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Majeed, Habeeb. "Supplementation to Improve Anticoagulation Control with Low Dose Vitamin K as an Adjuvant to Warfarin Therapy: A Double-blind, Placebo-controlled Randomized Controlled Trial." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23237.
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Vitamin K Antagonists [VKA] are the most frequently used oral anticoagulants in clinical practice; however, many patients fail to achieve adequate anticoagulation control. We conducted a randomized, placebo controlled, double blind study of Vitamin K1 (200mcg per day, Swanson Vitamins) in a population with predominantly venous thromboembolism aimed at evaluating its effectiveness in improving anticoagulation control in unstable patients. This study also aimed to evaluate the impact that clinical variables, patient anticoagulation knowledge, and genetic polymorphisms in genes known to impact warfarin and Vitamin K metabolism [VKORC1, CYP4F2, CYP2C9] had on anticoagulation control and intervention effectiveness. A total of N=54 patients were enrolled in the study over 15 months [January 2009 to June 2010]. Change score analysis and multivariate linear regression modelling of anticoagulation control measures were performed. No statistically significant reduction was observed in the Vitamin K1 arm for percent time in therapeutic range; however, reduction was observed in standard deviation of INRs [Change Score Vitamin K = -0.259, p=0.0261; Regression Model 95% C.I Beta Vitamin K = 0.38 to -0.08] during the intervention period. Adjusting for treatment group allocation, independent predictors of increased INR standard deviation included: >5 alcoholic drinks per week [95% C.I Beta = 0.04 to 0.41], self-reported dosing errors [95% C.I Beta = 0.13 to 0.47], and missed INR appointments [95% C.I Beta = 0.002 to 0.05]
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Bylund, Johan. "Cytochrome P450 enzymes in oxygenation of prostaglandin endoperoxides and arachidonic acid : Cloning, expression and catalytic properties of CYP4F8 and CYP4F21." Doctoral thesis, Uppsala University, Department of Pharmaceutical Biosciences, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-1066.
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<p>Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidoniacids.</p><p>The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemic analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy fatty acid metabolites in adult human liver microsomes.</p><p>19<i>R</i>-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian <i>CYP4</i> family genes, revealed expression of two nov P450 genes in human and ovine seminal vesicles. The full coding regions of the genes were cloned and the enzymes were expressed in a yeast system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced protein sequenceshowed that they had 74 % amino acid identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH<sub>1</sub> and PGH<sub>2</sub>) and three stable PGH<sub>2</sub> analogues int19-hydroxy metabolites. Oxygenation of these substrates was mirrored when incubated with microsomes isolated from human seminal vesicles. These results suggest that CYP4F8 is present in human seminal vesicles and that 19<i>R</i>-hydroxy-PGE is formed by CYP4F8-catalyze 19<i>R</i>-hydroxylation of PGH<sub>1</sub> and PGH<sub>2</sub>, followed by PGE synthase-catalyzed isomerization. Studies of catalytic properties of recombinant CYP4F21 suggest that 20-hydroxy-PGE may be formed by similar mechanisms in ovine seminal vesicles. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides.</p>
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Rieger, Michael A. "CYP4Z1 und CYP4Z2P: Identifizierung neuer Mitglieder der humanen Cytochrom P450 Familie mit präferentieller Expression in Brustdrüsengewebe und Mammakarzinom." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2004. http://nbn-resolving.de/urn:nbn:de:swb:14-1093525824984-91466.
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Bei der adjuvanten Immuntherapie soll das Immunsystem von Tumorpatienten gezielt gegen Mikrometastasen aktiviert werden, die nach der operativen Entfernung des Primärtumors im Körper verbleiben. Tumor-assoziierte Antigene (TAA) spielen dabei die zentrale Rolle. Um der Heterogenität eines Tumors in der Expression einzelner TAAs Rechnung zu tragen, werden in modernen Vakzinierungsstrategien Pools von verschiedenen TAAs eingesetzt. Für das Mammakarzinom sind aber bislang nur wenige TAAs bekannt. Auf der Suche nach unbekannten Genen mit Mamma- bzw. Mammakarzinom-restringierter Expression in der Transkriptomdatenbank GeneExpress® wurde ein EST gefunden, das nur in 2 % der getesteten weibl. Normalgewebe ohne Mamma mit einer marginalen mittleren Expression von 16 FE (Fluoreszenzeinheit) detektiert wurde, aber in 63 % der Mammanormalgewebe (159 FE) und in 61 % der Mammakarzinome (339 FE) exprimiert wurde. Das korrespondierende UniGene-Cluster gab erste Hinweise auf die Zugehörigkeit dieses neuen Gens zu der Cytochrom P450 (CYP) Familie. Durch computergestützte Homologievergleiche mit verwandten Mitgliedern dieser Familie in Kombination mit RT-PCR konnte die cDNA-Sequenz dieses neuen CYP ermittelt werden: Durch Amplifikation der Gesamtlängen-cDNA wurden drei Transkripte in der Mammakarzinomzelllinie SK-BR-3 gefunden, die von zwei neuen CYP Genen stammen, CYP4Z1 und dem Pseudogen CYP4Z2P. Die cDNA von CYP4Z1 kodiert für ein 505 As großes Protein, das aufgrund von Homologien einer neuen Subfamilie