Dissertations / Theses on the topic 'Inhibitor'
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Rodkey, Elizabeth A. "INHIBITOR RESISTANCE MECHANISMS AND INHIBITOR DESIGN IN ¿¿-LACTAMASES." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1354463033.
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Shkirskiy, Viacheslav. "Corrosion inhibition of galvanized steel by LDH - inhibitor hybrids : Mechanisms of Inhibitor Release and Corrosion Reactions." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066216/document.
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Le travail présenté essaie de comprendre les mécanismes de l’action d’un inhibiteur de corrosion présent dans un revêtement hybride sous forme de pigments intercalés dans les hydroxydes double lamellaires (HDL) pour la protection de l’acier galvanisé. Trois étapes clés ont été choisies pour ce travail : (1) l’identification d’un inhibiteur de corrosion hydrosoluble pour l’acier galvanisé avec une compréhension de sa réactivité, (2) la détermination des facteurs et des mécanismes contrôlant la libération de l’inhibiteur à partir d’HDL et (3) la compréhension des mécanismes de protection dans un système modèle avec le revêtement hybride contrôlé par la libération de l’inhibiteur et la réactivité d’inhibiteur. MoO42- a montré la meilleure efficacité d'inhibition comparable à CrO42- dans des solutions alcalines et neutres. L’effet inhibiteur de MoO42- a été associé à la formation d’un film riche en Mo(V). L'effet de cet anion sur la dissolution de l'acier à bas carbone a été également vérifié pour exclure la possibilité d'un effet d'accélération des espèces choisies. Les tests de lixiviation ont montré que la libération de MoO42- à partir d’HDL a été contrôlée par la nature des ions échangés à partir du support par un mécanisme d'échange d'ions à un pH neutre et par la dissolution du cadre de la LDH à un pH alcalin. La présence de seulement Cl- conduit à moins de 40% de libération de MoO42- après 24 h d'immersion alors que les additions des carbonates ont abouti à libération de 100% après 1 h. Les tests d'immersion ont montré léger effet d'inhibition du système de revêtement dans Cl- et une augmentation dans CO32- en accords avec le niveau plus élevé de MoO42- libéréThe current work was dedicated to the investigation of the fundamental mechanisms of the action of a layered double hydroxide (LDH) inhibitor hybrid coated systems for the corrosion protection of galvanized steel. The objective of the work was achieved by the realization of three milestones: (1) the identification of the effective water soluble inhibitor on Zn and steel substrates and the understanding the mechanisms of its action, (2) the revealing the factors and mechanisms controlling the release of the selected inhibitor from Zn2Al/-LDH hosts and (3) the understanding the mechanisms of coated system controlled by inhibitor release and its action. MoO42- showed the best inhibition efficiency comparable to CrO42- in alkaline and neutral solutions. The protective properties of MoO42- were assigned to the fast formation of Mo(V) film. The effect of MoO42- on the dissolution of low carbon steel was also verified to exclude the possible accelerating effect of chosen species. The leaching tests showed that MoO42- release from LDH was controlled by the nature of the exchanged ions from the media by ion-exchange mechanism at neutral pH and by the dissolution of the LDH framework at alkaline pH. The presence of only Cl- resulted in less than 40 % of MoO42- release after 24 hours of the immersion while the additions of the carbonates resulted in 100 % release after 1 hour. The immersion tests showed slight inhibiting effect of coated system in Cl and high in CO32- medias coherent with higher level of MoO¬42- released. The ways to control the inhibitor release and hence, the inhibition performance of coated systems were discussed in the vein of environment composition
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Hirst, Claire Elizabeth 1971. "Tissue distribution and regulation of the granzyme B inhibitor, proteinase inhibitor 9." Monash University, Dept. of Biochemistry and Molecular Biology, 2002. http://arrow.monash.edu.au/hdl/1959.1/8488.
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Roweth, Harvey George. "Mechanisms of platelet inhibition by the selective serotonin reuptake inhibitor citalopram." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275477.
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Background: Selective serotonin reuptake inhibitor (SSRI) antidepressants prevent serotonin (5-HT) uptake by the serotonin transporter (SERT). Since blood platelets express SERT, SSRIs may modify platelet function and the risk of cardiovascular disease. However, the beneficial or adverse effects of SSRIs on arterial thrombosis are poorly characterised and detailed in vitro experimental data is limited. The SSRI citalopram is a racemate, the (S)-isomer being the more potent SERT inhibitor. Although citalopram has been shown to inhibit platelets in vitro, it is unclear whether this is mediated via SERT blockade. Aim: To determine if citalopram inhibits platelet function via SERT blockade, or through a novel mechanism of action. Findings: 5-HT uptake into platelets was blocked by both citalopram isomers at concentrations that had no apparent effect on platelet function. Despite the (S)-citalopram isomer being the more potent SERT inhibitor, (R)-citalopram was equally potent at inhibiting other platelet functions. These findings strongly suggest that inhibition of platelet function by citalopram in vitro is not mediated by blocking SERT. Subsequent experiments identified two putative mechanisms for citalopram-mediated platelet inhibition: 1) citalopram did not inhibit calcium store release induced by the platelet agonist U46619, despite blocking subsequent Rap1 activation. A credible target for this inhibitory mechanism is the calcium and diacylglycerol guanine nucleotide exchange factor-1 (CalDAG-GEFI): 2) citalopram suppressed early protein phosphorylation within the GPVI pathway, resulting in the inhibition of subsequent platelet responses. Further experiments show that other commonly used antidepressants also inhibit platelets. As with citalopram, inhibition was only observed at concentrations above those required to block SERT, suggesting that alternative inhibitory mechanism(s) are responsible. Conclusions: Data presented in this thesis support two novel putative mechanisms of citalopram-induced platelet inhibition. These findings demonstrate that citalopram and other antidepressants inhibit platelets independently of their ability to block SERT-dependent 5-HT transport. The identification of thesemechanisms provides a pharmacological approach to develop novel antiplatelet agents based on current antidepressants.
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Guzanov, Pavel. "ERAP1 inhibitor development." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:e4369016-ab8f-445d-ab1a-71129b495a37.
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ERAP1 has been associated with a set of immune-mediated diseases, including ankylosing spondylitis. Currently, there is no selective and potent ERAP1 inhibitor available that would allow in-depth research of its functions and roles in diseases. Therefore, the aim of this work is to apply fragment-based drug design approach for the development of such inhibitor. A set of 12 structurally diverse hits was identified as a result of a screen containing 1200+ fragments using orthogonal ERAP1 activity assays. These hits can be used as a basis for development of a larger lead inhibitor, which requires structural information about their binding mode to ERAP1. Three different approaches have been tried in order to obtain such information about the binding mode. Unfortunately, ERAP1 crystallisation trials did not succeed, the potential reason being heterogeneity of the protein samples due to presence of several glycoforms. A set of 31 analogues of an IRAP inhibitor, which has been shown to inhibit ERAP1 activity, was synthesised and screened providing valuable structure-activity relationship information. Taken together, the screening campaign has resulted in a novel ERAP1 inhibitor with a single digit micromolar potency against ERAP1.
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Clarke, Tyler Brooke. "Studies on the inhibitor selectivity and inhibitory signal transfer of a-Isopropylmalate synthase." Thesis, University of Canterbury. Chemistry, 2013. http://hdl.handle.net/10092/11303.
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α-Isopropylmalate synthase (α-IPMS) is responsible for catalysing the first committed step in leucine biosynthesis. This pathway is found in plants and microorganisms, including pathogenic bacteria such as Mycobacterium tuberculosis and Neisseria meningitidis. α-IPMS catalyses a Claisen condensation reaction between α-ketoisovalerate (KIV) and acetyl coenzyme A (AcCoA) to form the product α-isopropylmalate (IPM). This enzyme undergoes feedback inhibition by the end product of the pathway, leucine. This regulation allows the control of the rate leucine biosynthesis.
This project focuses on the α-IPMS enzymes from M. tuberculosis and N. meningitidis (MtuIPMS and NmeIPMS). These α-IPMS enzymes are homodimeric in structure. Each monomer consists of a catalytic domain which comprises of a (β/α)8 barrel fold, two subdomains and a regulatory domain, to which the allosteric binding of the natural inhibitor leucine occurs. The mechanism by which the allosteric binding of leucine leads to a decrease in enzymatic activity is not yet fully understood.
Citramalate synthase (CMS) is responsible for catalysing the first committed step of threonine-independent isoleucine biosynthesis. This enzyme is extremely similar to α-IPMS in both the reaction which it catalyses and the catalytic and regulatory domain structure. CMS catalyses a Claisen condensation reaction between pyruvate and AcCoA to produce citramalate (CM). CMS is also feedback inhibited by the end product of its pathway, isoleucine.
The similarity between α-IPMS and CMS enzymes resulted in and examination of the inhibitor selectivity of MtuIPMS. Amino acids in the leucine binding site were altered to their counterparts in the isoleucine binding site of the CMS enzyme to see if the selectivity of the leucine binding site could be interchanged.
Results from this study show that it is possible to change inhibitor selectivity with a single amino acid substitution. However, changing the selectivity from leucine to isoleucine was unsuccessful. Instead, one of the MtuIPMS variants displayed significantly increased sensitivity to an alternative amino acid, norvaline. The MtuIPMS variants were expressed and purified using immobilised metal affinity chromatography and size-exclusion chromatography. These variants were then kinetically characterised and displayed similar binding affinities and turnover rates for the natural substrates to the wild-type enzyme. As expected changes to the leucine binding pocket had drastic effects on the sensitivity of the enzyme to its natural inhibitor. This work is described in Chapter 2 of this thesis.
