherapie eingesetzt werden. Die makrozyklischen Immunsuppressiva setzen sich im Gegensatz zu N,N-Dimethylphenylsulfonsäureamid allerdings aus zwei strukturellen Einheiten, einer Binde- sowie einer Effektordomäne, zusammen. Die Bindedomäne mit dem Pipecolinsäure-Grundgerüst ist für die Wechselwirkungen mit Mip und FKBP12 verantwortlich. Die Effektordomäne hingegen ist der aliphatische Teil der Makrozyklen, der erst durch die Bildung eines ternären Komplexes eine Immunsuppression hervorruft. Somit können Verbindungen vom Pipecolinsäure-Typ keine immunsuppressive Wirkung haben und stellen demnach optimale, neue Leitstrukturen für Mip-Inhibitoren dar. Aus diesem Grund wurden die zwei literaturbekannten, nicht-immunsuppressiven FKBP12-Inhibitoren A und B ausgewählt und in verschiedenen Docking-Studien untersucht. Das Molecular-Modelling zeigte, dass nur Verbindung B reproduzierbare Interaktionen mit Mip eingehen kann und demnach ein potenzieller Inhibitor ist. Um dies zu überprüfen, sollten beide Verbindungen A und B sowie eine Mischform hergestellt werden. Neben diesen Verbindungen wurden weiterhin Variationen an der Struktur vorgenommen. Alle Verbindungen wurden in einem In-vitro-Enzymassay gezielt auf ihre Mip-Interaktion untersucht. Die In-vitro-Untersuchungen zeigten, dass nur Pipecolinsäure-Derivate vom Sulfonsäureamid-Typ B die Mip-PPIase inhibieren, die besten Verbindungen sind 22b, 23a und 24a. Neben den enzymatischen In-vitro-Testungen wurden exemplarisch für die Verbindungen 1a, 12a, 22a und 22b HSQC-NMR-Experimente zur Bestimmung der Inhibitor-Proteinbindung durchgeführt. Für Verbindung 22b wurde zusätzlich die In-vivo-Wachstumshemmung der Legionellen mittels Gentamicin-Infektionsstudien ermitteltThe present work focused on the synthesis and characterization of potential inhibitors of the surface protein Mip of Legionella pneumophila. The gram-negative microorganism is the causative agent of Legionellosis, which may occur in two different progressive forms, the Pontiac fever, a flu-like respiratory disease, and the Legionnaires disease, a severe pneumonia with mortality rate of up to 30 %. Natural and man-made fresh water systems provide a biological habitat to the bacteria. From this environment they were transmitted into humans via technical vectors such as showers or air conditioners by inhaling the contaminated aerosols. Within the human lung, they affect alveolar macrophages and cause a pulmonary infection. Though, to affect the alveolar macrophages the microorganisms have first to cross the alveolar barrier, which consists of epithelial cells and an extracellular matrix. For this, the surface protein Mip, which represents the main virulence factor of the bacterium, is responsible. Mip forms a stable homodimer, which consists of two domains, a C- and an N-domain. While the N-terminus is responsible for the dimerization of the protein, the C-terminus exhibits the typical folding of a peptidyl-prolyl-cis/trans-isomerase (PPIase). The comparison of the amino acid sequence of the Mip-C-domain and the domain of different human PPIases shows a particularly high homology to FKBP12. This human PPIase belongs to the family of the FK506 binding proteins and plays an important role within the human immune system. Remarkably, known immunosuppressive FKBP12-inhibitors such as FK506 and Rapamycin are also able to inhibit the bacterial Mip. Furthermore, it was observed that the C-terminal Mip-PPIase binds to collagen IV, the main component of the human lung, and therefore, Mip is responsible for the transmigration of Legionella. Due to this fact, Mip-inhibitors should prevent a Legionellosis. To verify this hypothesis, the intention of this work was to develop novel lead structures for Mip-PPIase-inhibitors. By means of molecular modeling studies based on the NMR structure 2VCD N,N-dimethylbenzenesulfonamide was identified as a potential lead structure. Therefore, this compound as well as analogues were aimed to prepare. Although the macrolides FK506 and Rapamycin inhibit Mip, they cannot be used for the treatment of Legionellosis due to their immunosuppressive effects. Compared to N,N-dimethylbenzenesulfonamide the macro cyclic, immunosuppressive drugs consist of two structural domains, a binding domain and an effector domain. The binding domain with the pipecolic acid feature is responsible for the interactions with Mip and FKBP12, respectively, whereas the effector domain, the aliphatic part of the molecule, causes the immunosuppression by forming a ternary complex. Therefore, compounds of the pipecolic acid type cannot affect an immunosuppression and thus, represent optimal, novel lead structures. Due to this fact, the two common, non-immunosuppressive FKBP12-inhibitors A and B were analyzed by means of different docking studies with the result that only compound B is able to interact with Mip. Therefore, it was assumed that B must be a potential Mip-inhibitor. To verify this hypothesis, both compounds A and B as well as a hybrid of them should be prepared. In addition to these compounds, further structural variations were prepared. To determine the interaction with Mip, all synthesized compounds were analyzed by means of an in vitro enzyme assay. During the in vitro studies, it was observed that only pipecolic acid compounds of the sulfonamide type B inhibit the Mip-PPIase, the most promising compounds are 22b, 23a und 24a. To determine the inhibitor-protein binding, compounds 1a, 12a, 22a and 22b were analyzed by means of HSQC-NMR experiments. Furthermore, for compound 22b an in vivo determination of growth inhibition of Legionella by means of a Gentamicin infection assay was carried out
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Paul, Brian James. "DksA : a potentiator of regulation of transcription by small molecules in Escherichia coli /." 2005. http://catalog.hathitrust.org/api/volumes/oclc/62327078.html.