innerhalb der CYP4 Familie zugeordnet werden kann. Sowohl die Sequenz als auch die vorhergesagte Sekundärstruktur zeigen alle charakteristischen Merkmale eines funktionellen Mitglieds dieser Familie. Aufgrund einer Nonsensemutation in Exon 8 kodiert die cDNA von CYP4Z2P (1436 bp) für ein verkürztes, nichtfunktionelles P450 von 340 As, das zu 96% identisch mit P450 4Z1 ist. Außerdem wurde in SK-BR-3 eine Spleißvariante von CYP4Z1 identifiziert. CYP4Z1 (50,8 kb) und CYP4Z2P (57,3 kb) liegen auf Chromosom 1p33-p34.1 und bestehen aus 12 Exons mit konservierten Exon-Intron-Grenzen. CYP4Z2P ist aus einer inversen Duplikation von CYP4Z1 hervorgegangen. Mittels Realtime RT-PCR mit cDNA von 17 Normalgeweben von gepoolten Spendern und von Mammakarzinomen konnte die auf Brustdrüsengewebe restringierte Expression beider Gene demonstriert werden: Die Expression von CYP4Z1 war in Mammakarzinomgewebe 3,6-mal höher als in Mammanormalgewebe, 60-mal höher als in Lunge und 84-mal höher als in Leber. Alle anderen getesteten weibl. Normalgewebe zeigten eine noch geringere Expression. Ein ähnlich stringentes Expressionsprofil ergab die Analyse von CYP4Z2P, allerdings mit einer deutlich niedrigeren Expressionsstärke. Das Mamma-restringierte Expressionsverhalten von CYP4Z1 wurde durch einen ?Cancer-Profiling-Array? (241 Tumor-/Normalgewebepaare von 13 Gewebetypen) bestätigt. Damit konnte gezeigt werden, dass CYP4Z1 bei 52 % der getesteten 50 Brustkrebspatientinnen im Tumor versus peritumoralem Normalgewebe überexprimiert war. Mit einem spezifischen Kaninchen-Antiserum konnte die Expression von P450 4Z1 Protein sowohl in CYP4Z1-transduzierten Zelllinien als auch in Mammagewebeproben mittels Western-Blot nachgewiesen werden. Konfokale Laser-Scanning Mikroskopie von MCF-7 Zellen, die das Fusionsprotein CYP4Z1-EGFP exprimierten, und die subzelluläre Fraktionierung der CYP4Z1-Transduktanten zeigten P450 4Z1 als integrales Membranprotein des endoplasmatischen Retikulums (ER). Für die Lokalisation und die Zurückhaltung im ER ohne Recycling aus dem Prä-Golgiapparat sind die ersten 32 As erforderlich, was Studien mit unterschiedlichen Deletionsmutanten aus der N-terminalen Sequenz von 4Z1 zeigten. Die Entdeckung eines neuen Mamma-spezifisch exprimierten P450 Enzyms eröffnet neue Möglichkeiten sowohl in Hinsicht auf eine Immuntherapie von Brustkrebs als auch für die Entwicklung neuer Chemotherapeutika, die spezifisch durch P450 4Z1 am Tumorort umgesetzt werden können.
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Horley, Neill. "Molecular basis of CYP2B2 induction." Thesis, University of Nottingham, 1999. http://eprints.nottingham.ac.uk/10397/.
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Many structurally unrelated chemicals can induce members of the cytochrome P450 superfamily with phenobarbital (PB) being a typical example. PB induces CYP2B1/2, which are most highly expressed in the liver. Their mechanism of activation has not yet been elucidated, with advances hampered by the absence of a suitable cell culture system to mimic the in vivo PB-mediated induction. During this thesis a primary rat hepatocyte culture system has been developed which is highly responsive to PB at both RNA and protein levels. A sensitive and specific RNAse protection assay (RPA) has been used to demonstrate that CYP2B2 mRNA is highly inducible in vitro by PB. This response occurs in a time and dose-dependent manner. The use of RPA and Western blotting has demonstrated that this primary rat hepatocyte culture system supports the induction of CYP2B2 mRNA and protein levels by PB. Sequencing -1.4kb of the 5' flanking region of the CYP2B2 gene identified genomic regulatory elements and highlighted the location of the phenobarbital response element (PBRE). The PBRE was sub-cloned into various reporter constructs and transfection technology was used to determine its PB-mediated induction. A comparative study of the constructs generated in this thesis to that of a construct provided by Anderson's group (Trottier et al., 1995) was undertaken and no differences were found in their PB-responsiveness. The Anderson construct containing the PBRE was shown here to confer a 3.3-fold PB-mediated induction of the CYP2B2 gene by CAT reporter assays. This induction was shown to be both dose and time dependent. The induction is lower than that obtained by other workers due primarily to assay conditions which were not yet optimal. However, the effects of androstane on the constitutively active receptor (CAR) may also play a role in the small inductive response of the phenobarbital response element to PB.
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Peterson, Sabrina. "Effects of apiaceous vegetable constituents on CYP1A2 activity in humans and a yeast expression system : implications for CYP1A2-activated procarcinogens /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/6612.
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Hirashima, Takako. "Choroidal Vasculature in Bietti Crystalline Dystrophy With CYP4V2 Mutations and in Retinitis Pigmentosa With EYS Mutations." Kyoto University, 2020. http://hdl.handle.net/2433/253205.
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Dunning, Rebecca. "Insecticide detoxication in drosophila melanogaster : The role of Cyp6a2." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531811.
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Markov, Gabriel. "Evolution de la signalisation stéroïdienne chez les Métazoaires." Thesis, Lyon, École normale supérieure, 2011. http://www.theses.fr/2011ENSL0634/document.