The mechanism by which the regulatory signal is transferred from the allosteric leucine binding site to the catalytic site in α-IPMS is not fully understood. NmeIPMS variants were created based on preliminary molecular dynamic simulations which indicated that significant changes in residue contacts were associated with leucine binding. Chapter 3 describes studies that explore the effect of single amino acid substitutions of NmeIPMS. The NmeIPMS variants were expressed and purified similarly to MtuIPMS, using immobilised metal affinity chromatography and size-exclusion chromatography. Variants were subsequently characterised via mass spectrometry, differential scanning fluorimetry and kinetic assays. It was found that each variant generated retained sensitivity to leucine but displayed significant differences in the catalytic efficiencies with AcCoA. One of the generated variants also displayed a significant increase in thermal stability.
Results are drawn together in Chapter 4 along with future directions of this research. This chapter details knowledge gained into protein structure and allosteric mechanisms in this thesis.
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Menon, V. "Molecular and functional aspects of hydrolyases / inhibitors with emphasis on aspartic protease inhibitor." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2012. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2401.
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Miyazaki, Hiroshi. "Studies on Inhibitors of Plasminogen Activator Inhibitor-1(PAI-1) and Inhibitors of PAI-1 Production as Antithrombotic Agents." 京都大学 (Kyoto University), 2010. http://hdl.handle.net/2433/126818.
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Ahtyamova, Daria. "Cholinesterase inhibitor: pharmacological application." Thesis, Київський національний університет технологій та дизайну, 2019. https://er.knutd.edu.ua/handle/123456789/13033.
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McCready, Tara Lyn. "Inhibition of protein phosphatase-1 by endogenous inhibitor proteins and natural product toxins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0032/NQ46885.pdf.
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Chen, Ping. "Inhibitor adsorption study and the effect of inhibitor on kinetics of BaSO4 crystal growth." Thesis, Heriot-Watt University, 1995. http://hdl.handle.net/10399/1336.
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Poliakov, Anton. "Peptide-Based Inhibitors of Hepatitis C Virus NS3 Serine Protease: Kinetic Aspects and Inhibitor Design." Doctoral thesis, Uppsala universitet, Institutionen för naturvetenskaplig biokemi, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4127.
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Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs. In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies. A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values. The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases. Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond. The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.
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Schempp, Christina Maria. "The V-ATPase inhibitor archazolid." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-168586.
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Fighting metastasis is a major challenge in cancer therapy and novel therapeutic targets and drugs are highly appreciated. Resistance of invasive cells to anoikis, a particular type of apoptosis induced by loss of cell-extracellular matrix (ECM) contact, is a major prerequisite for their metastatic spread. Inducing anoikis in metastatic cancer cells is therefore a promising therapeutic approach.
The vacuolar H+-ATPase (V-ATPase), a proton pump located at the membrane of acidic organelles, has recently come to focus as an anti-metastatic cancer target. As V-ATPase inhibitors have shown to prevent invasion of tumor cells and are able to induce apoptosis we proposed that V-ATPase inhibition induces anoikis related pathways in invasive cancer cells.
In this study the V-ATPase inhibitor archazolid A was used to investigate the mechanism of anoikis induction in various metastatic cancer cells (T24, MDA-MB-231, 4T1, 5637). Therefore, cells were forced to stay in a detached status to mimic loss of cell-ECM engagement following treatment with archazolid.
Indeed, anoikis induction by archazolid was characterized by decreased expression of the caspase-8 inhibitor c-FLIP and caspase-8 activation, thus triggering the extrinsic apoptotic pathway. Interestingly, active integrin β1, which is known to play a major role in anoikis induction and resistance, is reduced on the cell surface of archazolid treated cells. Furthermore, a diminished phosphorylation of the integrin downstream target focal adhesion kinase could be demonstrated. The intrinsic apoptotic pathway was initiated by the pro-apoptotic protein BIM, increasing early after treatment. BIM activates cytochrome C release from the mitochondria consequently leading to cell death and is described as one major inducer of anoikis in non-malignant and anoikis sensitive cancer cells.
Of note, we observed that archazolid also induces mechanisms opposing anoikis such as proteasomal degradation of BIM mediated by the pro-survival kinases ERK, c-Src and especially Akt at later time points. Moreover, induction of reactive oxygen species (ROS) influences BIM removal as well, as moderate levels of ROS have second messenger properties amplifying cell survival signals. Thus, to antagonize these anoikis escape strategies a combination of archazolid with proteasome or ROS inhibitors amplified cancer cell death synergistically.
Most importantly, intravenous injection of archazolid treated 4T1-Luc2 mouse breast cancer cells in BALB/cByJRj mice resulted in reduced lung metastases in vivo.
To summarize this work we propose archazolid as a very potent drug in inducing anoikis pathways in metastatic cancer cells even though having learned that detachment together with treatment triggers multiple resistance mechanisms opposing cell death.
Hence, V-ATPase inhibition is not only an interesting option to reduce cancer metastasis but also to better understand anoikis resistance and to find choices to fight against it.
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Cao, Xianhua. "Simultaneously targeting hypoxic cancer cells by hsp90 inhibitor and glycolysis inhibitor in pancreatic cancer therapy." The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1173117669.
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Kreienbühl, Peter Lukas. "Protein phosphatase inhibitor okadaic acid alters cell shape and F-action distribution and inhibits /." Bern, 1992. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Reid, Anne Marie. "Raf-1 kinase inhibitor protein modulation of the cellular response to chemotherapeutic drugs and PDE5 inhibitors." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2497/.
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RKIP was initially discovered as an endogenous inhibitor of the ERK and NF-κB pathways,and was also shown to prolong the activation of GPCRs via inhibition of the GRK2 protein. Now increasing evidence has linked RKIP to a metastases suppressing and chemo-sensitising role in cancer cells.The chemo-sensitising effect of RKIP was investigated in a colon carcinoma cell line using a variety of chemotherapeutic agents from conventional agents to newer targeted therapies. Initial results suggested that role of RKIP in the modulation of chemotherapeutic drug response was at the level of apoptosis; there did not appear to be great observable effects in the cell proliferative response and the cell cycle distribution of the colon carcinoma cells after treatment with selected agents. Apoptosis modulation by RKIP occurred after treatment with doxorubicin, FasL, paclitaxel and TRAIL. TRAIL-treated colon carcinoma cells displayed increased cell death as the levels of RKIP within the cell were increased. In contrast, doxorubicin, FasL and paclitaxel-treated cells displayed a scaffold-like response as the levels of RKIP were increased in the cell; with WT RKIP-expressing cells being more sensitive to doxorubicin, FasL and paclitaxel-induced apoptosis than low or high RKIP-expressing colon carcinoma cells. There was no modulation of 5-FU, cisplatin and etoposide-induced apoptosis by RKIP. Indeed, these three agents did not appear to induce cell death in this colon carcinoma cell line. RKIP modulation of chemo-sensitivity has never been shown before in a colon carcinoma cell line and this is the first time that doxorubicin and FasL-induced apoptosis has been shown to be modulated by RKIP. Further, it is shown here, for the first time, that the modulation of chemotherapy-induced apoptosis by RKIP can change depending upon the cytotoxic drug employed as treatment. TRAIL and FasL, both members of the TNF super-family, were selected for further analysis due to the distinctive cell death responses observed as a consequence of the levels of RKIP within the cell. WT RKIP cells were sensitive to FasL treatment, and high RKIP cells were most sensitive to TRAIL administration. Increased sensitivity of high RKIP-expressing colon cells to TRAIL treatment appeared to involve up-regulation of the DR5 receptor; down-regulation of the anti-apoptotic molecule Bcl-xl; pIKK which activates the NF-κB pathway; and TRAF2 which has been shown to activate the NF-κB pathway. Whether RKIP directly interacts with these molecules is unknown however RKIP has been shown to bind upstream activators of the NF-κB pathway and another TRAF subtype TRAF6. YY1 expression was evident in the TRAIL-treated cells but the expression was unchanged as the levels of RKIP within the cell were altered. The FasL-treated cells also displayed decreased pIKK levels as the levels of RKIP were increased; it is possible that NF-κB was behaving as both pro- and anti-apoptotic within this cell line. Thus RKIP inhibition of the NF-κB pathway may have prevented FasL-induced apoptosis in the high RKIP-expressing colon carcinoma cells. The expression of TRAF6, which has been shown to bind RKIP, displayed a scaffold-like response with WT RKIP-expressing cells having the highest TRAF6 expression. This was also the case for the transcriptional regulator YY1, thus it is possible that both YY1 and TRAF6 were behaving in a pro-apoptotic-like manner in the WT RKIP-expressing cells. TRAF2 was also evident in the FasL-administered cells but the expression did not change regardless of the levels of RKIP within the cell. Overall, it appears that differential expression of TRAF adaptor proteins is responsible for the contrasting responses of TRAIL and FasL-treated cells with low, WT and high RKIP expression. Utilisation of particular TRAF adaptors or TRAF combinations by the TRAIL and Fas receptors may also account for the pro- and anti-apoptotic roles of the NF-κB pathway, and the recruitment or down-regulation of other proteins dependent upon the cell stimulus. How RKIP affects these proteins requires further investigation, however these results are exciting and novel, and strengthen evidence surrounding the role of RKIP in chemosensitivity. On another note, RKIP has been shown to bind the PDE5 inhibitor PF-3717842, therefore investigation of the effects of the PDE5 inhibitors sildenafil citrate and vardenafil citrate on RKIP inhibition of the ERK pathway in a colon carcinoma cell line were examined. The effects of the PDE5 inhibitors were compared to the cell migration inhibitor locostatin that has been shown to bind and inhibit RKIP, and prevent the RKIP-Raf-1 interaction. With TPA and EGF stimulation, locostatin appeared to act in a manner consistent with its known function as an RKIP inhibitor. The PDE5 inhibitors sildenafil citrate and vardenafil citrate displayed a similar trend to that of locostatin, although their effects on the ERK pathway were not as potent. It is possible that after EGF stimulation, the strong activation of B-Raf was over-shadowing the subtle effects of the drug treatments. Under growth conditions, the RKIP inhibitor locostatin did not appear to behave as an inhibitor of RKIP nor did the PDE5 inhibitors sildenafil citrate and vardenafil citrate. It is possible that the strong activation of various growth and proliferative cascades was impinging upon the ERK pathway, were overshadowing the drug effects, or resulting in off-target (RKIP-unrelated) effects of the drugs. In summary, the role of RKIP within the cell is becoming an increasingly exciting avenue of research and is consistently yielding new and interesting roles and interactions within the cell. Understanding and elucidating the roles of this intriguing protein within the cell will not only strengthen our knowledge of signal transduction regulation and modulation, but may also provide a new source of targeted therapy and means of manipulation in the treatment of cancer and chemotherapeutic drug resistance.