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Zhang, J., Lijun Shang, and A. Ma. "CFTR Potentiator PG-01 and Corrector KM-11060 can rescue hERG mutations trafficking." 2016. http://hdl.handle.net/10454/10856.
Full textAbstract:
yesType II congenitalLong QT syndrome (LQT2) is due to genetic mutations in hERG channel. Genetic or pharmacological factors could potentially affect hERG channel biogenesis and contributes to LQTS, for example, disease mutations G601S and T473P result in hERG trafficking deficiency [1,2]. Various rescue strategies for hERG dysfuction are being developed. Some correctors for CFTR channel have been reported to act indirectly on proteostasis pathways to promote folding and correction on hERG trafficking deficiency [3]. In this study, we tested the hypothesis that the CFTR corrector KM-11060 and the potentiator PG-01 may correct hERG mutation trafficking diseases. We use HEK293 cell line expressing a well-studied trafficking disease mutation G601S-hERG channel [4]. We treated cells with CFTR potentiator PG-01and corrector KM-11060, which function through different cellular mechanisms, and assessed whether correction occurred via immunoblotting. Whole cell proteins from HEK 293 cells expressing hERG channels were used for analysis [5]. Proteins were separated on 8% SDS-polyacrylamide electrophoresis gels for 1 hour, transferred onto PVDF membrane, and blocked for 1 h with 5% nonfat milk. The blots were incubated with the primary antibody (Santa Cruz Biotechnology) for 12-16 h at 4C temperature and then incubated with a donkey antigoat horseradish peroxidase-conjugated secondary antibody( Santa Cruz Biotechnology). Actin expression was used for loading controls. The blots were visualized using the ECL detection kit (Genshare).Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). Our results show that both KM-11060 (5, 10, 20uM) and PG-01(5, 15 uM) can correct G601S mutant alleles of hERG protein trafficking (Fig 1, 2). KM-11060 (20uM) but not PG-01(15 uM) enhance protein expression of wild type hERG channel (Fig 2). Further treatment on cells at low temperature with different drug concentration will be tested. Functional studies are also needed to test whether the drugs can correct the function of hERG mutation channel. These results could potentially provide novel insight into the correction mechanism of CFTR potentiator and also help to develop new treatment for LQT2.
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Tsao, Chang-Chi, and 曹昌麒. "Bamboo mosaic potexvirus satellite RNA encoded P20 potentiates satellite RNA movement." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/47118094066401263582.