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La signalisation stéroïdienne médiée par des récepteurs nucléaires est impliquée dans de nombreux processus ayant trait au développement des animaux. La compréhension de ces phénomènes est importante pour répondre à des questions de santé publique, d’agronomie ou de biologie de la conservation. Ceci nécessite de connaître et de mettre en relation l’évolution des récepteurs qui fixent ces stéroïdes et des voies de synthèse qui produisent les stéroïdes. Mon travail s’est articulé autour de trois grands axes. 1. La mise à jour des relations de parenté entre les récepteurs nucléaires impliqués dans la fixation des stéroïdes, mais aussi de ceux qui sont impliqués dans la régulation de la stéroïdogenèse, pour comprendre quand et dans quel contexte cette machinerie est apparue. 2. La démonstration que les enzymes impliquées dans la stéroïdogenèse étaient apparues indépendamment par recrutement d’enzymes à spécificité de substrat plus large impliquées dans la détoxification des xénobiotiques. 3. L'élucidation des relations de parenté entre des voies métaboliques, montrant que les voies de la stéroïdogenèse avaient évolué comme des voies de dégradation du cholestérol. Ces résultats aboutissent à un modèle dans lequel la signalisation hormonale des animaux à symétrie bilatérale serait l’héritière de voies de détoxification de molécules stéroïdiennes contenues dans leur alimentation. Ce modèle expliquerait le couplage entre l’accumulation de nutriments et la maturation sexuelle, ainsi que les nombreux dérèglements touchant à la fois le métabolisme et la reproduction dus aux perturbateurs endocriniens ou à certaines molécules thérapeutiques<br>Nuclear receptor mediated steroid signaling is involved in many processes in metazoandevelopment, such as puberty in vertebrates, molting in insects and entry into infective stage in some parasitic nematodes. Understanding those phenomena is important regarding public health, agronomical and conservation biology issues. This necessitates to know and to explore the interactions between the evolution of steroid-binding receptors and steroid-synthesizing pathways. My work was articulated around three major parts. First, using the historical expertise of the laboratory, I updated the relationships between nuclear receptors that are involved in steroid binding, but also from all those that are involved in steroidogenesis regulation, in order to elucidate when and in which context this machinery has arisen. Second, using a classical comparative genomic approach, I showed that the steroidogenetic enzymes have appeared independently by duplication from xenobiotic-metabolizing enzyme with a wider range of substrate specificity.Third, I explored the relationships between metabolic pathways using tools from comparative anatomy. This has confirmed and completed the previous results, showing that steroidogenetic pathways have evolved with the pattern of cholesterol degradation pathways.The synthesis of all these results has led to an evolutionary model where hormonal signaling in bilaterian animals has been inherited from the detoxification of dietary sterols. This model may explain the coupling between nutrient accumulation and sexual maturation, and also the link between metabolic disorders and endocrine disruption due to environmental chemicals or drugs
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Alavi, Hajar Karimi. "Development of mechanistic mathematical models for gene-mediated drug-drug interactions." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/development-of-mechanistic-mathematical-models-for-genemediated-drugdrug-interactions(b38da88a-bb2a-4667-9809-21a09c8feeeb).html.
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The glucocorticoid receptor (GR) is a member of the nuclear hormone receptors family and has been shown to exert significant effects on the induction of cytochrome P450 (CYP) enzymes responsible for the metabolism of many xenobiotics. CYP3A4/5 and CYP2C9 are important CYP enzymes which metabolise more that 60% of drugs. Induction or inhibition of the enzymatic activity and the levels of these enzymes can have significant effects on drug metabolism. Understanding the role of GR and other nuclear receptors, pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the mechanisms effecting CYP3A4/5 and CYP2C9 levels and activity can aid in the development of in vitro and in vivo models which have become a target for scientists in the clinic and the industry. The commonly prescribed synthetic glucocorticoid (GC) drug, dexamethasone (Dex), can induce GR, PXR and CAR and was used in this study to analyse its effects on the CYP enzymes studied. The hypothesis of this project was that changes in CYP3A4/5 and CYP2C9 gene expression affect drug metabolism and changes in gene expression of these CYP enzymes was under GR, PXR and CAR control, thus affecting the concentration and therapeutic activity of drugs metabolized by these enzymes during chronic use of GCs in conditions such as rheumatoid arthritis and asthma. This study aimed to measure mRNA, protein, ROS and enzymatic activity levels in human HepG2 hepatocytes treated with Dex for 120 h and analyze the results for various time points to produce a mathematical model. Our study has shown that changes in mRNA, protein and enzymatic activity levels of CYP3A4/5 and CYP2C9 in HepG2 cells were induced by Dex at sub-micromolar (0.1 µM) and supra-micromolar (1.5 mM) concentrations. The induction of CYP3A4/5 and CYP2C9 enzymes during 120 h treatment with Dex may be affected by the NRs studied; GR, phosphorylated GR, PXR and CAR protein levels were also shown to be induced by Dex. The efflux transporter, P-gp’s protein levels were also induced by 0.1 µM Dex, highlighting the importance of considering bioavailability of other drugs co-administered with Dex. The results of some of these laboratory experiments have been used to produce mechanistic mathematical models by MATLAB software with reference to previous studies in rats concentrating on the effects of steroids on GR. The models developed were not effective at the lower Dex concentration of 0.1 µM but were better modelled at the higher Dex concentration of 1.5 mM. The basic mechanistic models developed using HepG2 cells in this study can be utilised to design and conduct drug-drug interaction (DDI) analyses of the induction of CYP3A4/5 and CYP2C9 in other human liver cells and starting pre-clinical studies in animals to aid in drug development.
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Norman, Albin. "Co-localization of CYP4F22 and CERS3 in HeLa and HEKn cells could point towards metabolic pathway interactions." Thesis, Uppsala universitet, Institutionen för medicinska vetenskaper, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-300422.
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The skin is the largest organ in the body. Its function is to protect the body from potential harm and to maintain homeostasis. The epidermis is the outermost layer of the skin. Stratum corneum is the outermost layer of the epidermis, composed of corneocytes and surrounding lipids. The lipids are produced by different enzymes that all play a role in the formation and function of the skin permeability barrier. Mutations in genes coding for these enzymes can lead to barrier dysfunction and could cause autosomal recessive congenital ichthyosis (ARCI). Nine genes have been identified as ARCI-causative and two of them are CYP4F22 and CERS3. The purpose of this project was to study co-localization of CYP4F22 with CERS3 and also mutated CYP4F22 enzymes, by transfecting plasmids into HeLa and HaCaT cells and performing PLA on HEKn cells. Co-localization could indicate potential interactions and by studying these more in the future, novel treatment strategies can be developed for ARCI patients. Transfection attempts showed a low transfection grade of wild type genes in both HeLa and HaCaT cells. Tendencies towards co-localization was seen in both cell types and some HeLa cells showed strong correlation after image analysis. Transfection of mutated genes failed, unfortunately. PLA showed co-localization in normal keratinocytes. The obtained results indicated a co-localization, but results need to be confirmed by immunoprecipitation and immunoblotting in the future.
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Price, Claire Louise. "Candida CYP52 : alkane and fatty acid metabolism." Thesis, Swansea University, 2012. https://cronfa.swan.ac.uk/Record/cronfa42696.