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Haworth, Caroline Joanne. "Cloning and expression of a modified oryzacystatin inhibitor gene and an investigation of its inhibitory capabilities." Doctoral thesis, University of Cape Town, 1997. http://hdl.handle.net/11427/9485.
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Bibliography: leaves 128-146.Cysteine proteinase inhibitors have shown potential as biocontrol agents for the protection of plants against insect and pathogen attack. With the advent of protein and genetic engineering such inhibitors can now be modified in order to improve their effectiveness. Because cystatins have already been isolated from plants. they provide a good starting point for developing modifications which may improve their function as biocontrol agents. The purpose of this project, therefore, was to design a potentially improved analogue of the rice cysteine proteinase inhibitor, oryzacystatin I, through molecular modelling studies. The gene sequence for this modified protein was then synthesised and expressed for kinetic analysis and insect trial assays. A prediction of the oryzacystatin I (OC I) tertiary structure was made using Biograf software on an Evans and Sutherland workstation. This structure was based on the known structures of stefin B and chicken cystatin ho se co-ordinates are published in the Brookhaven data files. Chicken cystatin is one of the most potent inhibitors of papain in the cystatin superfamily. This is believed to be due, in part, to an increased binding of the cystatin to papain through its amino-terminal region with the residues Leu7 to Gly9 playing a particularly important role.
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Dabos, Maria Laura Belen. "Structural and functional insights into the substrate specificity of OXA-48-like carbapenemases." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS402/document.
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Les b-lactamines, grâce à leur efficacité clinique, sont parmi les antibiotiques les plus prescrits pour traiter des infections bactériennes. Cependant, leur utilité est compromise par la prolifération des b-lactamases (BLs) avec des profils d’hydrolyse de substrats très larges. La résistance induite par les BLs compromet également les b-lactamines les plus puissantes (c-à-d les carbapénèmes). OXA-48, une carbapénèmase de classe D (CHDL), a été initialement identifiée dans une souche de K. pneumoniae de Turquie en 2001. OXA-48 hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les céphalosporines de troisième génération (C3G). Cependant certains variants comme OXA-163 ou OXA-405 hydrolysent les C3G et pas les carbapénèmes. La comparaison de la structure tri-dimensionnelle d’OXA-48 avec d’autres CHDLs a révélé de petites différences principalement localisées dans les boucles qui relient des éléments de structure. Notamment, la boucle localisée entre les feuillets b5 et b6 semble jouer un rôle majeur dans l’hydrolyse des carbapénèmes.Afin de mieux comprendre la contribution de la boucle b5-b6 dans l’hydrolyse des carbapénèmes, nous avons étudié, grâce à des outils biochimiques et structuraux, des variants naturels ou synthétiques d’OXA-48 présentant des modifications dans la boucle: le rôle du remplacement de chaque AA de la boucle par une alanine, des délétions croissantes ou de l’augmentation de la taille de la boucle. Nous avons également réalisé l’échange de boucle entre OXA-48 et OXA-18, une oxacillinase inhibée par l’acide clavulanique et hydrolysant fortement les C3G mais pas les carbapénèmes. La protéine recombinante OXA-48loop18 hydrolysait les C3G, et conservait une activité significative d’hydrolyse des carbapénèmes. L’échange de boucle a permis l’élargissement du site actif, permettant l’accès à des b-lactamines possédant un radical volumineux (e.g. ceftazidime). De plus, le remplacement de chaque AA de la boucle par une alanine a relevé de faibles changements hydrolytiques. En réalisant des délétions croissantes d’AA soit en partant de la gauche de la boucle (Tyr-211 vers Pro-217) ou de la droite (Pro-217 vers Tyr-211), nous avons montré que l’activité d’hydrolyse des carbapénèmes diminuait avec la taille des délétions, alors que celle des C3G augmentait. Les délétions de 4 AA présentent les plus fortes activités hydrolytiques des C3G et une perte totale de l’activité carbapénèmase, excepté pour le simple mutant, OXA-48∆P217, qui présentait un profile d’hydrolyse avec une forte activité carbapénèmase et C3G. La cristallographie et la modélisation moléculaire ont montré une grande flexibilité de la boucle, permettant l’entrée de b-lactamines de tailles variables. De plus, l’étude de nouveaux variants d’OXA-48 a permis d’identifier des déterminants structuraux importants dans le profil d’hydrolyse observé. Ainsi, la délétion I215-E216 associée à la substitution R214K dans la boucle b5-b6 de OXA-517 permet une forte hydrolyse des carbapénèmes et des C3G. De même, dans OXA-519, une substitution V120L située à proximité de la boucle b5-b6, a pour conséquence une diminution de l’affinité pour tous les substrats. La chaine latérale plus encombrante de la L120 empêche l’insertion des b-lactamines, diminuant l’affinité de l’enzyme. Finalement, nous avons caractérisé OXA-535, la b-lactamase naturelle et chromosomique de Shewanella bicestrii JAB-1 qui, n’ayant uniquement 91,5% d’identité en AA avec OXA-48, présente le même profil d’hydrolyse. OXA-535 présentait 98.9% d’identité en AA avec OXA-436, codé par un gène plasmidique, suggérant ainsi que S. bicestrii portant le gène blaOXA-535 pourrait être le progéniteur du gène plasmidique blaOXA-436.Nos travaux ont montré le formidable pouvoir d’adaptation de OXA-48 à évoluer par mutation afin d’accommoder différents substrats, et comment la nature et la longueur de la boucle b5-b6 pouvait influencer sur la spécificité de substratAntimicrobial resistance is the most alarming emerging problem in infectious diseases. b-Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most frequently prescribed antibiotics used to treat bacterial infections. However, their utility is being threatened by the worldwide proliferation of b-lactamases (BLs). BL-mediated resistance does not spare even most powerful b-lactams, carbapenems, whose activity is challenged by carbapenemases. OXA-48, a carbapenem-hydrolyzing class D b-lactamase (CHDL) initially identified from a Klebsiella pneumoniae isolate from Turkey in 2001, has since spread globally with the isolation of more than 30 variants. Most OXA-48-like enzymes hydrolyze penicillins at high level, carbapenems at low level and lack significant expanded-spectrum cephalosporin (3GC) hydrolysis, others such as OXA-163 hydrolyze expanded-spectrum cephalosporins and poorly carbapenems. Comparison of OXA-48 tertiary structure with those of other CHDLs revealed small differences located mainly in the loops connecting secondary structure elements, which may vary in length and orientation. The loop located between the b5 and b6 strands (Tyr211 to Pro217) has been suggested to play a major role in carbapenem hydrolysis.To better understand the contribution of the b5-b6 loop in the carbapenem hydrolysis of OXA-48-like carbapenemases, we investigated, using biochemistry and structural biology, natural OXA-48 variants with changes in different loops, replaced each AA of the loop b5-b6 by alanines, performed increasing deletions or increased the size of this loop by replacing it with that of OXA-18, a clavulanic acid inhibited class D b-lactamase that presents activity against expanded-spectrum cephalosporins and none against carbapenems. The resulting OXA-48loop18 was able to hydrolyze expanded-spectrum cephalosporins and conserved partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an opening of the active site, being now accessible to b-lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements in the b5-b6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by changes in kcat. By increasing deletions in the b5-b6 loop, starting from Tyr211 to Pro217 and from the Pro217 to Tyr211, the activity against carbapenems decreased with the size of the deletion whereas the activity against ceftazidime increased. 4 AA deletions revealed the highest 3GC activity, except for one single AA mutant, OXA-48∆P217, with high level carbapenem and ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility of this loop allowing different sized b-lactams to enter the active site. Moreover, the characterization of three novel natural OXA-48 variants revealed structural features important in the observed hydrolysis profile. Thus, the I215-E216 deletion and R214K substitution in the b5-b6 loop of OXA-517 induced the hydrolysis of carbapenems and C3G at high level. In OXA-519, the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active Ser70 and the b5-b6 loop, and therefore overall higher Km values were observed compared to OXA-48. The bulkier side chain of L120 in OXA-519 hampers the approach of b-lactam substrate, resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally-encoded OXA-535 that is more distantly related to OXA-48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA-535 presented 98.9% of AA identity with the plasmid-mediated OXA-436 suggesting that the blaOXA-535 gene might be the progenitor of the plasmid-encoded blaOXA-436 gene.Taken together, our work illustrates the propensity of OXA-48 to evolve through mutations to accommodate different substrates in its active site and how the b5–b6 loop determines the specificity of the enzyme
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Furnish, Robin. "Evaluating Immune Modulatory Therapeutic Strategies for Diffuse Intrinsic Pontine Glioma." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595849080346532.