Full textAbstract:
碩士國立中興大學
農業生物科技學研究所
89
A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsidation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein (P20). The N-terminal arginine-rich motif of P20 is the RNA binding domain, which preferentially kinds at the 5’ and 3’ untranslated regions of satBaMV RNA. When the P20 open reading frame was replaced with sequence encoding chloramphenicol acetyltransferase (CAT), the accumulation level of BSCAT in systemic leaves of infected plants was only about 1/40-1/100 of that in inoculated leaves. Even though P20 is not required for satBaMV replication, the N-terminal or internal mutations of P20 caused a 21-50% decrease in the level of satBaMV accumulation in inoculated protoplasts when compared with the wild-type satBaMV. These results indicate that P20 may assist satBaMV replication, movement, or other functions. In this study, transgenic Nicotiana benthamiana plants that express the P20 were produced to investigate the effects of P20 on satBaMV movement. Complementary DNA of satBaMV that contains nt 59 to 836 was cloned in a plant expression vector, plasmid pKyLx7, and transformed to N. benthamiana by Agrobacterium-mediated transformation. R0 transformants selected were based on kanamycin resistance and verified by the PCR, Southern blot, Northern blot, and Western blot analyses. The ratio of kanamycin resistance in the F1 progenies was 3:1 which followed the Mendelian law. Transgenic lines which have homozygous satellite cDNA genotype were selected after analysis of F2 progenies. We have selected seedlings which have homozygous satellite cDNA genotype. The homozygous transgenic lines were challenged with BaMV and the virus titers were analyzed by indirect ELISA and western blots. The results showed that virus concentration in the transgenic lines was similar with the untransformed plants and the P20 level was not amplified by BaMV infection. Coinoculation of pCB and pCBSCAT onto untransformed plants, the BSCAT could move cell-to-cell in the inoculated leaves but failed to spread systemically. However, BSCAT could be detected in 16.6~80% of the upper uninoculated leaves of P20 transgenic plants 10 or 15 days after inoculation. Thses results indicat that the P20 transgenic plants can trans-complement the systemic movement of BSCAT. The ability that BSCAT spread from inoculated leaves to non-inoculation leaves was dependent upon the P20 protein in the transgenic plants. These results conclusively demonstrate that the P20 protein of satBaMV potentiates the movement of the satellite RNA.
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Hsieh, Tsung-Hsiu. "Deltamethrin, a Pyrethroid Insecticide, Potentiates Lipid Accumulation in 3T3-L1 Adipocytes." 2016. https://scholarworks.umass.edu/masters_theses_2/359.
Full textAbstract:
Obesity is a growing concern in the world today. As we ponder about the many causes of this global epidemic, we are driven to look at our food and the environmental toxicants that may contribute to obesity. Deltamethrin, being a common synthetic pyrethroid used in agriculture for pest control, is the primary insecticide this study explores to connect with obesity in 3T3-L1 adipocytes. To investigate the relationship between deltamethrin and adipogenesis, various concentrations were tested, 1nM, 10nM, 100nM, 1μM, and 10μM. The result indicated that higher concentration of deltamethrin had a direct impact on fat accumulation. These experiment results indicate that deltamethrin may potentiate adipogenesis in this model. Further in vivo studies will be needed to validate these findings and confirm the effects of deltamethrin on obesity.
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Mahmood, Hanan S. "Elucidating the Molecular Pathway through which L-Lactate potentiates NMDAR Signaling." Diss., 2019. http://hdl.handle.net/10754/660266.
Full textAbstract:
The role of L-Lactate has expanded from an energy metabolite to a signaling molecule in
neurons. Studies have shown that L-Lactate plays a role in neuroprotection and in
NMDAR-dependent long-term memory formation. The aim of this dissertation is to
characterize the role of L-Lactate as a signaling molecule and understand the molecular mechanism through which L-Lactate potentiates NMDAR signal. Using mass spectrometry, I monitored the time-dependent changes in the phosphoproteome of cortical neuronal cultures in response to Lactate. The phosphoproteomic analysis highlighted a number of cytoskeletal proteins involved in synapse remodeling as well as axon guidance that were regulated by L-Lactate. In addition, I found that L-Lactate
induced phosphorylation of proteins involved in the MAPK pathway, as reported in an earlier study. I hypothesize the involvement of CaMKII in this mechanism. CaMKII is one of the most abundant kinases in the brain and plays a role in learning and memory via interaction with NMDAR. Using CaMKII inhibitors and mutants of the NMDAR subunit GluN2B, the findings in this dissertation provide evidence for the involvement of CaMKII, specifically, the interaction between CaMKIIa and GluN2B, as a requirement for the L-Lactate mediated potentiation of NMDAR signal.
In addition, to gain insight into the evolution of lactate from a metabolite to a signaling
molecule, this study explores the evolution of glutamate as a signaling molecule in
multicellular organisms so it may serve as a model for evolution of metabolites like
lactate into signaling molecules. For this purpose, the model organism Hydra was used, since it belongs to phylum Cnidaria, evolutionarily one of the first phyla to have a
nervous system. In order to explore whether glutamate receptors, particularly, NMDAR
are functionally expressed in Hydra and are localized in neurons, a line of transgenic
Hydra expressing a calcium indicator (GCaMP6s) in neurons was generated. With the transgenic Hydra line, I attempted to measure the in vivo response of neurons in Hydra to glutamate. This study highlights several ground work experiments with an extensive discussion of implications and challenges and an outlook for future investigations.