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Cytochromes P450 are a superfamily of haem-thiolate proteins found in all kingdoms of life. To date 11294 enzymes have been identified and have been shown to be involved in the metabolism of a wide variety of substrates, including hydrocarbons and xenobiotics. In yeast and fungi the hydroxylation of alkanes is associated with a family of cytochromes P450 enzymes known as CYP52s. These enzymes are involved in the terminal hydroxylation of long-chain alkanes resulting in the production of alcohols, which can be further converted to form fatty acids and diacids. In vivo such hydrocarbons can be subjected to beta-oxidation for use in growth. Alternatively, the products formed by CYP52 catalysed hydroxylation in vitro can be used in biotechnological applications. They can be used as platform chemicals in the production of a number of industrial products, including plastics, fragrances and antibiotics. The p-oxidation of fatty acids has been less well documented for Candida albicans than for other Candida species, therefore it was the aim of this study to investigate a) did cytochromes P450 exist in C. albicans that could possibly fulfil this function and b) to definitively assign function to a single cytochrome P450. Using a bioinformatic approach, five putative CYP52s were identified in C. albicans. Of these CYP52s, Alk1 was shown to have the greatest homology to the archetypal alkane-assimilating CYP52, CYP52A3 from C. maltosa. ALK1 heterologous gene expression in the brewer's yeast Saccharomyces cerevisiae allowed growth on hexadecane (C16:0) as the sole carbon source. This showed for the first time that Alk1 is involved in the hydroxylation of long-chain alkanes as normally S. cerevisiae is unable to utilise alkanes for growth. This study has also shown that Alk1 is able to interact with sterol substrates suggesting a possible role for CYP52s in sterol metabolism, which was previously unknown.
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Wechsung, Vera. "Eignung von CYP1A2-Genvarianten als Prädiktoren des Serumspiegels von Olanzapin." kostenfrei, 2009. http://www.opus-bayern.de/uni-regensburg/volltexte/2009/1396/.
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Mkabayi, Lithalethu. "Molecular cloning and expression of equine CYP1A2 in Escherichia coli." Thesis, Rhodes University, 2017. http://hdl.handle.net/10962/4830.
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Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
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Becker, Markus. "Arterielle Hypertonie und Polymorphismus von Cytochrom P450 Epoxygenase CYP2J2 - eine Assoziation? /." Bochum, 2007. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000278424.
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Baer, Brian R. "Autocatalytic mechanism and functional consequences of covalent heme attachment in CYP4B1 /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8176.
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Graham, Richard Alan LeCluyse Edward L. "Biochemical and molecular characterization of beagle dog CYP4A." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,290.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.<br>Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Pharmacy." Discipline: Pharmacy; Department/School: Pharmacy.
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Hood, Steven Richard. "Isolation and characterisation of a human CYP4A gene." Thesis, University of Surrey, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359859.
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Nordmark, Anna. "Functional features of human cytochrome P450 1A2 with special focus on caffeine and melatonin metabolism /." Stockholm : [Karolinska institutets bibl.], 2002. http://diss.kib.ki.se/2002/91-7349-364-3/.
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Stoltz, Catherine. "Caractérisation d'une unité de réponse au phénobarbital du gène CYP2B2 du rat." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0019/NQ47588.pdf.
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Stark, Katarina. "Catalytic Properties and Tissue Distribution of Cytochrome P450 4F8 and 4F12 : Expression of CYP4F8 in Eye Tissues and Psoriatic Lesions." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5731.
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Tostões, Rui Manuel Lucas Gameiro Domingues. "Process engineering of liver cells for drug testing applications." Doctoral thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/7612.
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Dissertação para obtenção do Grau de Doutor em
Bioengenharia<br>The primary culture of human hepatocytes is a requirement in drug development tests. This
application is currently hampered by two problems: the limited proliferation of the hepatocytes and the rapid loss of liver-specific phenotype of these cells, when cultured in vitro. This thesis aimed at minimizing this latter issue by cultivating hepatocytes, as spheroids, in fully controlled bioreactors.
The state of the art of the primary cultures of hepatocytes is reviewed in Chapter 1, after a brief introduction to the liver physiology the drug development process.
The improvement of the bioreactor cultures of hepatocyte spheroids was initially done using freshly isolated rat hepatocytes; the effects of alginate microencapsulation, perfusion culture and their synergy on the maintenance of the hepatocyte spheroids liver-specific phenotype were assessed in Chapters 2 and 3; it was concluded that the perfusion culture and alginateencapsulation
had a positive synergic effect on such hepatic phenotype.
The perfusion bioreactor developed in Chapter 3 was used in Chapter 4 for the extended culture of freshly isolated human hepatocytes, as spheroids, from three different donors. These cultures responded to repeated dose drug treatments as expected from mature and differentiated hepatocytes, in up to 4 weeks culture time.
In Chapter 5, human embryonic stem cell-derived hepatic progenitors were cultured as spheroids and further differentiated into hepatocyte-like cells; the differential expression of hepatic genes between this spheroid population and a monolayer differentiated hepatocyte-like
cell population showed a more efficient differentiation under spheroid culture.
The bioengineering improvements of this thesis, as well as the future work, were discussed in Chapter 6.
This thesis has led to the establishment and validation of primary cultures of hepatocyte
spheroids, in perfusion bioreactors, which can be used for long-term, repeated dose tests in drug development.
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Pérez, Jacques. "Test à la caféine et évaluation de l'activité du CYP1A2 : cas de l'oméprazole." Paris 5, 1995. http://www.theses.fr/1995PA05P117.
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CURRAN, CHRISTINE PERDAN. "THE ROLE OF ARYL HYDROCARBON RECEPTOR AND CYP1A2 IN PCB-INDUCED DEVELOPMENTAL NEUROTOXICITY." University of Cincinnati / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1196254087.
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Heng, Yee M. "Genomic cloning and identification of a novel murine Cyp4a gene." Thesis, University of Nottingham, 1997. http://eprints.nottingham.ac.uk/10399/.