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Freeman, Thomas Charles. "Studies of pancreatic secretory trypsin inhibitor." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46304.
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Alotaibi, Fahad T. "Plasminogen activator inhibitor-1 in endometriosis." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/59961.
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Endometriosis is a disease that affects almost 10% of reproductive-age women, where 50 % of these women have pelvic pain with sexual intercourse. Deep endometriosis is defined as an endometriotic lesion penetrating to a depth of 5 mm or more, and is characterized by both fibrosis (forming nodules) and invasion (into structures such as the colon). Other groups have found that increased plasminogen activator inhibitor-1 (PAI-1) or (SERPINE1) expression was associated with fibrosis and tumor invasion. In addition, a previous study found that the SERPINE1 4G allele (and thus increased gene expression) was associated with increased pain in women with endometriosis, and other work suggested that SERPINE1 may be implicated in local neurogenesis. The objective of this thesis is to determine whether PAI-1 expression is associated with a) deep infiltrating endometriosis; and b) deep dyspareunia. We propose that increased PAI-1 expression will be associated with deep infiltrating endometriosis, and increased pain in endometriosis via an increase in local nerve fibers. We utilized immunohistochemical analysis using a validated PAI-1 antibody. In the first cohort, we examined PAI-1 expression in deep infiltrating endometriosis and compared to endometrioma, superficial endometriosis, and eutopic endometrium. In the second cohort, we examined PAI-1 expression in cul-de-sac endometriosis from women with or without deep dyspareunia. We found higher expression of PAI-1 in deep infiltrating endometriosis (n = 10) compared to superficial endometriosis (n = 10) (p = 0.031) and eutopic endometrium (n = 10) (p = 0.002). In the second cohort, we found lower expression of PAI-1 in women with more severe deep dyspareunia (r = - 0.352, n =35, p = 0.038). However, there was no association between PAI-1 expression and local nerve bundle density. In conclusion, we observed higher PAI-1 expression in deep infiltrating endometriosis, but lower PAI-1 expression in endometriosis from women with deep dyspareunia. Further research is needed to clarify the complexities of PAI-1 expression in endometriosis.Medicine, Faculty of
Graduate
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Izzi, Luisa. "CEACAM1 as a tumor cell inhibitor." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ55070.pdf.
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Holmes, David Ian Roderick. "Phage display of chymotrypsin inhibitor II." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283414.
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Gariani, Talal. "Design of serine protease inhibitor peptides." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267244.
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Boak, Lorraine Scott. "Factors that impact scale inhibitor mechanisms." Thesis, Heriot-Watt University, 2013. http://hdl.handle.net/10399/2670.
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The formation of mineral scales such as barium sulphate and calcium carbonate remains an issue for the oil industry, after many years of oil exploration. In the last 10 years, the difficulty in dealing with scale deposition has been accentuated by the appearance of more complex conditions, involving complicated well completions for deepwater or long sub-sea tiebacks. If scale control measures fail in these situations then long distances between the scale deposits and the production platform are present. Intervention into such systems is either impossible or extremely expensive. To combat such problems, the front end engineering design stage (FEED) now attempts to bring together multidisciplinary teams to provide a full risk assessment of all areas in which production chemistry problems might arise. Hence, benefits come from each discipline team having as much knowledge as possible available to them. This thesis aims to fuel this knowledge by developing a fundamental understanding of how various factors, conditions or environmental, impact scale inhibitor mechanisms, so that the results can be incorporated into the FEED process. Key areas affecting scale inhibitor operation were investigated. From these studies, a number of important findings can be highlighted. The presence of calcium was found to improve scale inhibitor (SI) performance, especially phosphonate types, whilst magnesium ions had little effect on polymeric performances and detrimentally affected the phosphonates’ inhibition efficiency (IE). These trends were related to the SI affinity for the divalent ions – polymer PPCA binds to calcium but shows incompatibility at [Ca2+] > 1000ppm - observed as low IE, whilst the phosphonate DETPMP binds with either ion but prefers calcium. Two inhibition mechanisms - nucleation and crystal growth blocking - were identified for different types of SI species and were illustrated using static IE tests relating IE to [SI] left in solution. High IE corresponds to high [SI] and similarly low IE with low [SI]. These initial results have since been investigated further in a additional study. An extensive range of phosphonate and polymeric scale inhibitor species can now be classified as i. either Type 1 or 2 (based on IE, Ca2+ and Mg2+ sensitivity ration and SI consumption tests) or ii. either Type A or B (based on compatibility/incompatibility with [Ca2+]= ~1000-2000ppm+). A requirement for both homogeneous and heterogeneous nucleation to be investigated for a scaling system was identified, as deposition kinetics can vary requiring different ii levels of SI. A [SI] falling below minimum inhibitor concentration (MIC), can promote surface scaling. Hence, scaling systems should be studied experimentally over a range of temperatures, to represent the conditions from sub-sea tiebacks to the production well. A model was developed from experimental data enabling the prediction of safe sulphate levels and mass of barite deposited. This model can be applied to un-seeded and seeded tests where, as expected, the foreign particles accelerated the reaction to equilibrium with the greatest deposition rate for barite over sand and for a higher surface area over a lower one. Both theoretical and experimental confirmation of each retention mechanism occurring in a porous medium was achieved. This adsorption/precipitation model has been incorporated into Squeeze VII, an in-house squeeze design software, to allow a better physical description of a squeeze treatment. The predictions of Squeeze VII have also been improved by using the more accurate data for the scale inhibitor return concentrations from core floods due to the better developed analysis techniques. The direct value of these improvements to industry is significant. These advances reduce OPEX costs and deferred oil production whilst giving the industry the opportunity of improved future lifetime predictions and operations.
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Christofakis, Steven. "SCRIBBLE: A POTENTIAL DUAL KINASE INHIBITOR." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/72.
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Extracellular signal-regulated kinases (ERKs) modulate cellular activities in response to extracellular stimuli and play important biological roles. Thus, perturbed kinase pathways induce pathological conditions, such as tumor development. Rit, a novel member of the Ras family GTPases, activase ERK6, and its over-expression confers tumorigenicity. We hypothesized the presence of scaffolding molecules specific to ERK6, similar to other known MAP kinases. We performed yeast two-hybrid assays using ERK6 as bait, and Scribble was identified as a binding partner. Scribble contains 16 LRR domains and four PDZ domains. We performed immunoprecipitation (IP) assays and discovered ERK2 as another binding partner. Surprisingly, no interaction was observed with the highly homologous MAP kinase, ERK1. No other representative kinases showed binding capabilities with Scribble. IP data confirmed that both ERK2 and ERK6 bind to Scribble through its LRR and PDZ domains. Deletion of ten aminoi acids from the C-terminus of ERK2 and ERK6 abolished these interactions. In vitro kinase assays indicated the kinase suppressing ability of Scribble. Focus formation assays were performed with RitQ79L and H-RasV12 as constitutive activators of ERK6 and ERK2, respectively, in the presence of Scribble. Results confirmed the role of Scribble as a tumor suppressor.
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Chee, Lai Yuen. "p53, a novel inhibitor of apoptosis." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6835.
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p53 is a transcription factor known to induce apoptosis via transactivating the expression of pro-apoptotic proteins and by directly activating the mitochondria apoptotic pathway. p53 is also found to be mutated in 50% of human cancers with some of these tumours overexpressing both wild type (WT) and mutant p53. Overexpression of the p53 protein has been implicated with the more aggressive nature of these tumour cells, suggesting a possible gain-of-function of such mutants. In this study, the involvement of p53 in apoptosis via the caspase pathway was investigated. WT-p53 and three mutant p53 with mutations at the 1) N-terminus (D42Y), 2) central domain (R175H) and 3) C-terminus (R337H) were selected. The data collected demonstrated that p53 was able to inhibit the cleavage of caspases early in the apoptotic pathway. In vitro assays showed that addition of recombinant WT and the mutant p53 inhibited the cleavage of both caspase-9 and caspase-3 and subsequently PARP, while overexpression of p53 in mammalian cells yielded the same inhibition profile in vivo. Conversely, removal of p53 via siRNA and immunodepletetion showed accelerated caspase-9 activity and cleavage. Immunoprecipitation experiments and recombinant assay systems suggest that the inhibition by p53 is targeted at the active cleaved caspase-9. In addition, the presence of p53 (WT or mutant) in p53-null cells were able to confer a higher survival rate. These data therefore demonstrate that p53 may have an additional anti-apoptotic role via the inhibition of caspase-9, which is often masked by its pro-apoptotic functions. This anti-apoptotic role could manifest itself in a cancer background overexpressing mutant p53 which has lost its transcriptional activity and hence its ability to induce apoptosis via the induction of pro-apoptotic genes such as PUMA and NOXA. This newly discovered role of p53 could potentially explain the aggressive nature of cancers which overexpress p53.
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He, Hua, and 何華. "Anti-tumor mechanisms of cyclooxygenase inhibitors and a c-Jun-N-terminal kinase inhibitor in gastrointestinal cancers." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30075245.
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Roever, Lisa. "Inhibitor Studies for 5’-ecto-nucleotidase (CD73)." Ohio University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1553892946798977.
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Noma, Naruto. "Inhibition of MMP-2-Mediated Mast Cell Invasion by NF-κB Inhibitor DHMEQ in Mast Cells." 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225446.
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Tuck, Benjamin. "Investigating Multispecies Biofilms on Steel Surfaces in Seawater and Biofilm Inhibition by a Novel, Multifunctional Inhibitor." Thesis, Curtin University, 2022. http://hdl.handle.net/20.500.11937/89066.
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Biofilm formation is a global, $multi-billion phenomenon spanning a plethora of stakeholders. This thesis investigates critical fundamental aspects of biofilm formation on steel and evaluates the efficacy of a novel, environmentally sustainable and multifunctional inhibitor compound developed through a broader Australian Research Council Discovery Project collaboration. Focused on sustainable and effective biofilm disruption, results from this thesis are used to expand fundamental knowledge and generate a targeted approach to biofilm mitigation that improves biocide function.
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Geng, Xinyan. "Investigations into how best to target FGFR2 mutant endometrial cancer." Thesis, Queensland University of Technology, 2017. https://eprints.qut.edu.au/123437/1/Xinyan%20Geng%20Thesis.pdf.
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Endometrial cancer (EC) is the fourth most common cancer in women in developed countries, such as North America, Europe and Australia. Patients with low-grade, early-stage disease usually have a favourable survival rate. However, patients that present at an advanced stage of disease have an average survival of only 12 months. Current treatments for these patients are radiation and chemotherapy, which offer limited clinical benefit. There is no efficient treatment for advanced EC. Improved therapeutic approaches are needed for the treatment of recurrent and metastatic endometrial cancer. Recent advances in cancer biology have resulted in the development of molecular targeted therapies.
The Fibroblast Growth Factors Receptor (FGFR) family and their ligands (fibroblast growth factors, FGFs) regulate a broad spectrum of physiological processes as well as tissue patterning and organogenesis during embryogenesis. Abnormally activated FGFRs have been identified in various cancers and are emerging as potential therapeutic targets. The Pollock laboratory and other groups have demonstrated that 10-20% of endometrioid ECs carry FGFR2 mutations that may be a novel therapeutic target in endometrial carcinoma.
Preclinical studies show that inhibition of FGFR can inhibit EC cell growth in vitro. However, FGFR inhibitors are not as efficient at inhibiting tumour growth in vivo. We aim to find a way to improve the efficacy of FGFR inhibition in cancer treatment. About 90% of EC patients harbour genetic aberrations in the components of the PI3K/AKT pathway which indicates this signalling pathway plays an important role in the development of EC. Work from our lab demonstrates that inhibition of FGFR results in abrogation of MAPK activation in sensitive EC cells, however, the PI3K/AKT signalling pathway remains unaffected. PI3K/AKT signalling plays a vital role in cancer cell proliferation and survival, furthermore crosstalk between the MAPK and PI3K/AKT signalling pathways is associated with resistance to targeted therapies. Thus, the first aim of this study was to examine whether combination of the FGFR inhibitor (BGJ398) with various different PI3K inhibitors was synergistic in FGFRi sensitive EC cells.
We present data that the combination of the pan-FGFR inhibitor (BGJ398) with pan-PI3K inhibitors (GDC-0941, BKM120) or a p110α-selective PI3K inhibitor (BYL719) was synergistic in inhibiting cell growth. Significantly more cell death and inhibition of long-term cell survival was observed in the combination treatments compared to each of the single drug treatments. Importantly, these effects could also be observed at lower concentrations. This study is the first to indicate that partial inhibition of the PI3K signalling pathway could significantly increase cell death when combined with the FGFR inhibitor BGJ398 in FGFR2 mutant EC cells. These data provide evidence that sub-therapeutic doses of PI3K inhibitors could enhance the efficacy of anti-FGFR therapies and a combination therapy may represent a superior therapeutic treatment in FGFR2 mutant EC patients. The in vivo work (conducted by Dr Vanessa Bonazzi) shows that the combination of BGJ398 and GDC-0941 and BYL719 resulted in tumour regression, while single drug treatment only slowed tumour growth. Interestingly, BYL719 alone resulted in increased tumour growth in tumour xenografts of AN3CA but not JHUEM2.
In the first results chapter we further investigated the mechanism of enhanced cell death from the combination of BGJ398 and PI3K inhibitors. The activation of ERK and AKT has been inhibited by the combination of BGJ398 and PI3K inhibitors. However, the combination of the MEK inhibitor trametinib and the PI3K inhibitors induced less cell death than inhibition of the FGFR and PI3K signalling pathways. BGJ398 but not trametinib or GDC-0941 inhibited the activity of PLCγ1. We have also found trametinib up-regulated PLCγ1 activity, which is a novel finding in the field. We next employed several pharmacological inhibitors to investigate whether PLCγ1 is involved in the cell death observed following the combination of BGJ398 and GDC-0941 treatment. As there is no PLCγ1 inhibitor available currently, we used two different pan-PLC inhibitors, manoalide and U73122. Co-inhibition of the MAPK, PI3K/AKT and PLC signalling recapitulated cell growth inhibition seen with the combination of FGFR and PI3K inhibitor in both cell lines. Cell death induced by the combination of PLC inhibitors with trametinib and GDC0941 was similar as the combination BGJ398 and GDC0941 in AN3CA, but significantly less than the combination BGJ398 and GDC0941 in JHUEM2. Unfortunately, Western blotting was unable to show inhibition of PLCγ1 bringing into question whether these PLC inhibitors inhibited PLC function sufficiently, and whether the phenotypic effects of manoalide and U73122 when added to the trametinib and GDC0941 combination are due to inhibition of PLCγ1.
The second results chapter reports efforts to identify the mechanism of intrinsic resistance to FGFR inhibition in EC cell lines carrying FGFR2 activating mutations but showing intrinsic resistance to FGFR inhibition (EI, EN1078D, and MFE319) with comparisons to the two sensitive EC cell lines (JHUEM2 and AN3CA). We have observed sustained activation of ERK in the resistant cells after treatment with an FGFR inhibitor, while ERK was inhibited in the sensitive cells. Inhibition of the MAPK signalling pathway could not sensitise the resistant cells to FGFR inhibition. Although several other receptor tyrosine kinases (RTKs) were hyperactivated in these cells, pharmacological inhibition did not show they were reliant on these RTKs. Co-inhibition of these kinases did not sensitise these cells to BGJ398. Knockdown of FGFR2 by shRNA in the sensitive cells induced moderate cell death, but limited cell death in the resistant cells. Interestingly, co-inhibition of the MAPK, PI3K/AKT and PLC signalling pathways has induced markedly less cell growth inhibition in the resistant cells compared to the sensitive cells, suggesting the resistant cells are less dependent on these central signalling pathways than the sensitive cells. Western blotting results showed that FGFR2 expression was considerably lower in the resistant cells than in the sensitive cells. Based on these results we have concluded that FGFR2 mutation status is not the only factor that determines sensitivity to FGFR inhibition, high expression of mutant FGFR2 is also important. This is a novel finding in the field and one which could guide patient select criteria in future clinical trials. Lastly, we show that FGFR2 knockdown in medium containing 10% FBS has little impact on downstream ERK phosphorylation whereas pan FGFR inhibition with BGJ398 could totally abrogate ERK phosphorylation. In cells grown overnight in serum starved conditions, FGFR2 knockdown did reduce downstream ERK phosphorylation but not to the same extent as pan FGFR inhibition in full growth medium. These data suggest that inhibition of FGFR2 alone is insufficient and that inhibition of multiple FGFRs will be more effective as a cancer treatment.
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Nakahira, Marcel. "Caracterização físico-química e estrutural do SbKI, um inibidor de serinoproteases de sementes de barbatimão (Stryphnodendron barbatiman)." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-01042014-174549/.