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The mouse cytochrome P450 4a family of genes is poorly characterised. This thesis describes a detailed study of these genes. Using primers derived from exon 1 of the mouse cyp3a10 cDNA sequence, a 1.4kb genomic fragment was cloned which contained the promoter region of the gene. Additionally, a lambda genomic library was screened with probes derived from the partial cDNA clones of the mouse cyp4a10 and cyp4a12. Originally, two classes of phage lambda clones were isolated. One clone, lambda 16, was partially sequenced and was found to contain the 3' end of the cyp4a12 gene. The second clone, lambda 4, was subcloned and found to be distinct from previously known murine cyp4a genes. This novel gene has been designated cyp4a14. To obtain the full cyp4a14 gene, the genomic library was rescreened. Lambda clone 12 was subsequently isolated from the library, which was found to contain exons 1 to 5 of Cyp4a14. In order to study the promoter region of cyp4a14, a pcr-based approach was undertaken to clone 4.4 kb upstream of exon 1 of the gene and a 1.2kb fragment has been sequenced. Cyp4a14 RNA was also found to be highly induced by a peroxisome proliferator, methylclofenapate. Primer extension analyses were performed to determine the start of transcription of Cyp4a14, which was mapped to a single T nucleotide 26bp upstream of the putative start site of protein translation. The promoter region of Cyp4a14 is highly similar to the corresponding regions of the rat CYP4A2 genes. however, similarity is dramatically reduced about 350bp upstream of the start of transcription, which coincides with the presence of two 358bp repeats in teh CYP4A2 gene. The promoter also contains a highly conserved 19bp element which is also found in the promoter regions of the CYP4A1 and CYP4A2 genes. Cyp4a14 is a novel member of the murine Cyp4a subfamily. It contains 12 exons and is highly similar to the rat CYP4A2/3 genes. However, there is high conservation in exon 3 between Cyp4a14 and cyp4a3, which makes it marginally more related to this gene. In addition, cyp4a14 shows very high similarity in exons 4,8, 11 and 12 with other known CYP4A genes. Correspondingly, the peptide sequences encoded by these exons are highly conserved. Exon 8 encodes a 16 amino acid motif, LRAEVDTFMGEGHDTT, which is highly conserved in the CYP4 family. Exons 22 and 12 encode the well known RNCIG motif, containing the haem-binding domain, which is the proposed signature for a P450 protein. Exon 4 in the CYP4A genes encodes for a highly conserved, but previously unreported motif, HRRMLLTPGFHYDIL. Primary sequence alignments indicate that these motifs map very close to or within predicted substrate recognition sites and thus could have an important role in the enzyme activities of the CYP4A enzyme. The identification of Cyp4a14 also enables the analysis of the evolution of the murine Cyp4a subfamily. The mouse has 4 genes in the Cyp4a subfamily; however, the closely related rat has four CYP4A members. As the rat CYP4A2, which is very similar to CYP4AA3, was not to have a homologue in the mouse, it must have arisen after the mouse lineage had diverged from the rat in evolutionary history. As all three mouse Cyp4a genes are significantly more similar to their equivalents in the rat than to other murine Cyp4a genes, the gene duplication events giving rise to these three genes must have occurred before the mouse had diverged from the rat. Phylogenetic analyses of the CYP4A, CYP4B and CYp4F subfamilies demonstrate that CYP4A genes are more similar to the CYP4B family than to the CYP4F subfamily. This suggests that the gene duplication event giving rise to the CYP4A and CYP4B genes must be a more recent event than that giving rise to the CYP4F subfamily. In addition, the CYP4A subfamily of genes is more divergent than the CYP4B subfamily across species.
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Brito, Rodrigo Bernini de. "Farmacogenética em psiquiatria: influência dos polimorfismos CYP1A2*1F e CYP2C19*17 na refratariedade ao tratamento à clozapina e ao escitalopram." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/5265.
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Previous issue date: 2015-08-26<br>The aim of pharmacogenetics is to understand the hereditary basis of
therapeutic response and side effects of pharmacological agents for each individual.
Antipsychotics and antidepressants are effective drugs for schizophrenia and major
depressive disorder (MDD) treatment, respectively. Although a number of patients
respond satisfactorily to antipsychotics and antidepressants, 20-40% of them present
inadequate response, and the treatment with ineffective medication may take weeks
of unremitted illness, potential adverse drug reactions and nonadherence to
treatment. This study aims to identify polymorphisms in genes that potentially
influence the treatment response to clozapine in schizophrenic patients and the
treatment with escitalopram in MDD patients. This approach involved the study of
CYP1A2 and CYP2C19 genes related to the metabolism of these drugs and which
may to affect the efficacy of treatment. It was studied 54 schizophrenic patients
taking clozapine and 31 patients with MDD treated with escitalopram, both for long
term. The investigated polymorphisms, CYP1A2*1F in schizophrenic patients and
CYP2C19*2 and CYP2C19*17 in depressive patients, were analyzed by polymerase
chain reaction (PCR), followed by sequencing (CYP1A2*1F) or by restriction
fragment length polymorphism (RFLP) (CYP2C19*2 and *17) techniques. The results
pointed for the association between CYP1A2*1F polymorphism and super-refractory
clozapine treatment and for the association between CYP2C19*17 polymorphism
and the decreased response to escitalopram treatment. No association was
observed between CYP2C19*2 and the response to escitalopram treatment. These
findings suggest that these genetic variants have an important influence on the
treatment effectiveness of antipsychotics and antidepressants in psychiatric
disorders, as schizophrenia and MDD. The pharmacogenetics may be useful to the
psychiatrists helping in the choice of drugs and doses more efficient for each patient,
reducing suffering and costs and contributing to improve the quality of life for patients
and families.<br>A farmacogenética busca compreender a base hereditária da variabilidade da
resposta e dos efeitos adversos dos agentes farmacológicos entre os indivíduos. Os
medicamentos antipsicóticos e antidepressivos são utilizados em tratamentos
bastante efetivos para a esquizofrenia e transtorno depressivo maior (TDM),
respectivamente. Embora boa parte dos pacientes responda às terapias com
antipsicóticos e antidepressivos, 20-40% mostram resposta inadequada, e o custo
de cada tentativa de medicação não-efetiva para os pacientes pode levar a semanas
de permanência da doença, ocorrência de efeitos adversos potenciais e nãoaderência
ao tratamento. Este estudo teve como objetivo identificar polimorfismos
genéticos que podem potencialmente influenciar a resposta ao tratamento à
clozapina em pacientes esquizofrênicos e ao escitalopram em pacientes com TDM.