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Os inibidores de proteases desempenham nas plantas funções como: defesa contra ataque de predadores de sementes, regulação de enzimas endógenas e fontes de proteínas e aminoácidos. Muitos destes inibidores são utilizados em estudos bioquímicos, bem como no tratamento de patologias humanas como inflamação e câncer. Neste trabalho, um inibidor de serinoprotease, presente na semente de Stryphnodendron barbatinan (barbatimão), foi purificado, caracterizado e denominado SbKI. Sementes de barbatimão maduras foram trituradas, até a obtenção de uma farinha, e esta foi suspensa em PBS, pH 7,4 (1 :5 m/v), sob agitação por 14 horas a 4°C. O extrato foi centrifugado, filtrado e tratado com PVPP, sendo denominado EB, o qual apresentou inibição da coagulação sanguínea e da atividade de algumas serinoproteases. O inibidor SbKI foi purificado utilizando-se três procedimentos cromatográficos: cromatografia de exclusão molecular (Superdex-75, 10/30), troca iônica (Mono-S HR, 5/5), ambas acopladas em um sistema &TA Purifier e fase reversa (C-18, Waters 250 x 4,6mm) acoplada a um sistema HPLC. Em cada etapa de purificação a presença do inibidor foi monitorada pelos testes de atividade inibitória da tripsina e da coagulação, ambos in vitro. SDS-PAGE, sob condições redutoras, mostrou que o inibidor é formado por duas cadeias polipeptídicas (cadeia pesada e leve) unida por ligação dissulfeto. As cadeias foram separadas pela cromatografia de &se reversa após serem reduzidas e alquiladas. Suas seqüências N-terminais foram determinadas pela degradação de Edman, em seqüenciador automatizado, apresentando alta identidade seqüencial com inibidores do tipo Kunitz de outras leguminosas. A determinação da massa/molecular do inibidor e de suas cadeias isoladas, foram determinadas por espectroscopia de massa (LCtESI-MS system) mostrando massas moleculares de 19.570Da7 15530Da e 4040Da, respectivamente. A espectroscopia de dicroísmo circular (CD) revelou que o inibidor é formado predominantemente por elementos beta e estruturas desordenadas. SbKI foi estável a variações de pHs (2-12) e temperaturas extremas e a temperatura de transição foi calculada em 73,3\" C. A determinação das constantes de inibição (KI) foi realizada para as serinoproteases tripsina (KI = 5,5 nM) e calicreína plasmática (KI = 1,l nM).Proteinase inhibitors perform many beneficia1 roles in plants such as defense against the attack of seed predators, regulation of endogenous enzymes and sources of proteins and amino acids. Many inhibitors are used in biochemistry research, as well as human pathology treatment such as inflammation and cancer. In this work, a serino proteinase inhibitor found in Stryphnodendron barbatiman seeds (barbatimão) was purified, characterized and denoted SbKI. Mature barbatimão seeds were ground and suspended in PBS pH 7.4 (15 wlv) and stirred for 14 hours at 4OC. The suspension was centrifuged, filtered and treated with PVPP and denoted EB. This EB inhibited blood coagulation and some serine proteinases activities. The inhibitor SbKI was purified by three chromatography step: molecular exclusion (on Supredex-75, 10/30), ion exchange (on Mono-S, 5/5), both connected to AKTA Purifíer System and reversed phase (on C-18, Waters 250 x 4.6 mm) connected to HPLC System. In each purification step the presence of inhibitor was monitored, in vitro, by trypsin and coagulation inhibitory activity. SDS-PAGE, reduced conditions, showed two polypeptide chains (heavy and light chains) linked by one disulphide bridge. The chains were separated by reversed phase chromatography aíter reduced and alquilated. The N-terminal sequence were performed on automated protein sequencer by Edman degradation and showed homology with Kunitz type inhibitors from Leguminosae. Molecular weight of inhibitor and its chains were determined by mass spectrometry (LC/ESI-MS System) and showed molecular weight of 19.570Da, 15.530Da and 4040Da, respectively. Circular dichroism spectroscopy showed SbKI is constituted predominantly by P elements and unordered structures. SbKI was stable over extreme ranges of pH (2-12) and temperature and the transition temperature 73.3\"C investigated by CD and fluorescence emission spectroscopies. Inhibition constants (Ki) were determined by typsin (Ki = 5.5 nM) and human plasmatic kallikrein (Ki = 1.1 mM)
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Dawson, Sally. "Genetic variation at the plasminogen activator inhibitor-1 locus and its effect on plasminogen activator inhibitor-1 expression." Thesis, Imperial College London, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298676.
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McLaren, Lorna J. "The mouse protein phosphatase inhibitor-1 gene." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/24958.
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Intracellular proteins are phosphorylated by kinases and dephosphorylated by phosphatases. Protein phosphatase inhibitor-1 (I-1) inhibits protein phosphatase-1 (PP-1), thereby increasing the phosphorylated state of proteins in the cell. The initial aim of the work reported here was to determine whether I-1 is a kidney 'stem' cell marker (Svennilson et al., 1995); if so, it would be the first unique marker for these cells. The project involved characterizing the protein coding region of the mouse I-1 gene, determining the I-1 protein expression pattern in the developing mouse embryo and analysing potential promoter elements of the I-1 gene. A mouse genomic library was screened using a mouse cDNA clone homologous to the published rat I-1 mRNA and two types of clones with positive sequence homology to the rat mRNA sequence were isolated. These overlapping clones were sequenced, and analysis showed that the predicted mRNA and protein sequences contain 513 nucleotides and 171 amino acids, respectively, with both sharing over 95% homology with the known rat sequences. Further comparison of the predicted mouse protein I-1 sequence to those of rabbit, rat and human showed that there is strong homology across species, and that all share motifs which are important for inhibiting PP-1 (a KIQF sequence at amino acid positions 9-12 and a threonine at position 35). The mouse I-1 gene contains seven protein-coding exons which lie in a 7 kb region of DNA on chromosome 15, band F. Svennilson et al. (1995) detected I-1 expression in peripheral metanephric mesenchyme (nephron progenitor) cells in rate sections using RNA in situ hibridization. With this in mind, the mouse protein expression pattern was determined using wholemount tissue, a commercially available anti-I-1 antibody and fluorescent confocal microscopy. The results showed that I-1 is not a 'stem' cell marker in the developing kidney, but is restricted to the peripheral epithelial layer of cells of the kidney i.e. the mesothelium.
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Montgomerie, Harry. "Novel inhibitor chemistry for oilfield scale application." Thesis, University of Huddersfield, 2014. http://eprints.hud.ac.uk/id/eprint/24277/.
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The body of work presented here is focussed on five published papers which address solving inorganic scale problems experienced by the North Sea oil industry with a focus on the Norwegian Continential Shelf (NCS) over the last ten years or so. The degree of increasing difficulty in addressing issues of serious barium sulphate scaling, in the reservoir and wellbore areas, complicated by seawater breakthrough, the authorities demand for improvement in environmental properties of the chemistries deployed, the cost driven push for longer treatment life and with the increasing awareness of the damaging effects of deploying water based scale inhibitors into water sensitive reservoirs, is described in detail. The work focuses on industry developed laboratory test methods, synthesising novel chemistries and developing improved deployment designs as a means of solving these significant problems. The work is not a pure chemistry programme, it is an applied chemistry study focused on solving reallife oilfield scale problems. The project(s), planning and strategy involved leveraging knowledge and input from across the disciplines of production chemistry, petroleum engineering and near wellbore region modelling. It should be noted the real driver for the work was the inability of the available commercialised chemistries to resolve the problems. The resultant loss of production and associated remedial treatments were of a significant financial and environmental cost to the industry. The papers cover the design testing and deployment of co-polymers and terpolymers to produce innovative molecules which offer higher performance scale inhibition and life of treatments in actively producing oil wells. The new molecules meet the environmental requirements of biodegradation, bioaccumulation and toxicity. The projects offered are under the following headings: Enhanced inhibitor squeeze treatment life through "Bridging" Field experiences in Application of Inhibitor Interactive Packages Resulting in Increased Squeeze Life. Development of multi-functional chemicals for efficient "fines" control and squeeze life enhancement in producing oil wells. Development of highly efficient and environmentally friendly scale inhibitor molecules for oilfield use, including reference to more efficient placement technology. Oil soluble scale inhibitor development. The papers describe synthesis, laboratory testing and field deployment experiments leading to adoption of the new technology by multi-national oil companies. In most cases patents have also been granted. The value added of the work is also quantified in both environmental and financial aspects.
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Davies, Glyn Daniel. "Inhibitor studies on para-aminobenzoic acid synthase." Thesis, University of Cambridge, 2003. https://www.repository.cam.ac.uk/handle/1810/265461.
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Para-aminobenzoic acid (PABA) synthase is a three-subunit enzyme system that catalyses the conversion of chorismic acid top-aminobenzoic acid in plants and microorganisms. P ABA is then incorporated into folic acid an essential nutrient for mammals and utilised in the transfer of one-carbon units. For this reason it is a potential target for herbicidal and antibiotic development. A glutamine amidotransferase and ADC synthase form a heterodimeric complex and carry out the amino substitution reaction to yield 4-amino-4-deoxychorismate (ADC). A separate subunit, ADC lyase, then carries out the elimination of pyruvate to yield P ABA. My research describes a number of advances in the purification and assay system used and the organic synthesis of inhibitors of ADC synthase. The purification and characterisation was carried out of newly cloned 6-His tagged PABA synthase enzymes from E. coli. engineered to overexpress these proteins. The assay system used was refined and substrate kinetic data obtained. Results were compared to 'wild-type' enzyme and data obtained when the assay system has ammonium in place of glutamine and glutamine amidotransferase. The crystal structures of 'wild-type' ADC synthase and ADC Lyase enzymes are only recently available in the literature. Inhibition data for compounds synthesised was obtained initially via a fluorescence screen and then to greater accuracy via a continuous UV assay. My inhibitor studies revealed a close structural analogue of 4-amino-4- deoxychorismate, 4-amino-3-[1-carboxyethoxy] benzoate as the best inhibitor (ea. 20 ?M) of P ABA synthase so far. The synthesis of a number of aromatic compounds to act as m1m1cs of ADC was accomplished. An aromatic nitro group was reduced to give an amine using tin(II) chloride and hydrogenation reactions. Other forms of nitrogen protection using carbamates and bissilyl protection were also utilised. The formation of an enol pyruvyl group was accomplished in an original manner using lithium diisopropylamine to deprotonate a proton alpha to an ester. This was followed by selenation and elimination/oxidation to form the required methylene group as an a,,B-unsaturated ester. Alternative methods, involving a rhodium catalysed hydroxy insertion followed by either a Wadsworth-Horner-Emmons modified Wittig reaction with formaldehyde or alkylation with Eschermosers' Salt, were also carried out. Further synthesis of ADC and chorismate analogues was performed using the aza-Cope and oxy-Cope rearrangements of oxygen-alkylated hydroxybenzoates and N-alkylated aromatic amines. The organic synthesis of an inhibitor containing vinyl fluoride functionality has been the focus of previous studies. The biosynthesis of 6-fluorochorismate led to the discovery that this species was an irreversible inhibitor of the P ABA synthase system. Synthesis of a vinyl fluoride was achieved via a vinyl triflate and vinyl trialkylstannane utilising a lithium cuprate reagent. Fluorination was carried out using XeF2 in the presence of a catalytic amount of Ag(OTf). The mild conditions achieved form a foundation for the synthesis of more complex 6-fluorochorismate analogues. Further synthesis was carried out to try to synthesise a Michael acceptor for an enzyme active-site nucleophile based on a proposed mechanism for the irreversible inhibitor. Dianion alkylation chemistry using �sodium hydride followed by n-BuLi was used to control regioselectivity in the alkylation of a cyclic ,8-ketoester. In a one-pot synthesis, selenyl elimination/oxidation of the alkylated cyclic ,8-ketoester was used to form an extended delocalised system with the potential to act as a Michael acceptor.
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Mahmoodi, Niusha. "Indole prenyltransferases : mechanistic studies and inhibitor design." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/55872.
Full textAbstract:
The cyclic dipeptide N-prenyltransferase (CdpNPT) catalyzes the reverse C-3 prenylation of a variety of cyclic dipeptides and benzodiazepinediones. A previous study misassigned the structure of the product of this reaction. In this work, the true product of the CdpNPT-catalyzed reaction between cyclo-L-Trp-L-Trp and dimethylallyl diphosphate (DMAPP) is identified as a C-3 reverse prenylated species. Furthermore, the non-enzymatic Cope/aza-Cope rearrangement of the CdpNPT product was examined under acidic conditions. Our results indicated that only the aza-Cope rearrangement onto the N-1 position of the indole ring can occur and no Cope rearrangement onto the C-4 position was observed. These results suggest that in the absence of an enzyme active site, the aza-Cope rearrangement is preferred over the Cope rearrangement.
Brevianamide F prenyltransferase (FtmPT1) catalyzes the C-2 normal prenylation of brevianamide F (cyclo-L-Trp-L-Pro). A mechanism involving a direct C-2 attack was proposed for this reaction. However, the structural analysis of FtmPT1, as well as studies of alternate substrates and mutant enzymes suggested that a different mechanism involving an initial C-3 reverse prenylation followed by a rearrangement may be operative. In this work, we investigated the reactivity of FtmPT1 with tryptophan and cyclo-L-Trp-L-Trp, as well as two alternate substrates: 5-hydroxybrevianamide F and 2-methylbrevianamide F. The isolated products were reverse prenylated at C-3 and normal prenylated at N-1, C-2, C-3, or C-4. The formation of these products can be rationalized through mechanisms involving either an initial C-3 normal or C-3 reverse prenylation as the first step of catalysis.
4-Dimethylallyltryptophan synthase is an aromatic prenyltransferase that catalyzes an electrophilic aromatic substitution reaction between DMAPP and L-tryptophan. The reaction is believed to proceed via the dissociation of DMAPP to form a dimethylallyl cation/phosphate ion pair. An inhibitor containing a guanidinium moiety appended to a phosphorylated phosphonate was designed in order to mimic the transition state for the dissociation of DMAPP into an allylic carbocation and pyrophosphate. This compound was found to serve as a potent competitive inhibitor (submicromolar Ki value) of the enzyme 4-DMATS.Science, Faculty of
Chemistry, Department of
Graduate
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Cho, Park 1975. "The Cap-binding inhibitor of translation, d4EHP /." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111819.
Full textAbstract:
In eukaryotes, the initiation phase of protein synthesis or translation is a multi-step process that culminates in the positioning of the SOS ribosome at the initiation codon of a messenger RNA (mRNA). Recognition of the cap structure by eukaryotic initiation factor 4F (etF4F; composed of three subunits: the cap-binding protein e1F4E, the RNA-helicase eIF4A and the scaffolding protein eIF4G) facilitates this process. The ability of eIF4F to bind to the cap, as a result of the Cap:eIF4E interaction is of particular importance, as it is the major target of translational regulatory mechanism.Early embryogenesis requires the activity of various maternal determinants called morphogens, whose spatial and temporal expressions are tightly regulated at the level of translation. Positional information encoded within these factors is thus important for the establishment of body polarity. For instance, in Drosophila, when maternal Caudal (Cad) and Hunchback (Hb) proteins are allowed to accumulate inappropriately in an embryo, anterior and abdominal segmentations are blocked. Hence, the precision of Cad and Hb expression domains is critical for normal development.
An eIF4E-related protein called eIF4E-Homologous protein (4EHP) was first described in 1998. However, the function, if any, of 4EHP in translation has been elusive, since it does not interact with any known initiation factors. In order to elucidate its biological function, the power of Drosophila genetics was used. In this thesis, I show that the Drosophila homolog of 4EHP (d4EHP) interacts with Bicoid (Bcd) and Brain tumor (Brat) proteins to inhibit the translation of maternal cad and hb mRNAs. Simultaneous interaction of d4EHP with the cap and Bcd or Brat results in mRNA circularization, which renders cad and hb mRNAs translationally inactive. This example of cap-dependent translational control that is not mediated by eIF4E defines a new paradigm for translational inhibition involving tethering of the mRNA 5' and 3' ends.
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Sirry, Baheya. "Regulation of the translational inhibitor 4E-BP1." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79128.
Full textAbstract:
Regulation of translation has long been recognized as a crucial event in the control of gene expression. It allows for rapid changes in gene expression in response to a wide-range of extracellular stimuli. Variations in nutrient availability for instance are detected and transmitted through the protein kinase FRAP/mTOR to key translational regulators such as the CAP-dependent translation inhibitor 4E-BP1. 4E-BP1 function is regulated mainly by phosphorylation. The focus of this thesis is to better understand signalling involved in 4E-BP1 regulation.We first decided to examine 4E-BP1 phosphorylation against the background of an embryonic lethal mutant of FRAP/mTOR, the flat-top mutant, in order to elucidate the effect of this point mutation on FRAP/mTOR function. Our results revealed that signalling leading to 4E-BP1 phosphorylation is impaired in these mutants.
We were then interested in establishing 4E-BP1's role in the cell cycle by examining its phosphorylation state during mitosis. This study was based on the finding that CAP-dependent translation is inhibited in mitotic cells. Using 2-dimensional gel electrophoresis we detected two new isoforms of 4E-BP1 during mitosis.
The above studies demonstrate the essential role of 4E-BP1 in mammalian development and the cell cycle, emphasizing the need to better understand mechanisms involved in its regulation.
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Patient, Michaela Eileen. "Rcd, a ColE1-encoded cell division inhibitor." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309210.
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Duncan, S. J. "Structural studies on a p53-MDM2 inhibitor." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598682.
Full textAbstract:
The 53 kDa phosphoprotein, p53 plays a critical role in the regulation of cell proliferation and development of genetic abnormalities by inducing G1 arrest or apoptosis in response to DNA damage. It also forms a stable complex with the MDM2 protein, in which state, the transactivation domain of p53 is concealed from the transcriptional machinery and is unable to induce G1 arrest or apoptosis. p53 activates the transcription of the mdm2 gene, so the levels of MDM2 and the activity of p53 are kept under the control of an autoregulatory feedback loop. When overexpressed, MDM2 acquires tumorigenic potential. It is possible that tumours expressing abnormally high levels of MDM2 could be treated with small molecules that disrupt the p53-MDM2 interaction, thereby restoring normal function to wild-type p53. A peptidic secondary metabolite from Microdochium caespitosum has been identified as an inhibitor of the p53-MDM2 interaction. The primary structure of this natural product, chlorofusin, has been determined by NMR, mass spectrometry and chiral GC-MS. The three-dimensional structure has been calculated with random simulated annealing, based on NOE and coupling restraints. An investigation into the biosynthetic origin of the chromophore of chlorofusin was then carried out with 13C-labelled sodium acetate feeding experiments. Finally, the N-terminal regions of both MDM2 and p53 were expressed for SPR binding assays with chlorofusin, to further investigate chlorofusin's mode of action. It was shown that chlorofusin binds to the N-terminal region of MDM2. As an antagonist of the p53-MDM2 interaction, chlorofusin could be useful in the design of new drug candidates for the treatment of tumours arising from the overexpression of MDM2.
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Panahloo, Archia. "Plasminogen activator inhibitor-1 and cardiovascular risk." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286442.
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Bevan, A. W. "Specificity of inhibitor binding to dihydrofolate reductase." Thesis, University College London (University of London), 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.352532.
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Potter, Garrett. "Chemical synthesis of a mimetic heparanase inhibitor." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/chemical-synthesis-of-a-mimetic-heparanase-inhibitor(6802b624-c3c0-4209-9c6d-bbcebf8e2d0b).html.
Full textAbstract:
Heparanase (Hpa1) is an enzyme overexpressed in nearly all cancers, typically at the tumour growth front. It cleaves proteoglycan heparan sulfate (HS) chains to release growth factors necessary for tumour growth. While some carbohydrate-based mimetic inhibitors have progressed to advanced clinical trials, new inhibitors and tools to further investigate heparanase are of continued interest. This thesis proposes a HS mimetic trisaccharide sequence that can bind Hpa1 and is suitable both for biological evaluation and inhibitor development. Synthetic work was then undertaken toward the progression of this moiety. Exploring building blocks applicable to the trisaccharide, conformationally-locked glucose derivatives were developed. This included the introduction of a conformational switch that resulted in the isolation of constrained half-chair conformers. The synthetic work toward trisaccharide formation also evaluated the utility of 1,2-cyclohexane-diacetal as a protecting group with glucuronic acid. The disarming qualities of these moieties were assessed, leading to the development of alternate routes. A more linear approach resulted in the formation of important disaccharide building blocks that contribute toward the synthesis of the core trisaccharide, including isolated 1,2-orthoesters. Further development of the chemistry established herein should allow for the formation of the desired core trisaccharide, while contributions have additionally been made toward its tool functionalisation and use in multivalent schemes.