Essa abordagem envolveu o estudo de genes das enzimas metabolizadoras de
fármacos CYP1A2 e CYP2C19, potencialmente envolvidas no metabolismo desses
fármacos e que podem afetar a eficácia do tratamento. Foram estudados 54
pacientes esquizofrênicos em uso de clozapina e 31 pacientes com TDM em
tratamento com escitalopram, ambos por longo prazo. Os polimorfismos
investigados, CYP1A2*1F em pacientes esquizofrênicos e CYP2C19*2 e
CYP2C19*17 em pacientes depressivos, foram estudados por métodos baseados na
reação em cadeia da polimerase (PCR) seguido por sequenciamento (CYP1A2*1F)
ou pela técnica de polimorfismo no comprimento de fragmentos de restrição (RFLP)
(CYP2C19*2 e *17). Os resultados encontrados apontam a associação do
polimorfismo CYP1A2*1F com a super-refratariedade ao tratamento à clozapina e a
associação do polimorfismo CYP2C19*17 com resposta diminuída ao tratamento
com escitalopram. Não foi encontrada associação entre o polimorfismo CYP2C19*2
e a resposta ao tratamento ao escitalopram. Os resultados obtidos sugerem que as
variantes genéticas CYP1A2*1F e CYP2C19*17 podem desempenhar um papel
importante na efetividade do tratamento com antipsicóticos e antidepressivos em
transtornos psiquiátricos incapacitantes como a esquizofrenia e o TDM. A
farmacogenética pode auxiliar na psiquiatria como ferramenta para a escolha de
medicamentos e doses mais adequadas para cada paciente, diminuindo o
sofrimento, reduzindo custos e trazendo qualidade de vida a pacientes e familiares.
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Bendriss, El Khalil. "Expression et variation des activites n-acetyltransferase et cyp1a2 mesurees par la cafeine chez l'homme." Besançon, 1994. http://www.theses.fr/1994BESA3702.
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Boer, Jason. "Mechanistic investigation of cytochrome P450 1A2 catalyzed metabolism of 8-alkylxanthines /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8161.
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Labbé, Line. "Métabolisme de la mexilétine chez l'humain, rôle du CYP1A2 et interaction avec la propafénone." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0016/NQ55817.pdf.
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Spallek, Bastian [Verfasser]. "Antiarrhythmische Wirkung einer transgenen Überexpression des humanen CYP2J2 in Mausmodellen der Herzhypertrophie / Bastian Spallek." Berlin : Freie Universität Berlin, 2012. http://d-nb.info/1028497466/34.
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Russo, Anelise. "Expressão de genes da família CYP450 e outras oxigenases em câncer de cavidade oral." Faculdade de Medicina de São José do Rio Preto, 2015. http://hdl.handle.net/tede/258.
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Previous issue date: 2015-11-23<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES<br>Introduction: Susceptibility to enviromental agents as well as its adverse effects health depend on the personal genetic profile. Some individuals can present increased risk to develop cancer due to differences in biometabolism. Changes in the biotransformation mechanism of endogenous and exogenous compounds by oxidation reactions may be related to the tumorigenesis process. Differential expression of the genes involved in this xenobiotic metabolizing, such as cytochrome P450 (CYP) family members and others oxygenases, can change the activation process of toxic agents and lead to the oral cavity tumor development. Therefore, the individual differences of gene expression of this pathway associated to smoking and drinking can present significant risk factor for neoplasia. Objective: To identify the pattern of the gene expression involved in the biotransformation mechanism of endogenous and xenobiotic compounds in oral cavity tumors, aiming at identifing susceptibility biomarkers to this cancer type. Methods: Eight tissue samples of patients with oral cavity squamous cell carcinoma and eight adjacent non-tumor tissue samples were used. Expression of 92 genes CYP450 family and other oxygenases was quantifited by duplicate reactions of real time qPCR using “TaqMan® Array Human CYP450 and other Oxygenases 96-well fast plate” (Applied Biosystems). For statistical analysis was performed D'Agostino & Pearson omnibus normality test, followed by One-sample T test (data that showed normal distribution), and Wilcoxon signed rank test (data that did not present normal distribution) using GraphPad Prism v.5 program. Correction for multiple testing of Benjamini-Hochberg False Discovery Rate was applied to correct false positives. Bioinformatics tool was used to understand the biological system functions and to associate the differential expressed genes with specific metabolic pathways. Results: Of the 96 investigated genes involved in biotransformation mechanism (excepting the four reference genes), 12 genes showed differential expression in carcinoma tissue of oral cavity squamous cell type compared to non-tumor tissue (p<0.05). Only CYP27B1 gene presented increased expression in the oral cavity tumors, whereas CYP27A1, CYP2E1, CYP2R1, CYP2J2, CYP2U1, CYP4F12, CYP4X1, PTGIS, ALOX12, CYP4B1 and MAOB genes showed reduced expression. After correction by multiple tests, only PTGIS gene presented differential expression, with reduced expression. After data survey bioinformatics, five proteins were observed involved in the metabolism of arachidonic acid associated with important inflammatory processes in carcinogenesis (CYP2E1, CYP2J2, CYP2U1, ALOX12 and PTGIS). Conclusion: Genes involved in the carcinogens oxidation reactions showed differential expression in tumors of oral cavity. The enzymes encoded by these genes play an important role in the arachidonic acid metabolism, only pathway related to PTGIS enzyme, significant after analysis of statistical correction. The arachidonic acid and/or metabolites derived this pathway may modulate others metabolisms in which these enzymes are involved and can influence the regulation of important physiological mechanisms in tumorigenesis process.