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Campbell, A. F. "Protein engineering of chymotrypsin inhibitor II (C12)." Thesis, Imperial College London, 1988. http://hdl.handle.net/10044/1/46983.
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陳恒琦. "Studies on the Inhibitory Mechanism of Angiogenesis Inhibitor, endostatin." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/08355482627008954517.
Full textAbstract:
碩士國立中山大學
生物科學研究所
89
Antiangiogenic tomor therapies have attracted intense interest for their broad-spectrum action, low toxicity, and in the case of direct endothelial targeting, and absence of drug resistance. Among the growing list of antiangiogenic agents, endostatin has attracted most attention and been under the spotlight of numerous debates. Like other angiogenesis inhibitors, endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on cencer cells. Above all, therapy of experimental cancer with endostatin in rodents leads to tumor dormancy and does not induce resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E.coli expression vector to express and generate large quantities of recombinant GST-fused endostatin (GST-endostatin). Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting various endothelial cell lines including HUVEC, EA.hy926 and BAEC with IC50~20nM. Flow cytometry analysis indicated GST-endostatin induced apoptosis in EA.hy926 cells toward chemoattractant bFGF with IC50~0.5nM. Further more, GST-endostatin inhibited in vivo angiogenesis in chicken chorioallantoic membrane and suppressed tumor growth in mice bearing Lewis lung carcinoma cells. After functional characterization of GST-Z endostatin, we decided to use GST-endostatin and EA.hy926 cells as a model system to study the inhibitory mechanism of endostatin in endothelial cells. By using fura-2 fluorescence probe, GST-endostatin was shown to elevate the cytosolic calcium in dose-dependent manner from extracellular source. Chelation of extracellular Ca2＋by EGTA or inhibition of calcium channel by nifedipine abolished the cytotoxic effect endostatin, suggesting the calcium rise by endostatin play an important role in its inhibitory mechanism. Besides, endostatin also stimulated activity of a large- conductance calcium-activated potassium (Bkca) channel, further supporting endostatin initiated serial changes in ion channels activities in endothelial cells. Respiratory enzyme activities and endogenous ATP synthesis in endothelial cells were significantly inhibited by GST-endostatin treatment, indicating GST-endostatin depleted the energy source for endothelial cells. In summary, present study demonstrated GST-endostatin caused dramatic changes in electrophysiologic properties and decreased endogenous ATP synthesis in endothelial cells, which may participate in its inhibitory mechanism.
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Chen, Heng-Chi James, and 陳恒琦. "Studies on the Inhibitory Mechanism of Angiogenesis Inhibitor, Endostatin." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/20681125193868341554.
Full textAbstract:
碩士國立中山大學
生物科學系研究所
88
Antiangiogenic tomor therapies have attracted intense interest for their broad-spectrum action, low toxicity, and in the case of direct endothelial targeting, an absence of drug resistance. Among the growing list of antiangiogenic agents, endostatin has attracted most attention and been under the spotlight of numerous debates. Like other angiogenesis inhibitors, endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on cancer cells. Above all, therapy of experimental cancer with endostatin in rodents leads to tumor dormancy and does not induce resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E.coli expression vector to express and generate large quantities of recombinant GST-fused endostatin (GST-endostatin). Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting various endothelial cell lines including HUVEC, EA.hy926 and BAEC with IC50 ~ 20 nM. Flow cytometry analysis indicated GST-endostatin induced apoptosis in EA.hy926 cells. GST-endostatin also inhibited the cell migration of EA.hy926 cells toward chemoattractant bFGF with IC50 ~ 0.5 nM. Further more, GST-endostatin inhibited in vivo angiogenesis in chicken chorioallantoic membrane and suppressed tumor growth in mice bearing Lewis lung carcinoma cells. After functional characterization of GST-endostatin, we decided to use GST-endostatin and EA.hy926 cells as a model system to study the inhibitory mechanism of endostatin in endothelial cells. By using fura-2 fluorescence probe, GST-endostatin was shown to elevate the cytosolic calcium in dose-dependent manner from extracellular source. Chelation of extracellular Ca2+ by EGTA or inhibition of calcium channel by nifedipine abolished the cytotoxic effect endostatin, suggesting the calcium rise by endostatin play an important role in its inhibitory mechanism. Besides, endostatin also stimulated activity of a large- conductance calcium-activated potassium (Bkca) channel, further supporting endostatin initiated serial changes in ion channels activities in endothelial cells. Respiratory enzyme activities and endogenous ATP synthesis in endothelial cells were significantly inhibited by GST-endostatin treatment, indicating GST-endostatin depleted the energy source for endothelial cells. In summary, present study demonstrated GST-endostatin caused dramatic changes in electrophysiologic properties and decreased endogenous ATP synthesis in endothelial cells, which may participate in its inhibitory mechanism.
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Hua, Tzu-Yu, and 華梓佑. "Structures of NP exonuclease-inhibitor complex reveal the unique inhibition mechanism by a covalent bond between cysteine and inhibitor." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/b95fht.
Full textAbstract:
碩士國立交通大學
生物資訊及系統生物研究所
105
The Nucleoprotein exonuclease ( NP exonuclease ) of Lassa virus is involved in viral genomic RNA encapsidation, viral RNA synthesis and host immune evasion. NP exonuclease is constituted by N-terminal and C-terminal domains, of which the main function of N-terminal domain is to capture the 5' cap of mRNA in the host cell for carrying out transcription and replication of its own viral RNA; the C-terminal domain of NP exonuclease belongs to the DEDDh exonuclease family. The C-terminal domain is used to degrade pathogen associated molecular patterns generated by infecting host cells, like RNA; and thus further make IRF-3 transcription factor unable to enter the nucleus to induce interferon synthesis of immune system. If the highly conserved amino acids in the C-terminal domain are mutated, it will cause a decline in ability for Lassa virus to escape the host immune system, in other words, the C-terminal domain plays an important role in virus infections; hence the C-terminal domain of NP exonuclease can be used as a target for anti-viral drug research. In this study, we found the inhibitor candidates for NP exonuclease by literature reviews and computational molecular docking program. Through the nuclease activity assay, we identified several inhibitors with high inhibition efficiency, such as ATA、PCMPS、PHMB、PV6R and NCI35. We also determined the crystal structures of apo-NP exonuclease protein structure and NP exonuclease-PCMPS complex in which PCMPS was covalently bound to the cysteine ( C409 ) in the C-terminal domain, indicating that the covalent bond is critical to suppress the activation of NP exonuclease. Our biochemical experiments and two crystal structures reveal a unique inhibitory mechanism of PCMPS through covalent linkage to the NP exonuclease, and this work could be applied to the development of antiviral drugs.
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Lapinska, Karolina Eva. "Anti-ovarian cancer effects of histone deacetylase inhibitors and calpain inhibitor." Thesis, 2016. https://hdl.handle.net/2144/14609.
Full textAbstract:
Ovarian cancer is the leading cause of death among gynecologic malignancies. The risk of developing ovarian cancer in a woman’s lifetime is 1 to 2 in 100. This high rate of development and death from the tumorigenesis is a result of its asymptomatic manifestation. Ovarian cancer is usually found in its advanced stage; therefore the survival rate is lower than for other types of cancers. The most common type of ovarian cancer, serous epithelial ovarian cancer, arises from the surface epithelium of the ovary and less frequent from the fallopian tubes or uterus. The treatment of surgery is limited by the fact that most ovarian cancers are detected after they have metastasized. Chemotherapy is often difficult because of the lack of sufficient target specific drugs. Typically, platinum in combination with other drugs is provided as the standard treatment. These combinations exhibit higher toxicity, are often not target specific, and frequently despite treatment, the tumor relapses. Current studies suggest epigenetics plays a significant role in carcinogenesis by the silencing of tumor suppressor genes (TSG). Histone modifications and the methylation of specific cytosine phosphate guanosine (CpG) residues in the upstream region of genes silence the TSG. Many clinical trials are in progress to develop combination therapies utilizing histone deacetylase inhibitors (HDACi), and DNA methyl transferase I (DNMTI) inhibitors, in combination with other cytotoxic agents. HDACi are known to be effective against different types of leukemia’s, such as Cutaneous T-cell Lymphoma; however, they are not as effective against solid tumors when used as a single agent. Our laboratory was one of the first to demonstrate that HDACi, in addition to its known property to increase histone acetylation, additionally decrease CpG island methylation in the upstream region of TSG. This demethylation causes re-expression of TSG. Our laboratory hypothesizes that re-expression of TSG sensitize cancer cells to other cytotoxic drugs. In an effort to develop improved therapy for ovarian cancer, we employed a combination therapy, which includes epigenetic drugs, HDACi, in combination with calpain protease inhibitor, calpeptin. Calpain is a ubiquitous protease usually activated in cardiovascular diseases and cancer cells. The present study discerns that combination of HDACi and calpeptin produce more than additive growth inhibition of diverse ovarian cancer cells. HDACi re-expressed TSG. Additionally, the observed growth inhibition of ovarian cancer cells was caused by cell-cycle arrest, induction of apoptosis, followed by autophagy. The phosphorylation of growth promoting signaling protein, Mitogen Activated Protein Kinase 1 (ERK), was inhibited. In addition, the inhibitors also partially inhibited phosphorylation of anti-apoptotic protein V-ask Murine Thyomoma Viral Oncogene Homolog 1 (Akt). Collectively, the outcome of this study suggests that epigenetic drugs (HDACi) sensitize the diverse ovarian cancer cell lines by re-expression of TSG, followed by cell death, when treated in combination with calpain inhibitor, calpeptin.