<br>Introdução: A suscetibilidade aos agentes ambientais e seus efeitos adversos à saúde dependem do perfil genético individual. Alguns indivíduos podem apresentar risco aumentado de desenvolver o câncer devido às diferenças no biometabolismo. Alterações no mecanismo de biotransformação de compostos endógenos e exógenos por reações de oxidação podem estar relacionadas com o processo de tumorigênese. Expressão diferencial de genes envolvidos nesse metabolismo de xenobióticos, tais como, os membros da família do citocromo P450 (CYP) e outras oxigenases, podem alterar o processo de ativação de agentes tóxicos e levar ao desenvolvimento do tumor de cavidade oral. Desse modo, as diferenças individuais de expressão gênica desta via associadas ao tabagismo e etilismo podem apresentar-se como significante fator de risco para neoplasias. Objetivo: Identificar o padrão de expressão de genes envolvidos no mecanismo de biotransformação de compostos endógenos e xenobióticos em tumores de cavidade oral, visando identificar biomarcadores de suscetibilidade para este tipo de câncer. Casuística e Métodos: Foram analisadas oito amostras de tecido de pacientes com diagnóstico patológico de carcinoma espinocelular de cavidade oral e oito amostras de tecidos não-tumorais adjacentes. A expressão de 92 genes da família CYP450 e outras oxigenases foi quantificada por reações em duplicata de PCRq em tempo real por meio do ensaio TaqMan® Array Human CYP450 and other Oxygenases 96-well fast plate (Applied Biosystems). Para análise estatística, foi realizado teste de normalidade de D'Agostino & Pearson omnibus normality test, seguido dos testes One-sample T test (dados com distribuição normal) e Wilcoxon signed rank test (dados que não apresentaram distribuição normal) no programa GraphPad Prism v.5. A correção para múltiplos testes de Benjamini-Hochberg False Discovery Rate foi aplicada para corrigir falsos positivos. Foram utilizadas ferramentas de bioinformática para compreender as funções do sistema biológico e associar os genes diferencialmente expressos com vias metabólicas específicas. Resultados: Dentre os 96 genes investigados envolvidos no mecanismo de biotransformação (excetuando-se os quatro genes de referência), 12 genes apresentaram expressão diferencial em tecido de carcinoma do tipo escamoso de cavidade oral, comparado ao tecido não-tumoral (P<0,05). Somente o gene CYP27B1 apresentou expressão aumentada nos tumores, enquanto os genes CYP27A1, CYP2E1, CYP2R1, CYP2J2, CYP2U1, CYP4F12, CYP4X1, PTGIS, ALOX12, CYP4B1 e MAOB apresentaram expressão reduzida. Após a correção para múltiplos testes, apenas o gene PTGIS apresentou expressão diferencial nos tumores. Após o levantamento de bioinformática, foram observadas cinco proteínas envolvidas no metabolismo do ácido araquidônico, associado com processos inflamatórios importantes na carcinogênese (CYP2E1, CYP2J2, CYP2U1, ALOX12 e PTGIS). Conclusão: Genes que participam de reações de oxidação de carcinógenos apresentam expressão diferencial em tumores de cavidade oral. Enzimas codificadas por esses genes possuem papel importante no metabolismo do ácido araquidônico, única via relacionada à enzima PTGIS, significante após análise de correção estatística. O ácido araquidônico e/ou os metabólitos provenientes desta via podem modular outros metabolismos, nos quais essas enzimas atuam e podem influenciar a regulação de mecanismos fisiológicos importantes no processo de tumorigênese.
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Gómez, Martín María del Carmen. "Influència d´alguns anestèsics i analgèsics en l´activitat citocrom P45O hepàtica (CYP450) de rata." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/104268.
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Els xenobiòtics són compostos exògens als éssers vius, i encara que no formen part de la seva bioquímica normal, s´incorporen a les seves vies metabòliques. Els xenobiòtics entren a l´organisme per diferents vies: oral (p.o.), intravenosa (i.v.) subcutània (s.c.), intramuscular (i.m.), respiratòria, etc. Els xenobiòtics es poden classificar segons el seu origen i segons els seus efectes. Els organismes, per la seva part, han desenvolupat sistemes de detoxificaxió per eliminar-los. En aquest treball, s´estudien xenobiòtics que tenen o estan desenvolupats per tenir una activitat terapèutica (fàrmacs). Aquests compostos són normalment de naturalesa lipofílica, de forma majoritària amb capacitat de difondre per les membranes cel.lulars, encara que alguns interaccionen amb transportadors específics. Un cop dins de la cèl.lula són normalment difícils d´eliminar. Per aquest motiu, els organismes transformen els xenobiòtics mitjançant processos metabòlics, i així faciliten la seva eliminació. En aquest procés de detoxificació intervenen un conjunt d´enzims poc específics que reconeixen una àmplia gamma de compostos. Les reaccions de metabolisme d´aquests enzims s´anomenen reaccions de biotransformació de fase I, i reaccions de conjugació o de fase II. En el present treball s´estudien les reaccions de fase I, que són processos d´oxidació, reducció i hidròlisi que es donen a temperatura fisiològica. El Citocrom P450, CYP450, és el principal complexe enzimàtic encarregat de les reaccions de fase I, i es caracteritza per la gran varietat de processos que poden catalitzar així com la quantitat de substractes diferents que poden metabolitzar.
L´estudi es porta a terme mitjançant experiments in vitro en un sistema d´incubacions en microsomes de fetge de rata. Les isoformes on s´estudien les possibles interaccions en la seva activitat són: CYP1A1/2, CYP2A1/2, CYP2B1/2, CYP2C, CYP2C11, CYP2D1, CYP2E1 i CYP3A1/2. Per assolir aquest objectiu global, es va obtenir i caracteritzar el sistema experimental (microsomes de fetge de rata), es van posar a punt sistemes analítics per avaluar les cinètiques enzimàtiques, i es van desenvolupar models cinètics pel tractament de les dades experimentals.
32
Bulsara, Daksha. "The effects of Poly IC and human interferon #alpha# on rat hepatic CYP4A1 and CYP2E1." Thesis, University of Surrey, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334347.
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Parmentier, Jean-Hugues. "Répression des cytochromes P450 par les Interleukines-1 et 6 : contrôle de l'expression du CYP4A1." Nancy 1, 1995. http://www.theses.fr/1995NAN10455.
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Yao, Caiping. "Comparison of in vitro and in vivo inhibition potencies of fluvoxamine toward CYPIA2 and CYP2C19 /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/7967.
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Chen, Chiung-Kuang. "Scanning Chimeragenesis: The Approach Used to Change Monoxygenase Cytochrome P450 BM3 into ω-Hydroxylase CYP4C7". Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/195451.
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It is believed that the specificity of cytochrome P450 is determined by a specific set of protein fragments that form the Substrate Recognition Site (SRS-1) and are responsible for a particular orientation of the bound substrate relative to the activated oxygen atom. Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, is known for its high catalytic activity. Wild type BM3 catalyzes the oxidation of medium and long chain fatty acids (C12-18), and of farnesol, but the two form different metabolites, forms 9-hydroxyfarnesol and 10,11- and 2,3-epoxyfarnesol in a ratio of 3:3:2 and 12-hydroxyfarnesol, respectively. CYP102A1 and CYP4C7 share a common substrate, farnesol. Therefore, CYP4C7 has become the target for homologous replacements in CYP102A1. CYP4C7 from Diploptera Punctata (Pacific Beetle Cockroach) only catalyzes farnesol to produce 12-hydroxyfarnesol as its primary metabolite, with no activity towards fatty acids. By using the technique of scanning chimeragenesis, in this work three generations of chimeras have created twenty chimeric proteins. By starting with CYP102A1 as the experimental model and employing sequential rounds of selective mutagenesis, the third generation mutant C(78-82,F87L,328-330) was produced, which catalyzed the 12- and 15-hydroxylation of farnesol as its major products in a 3:1 ratio with a hundred-fold increase in catalytic activity compared to the wild type CYP4C7, and a two-fold increase over CYP102A1. Based on the activity assay results for the chimeric proteins with substrates geranyl-geraniol, 10,11-epoxymethylfarnesoate (JH III), methylfarnesoate, farnesol, geraniol, 3,7-dimethyl-1-octanol, and lauric and palmitic acids, most chimeric proteins showed a change in substrate selectivity and/or regiospecificity. Scanning chimeragenesis can be used as a tool to not only study the relationship between the protein fragments that form the substrate binding site, but also to help elucidate the roles of substrate selectivity and regiospecificity among any two cytochromes P450. Furthermore, this investigation has resulted in the production of highly efficient chimeric enzymes that have previously evaded other methods of sequence modification by mutagenesis or directed evolution and chemical synthesis.
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Mascarenhas, Rita de Cássia Gonçalves. "Avaliação da atividade da CYP1A2 com excreção de cafeína na urina de pacientes com esquizofrenia com prescrição de clozapina." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2010. http://hdl.handle.net/10183/27788.
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Introdução: A esquizofrenia é uma doença mental que evolui para a cronicidade em mais de 80 % dos casos, caracterizada por distorções do pensamento, delírios bizarros, alterações na senso-percepção e respostas emocionais inadequadas, que podem levar o paciente a algum grau de deterioração. É uma das formas mais importantes de doença psiquiátrica, porque afeta pessoas jovens, com evolução em geral para incapacitação funcional e prejuízo social. A farmacoterapia tem provado ser o ponto chave na terapêutica da esquizofrenia. Embora não curativas, as drogas antipsicóticas se estabeleceram como o tratamento primário para todos os estágios da doença, possuindo efeito clínico significativo em cerca de 80 % dos casos,os demais apresentam a chamada forma refratária, que responde em 60 % dos casos a medicação denominada Clozapina.A CYP1A2 é a principal enzima hepática de metabolização da clozapina, e o conhecimento prévio de sua atividade enzimática pode possibilitar a personalização da terapia medicamentosa, permitindo ao médico escolher o fármaco e a dose que melhor se adapte ao perfil de metabolização do paciente, aumentando desta forma a eficácia do tratamento e a redução do aparecimento dos efeitos adversos descritos para a clozapina. Objetivo: Avaliar a excreção de cafeína inalterada na urina, como um indicativo da atividade da CYP1A2, através da utilização de cafeína como fármaco de prova. Metodologia: Foram estudados 20 adultos portadores do diagnóstico DSM-IV e CID-10 de Esquizofrenia, com forma refratária, estabilizados, em uso continuado de clozapina há pelo menos 12 meses. Foi administrado 200mg de cafeína com coleta de urina 6 horas após, e dosagem através de uma técnica de cromatografia gasosa. Não foi evidenciada correlação entre a dose de clozapina administrada e a cafeína detectada, a qual deveria ser negativa, o que pode ser explicado pelo tamanho da amostra. Porém, a excreção de cafeína apresentou diferença (P=0,019) entre homens e mulheres, acompanhando o descrito na literatura para a atividade da CYP1A2.
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Robinson, Jacob. "Characterisation of novel cytochrome P450 fusion systems." Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-novel-cytochrome-p450-fusion-systems(5b0847b2-8a5d-427d-9434-11a50f24311c).html.
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The biophysical and spectroscopic characterisation of two novel P450 fusion enzymes is reported. The first of these is CYP102A3, which is a fusion of P450 haem and cytochrome P450 reductase (CPR)-like domains and functions as a catalytically self-sufficient fatty acid hydroxylase in its host organism Bacillus subtilis. The elucidation of structural aspects of the isolated haem domain of CYP102A3 (HDCYP102A3) is described. This reveals a strong homology between HDCYP102A3 and the haem domain of the related, well studied enzyme CYP102A1 (known as BM3). Examination of the substrate binding and redox properties of HDCYP102A3 reveals variations in substrate selectivity and the influence of substrate binding over the haem-iron redox potential compared to BM3. Of particular note is the apparent cooperative binding profile displayed for some branched chain fatty acid substrates with CYP102A3. The second system characterised is CYP116B1 from Cupriavidus metallidurans, a P450 fusion with a reductase domain that resembles phthalate dioxygenase reductase (PDOR). The purification of the intact CYP116B1 enzyme, and also of its isolated haem domain (expressed from the relevant gene section), is optimised and biophysical characterisations are reported. The haem iron redox potential is found to be unusually positive (-85 mV) and the influence of thiocarbamate herbicide substrate binding upon this potential is found to be minimal, unlike the case in CYP102A£ with its fatty acid substrates and likely as a consequence of the relatively small degree of shift in haem-iron spin-state towards the high-spin form. From a panel of eight potential substrates for CYP116B1, six were found to stimulate NADPH oxidation, but only two of these were themselves oxidised by the enzyme, with hydroxylated products observable. The genetically dissected reductase domain of CYP116B1 was also expressed and purified, and kinetic studies of the reductase domain revealed a preference for NADPH over NADH coenzyme, and enables comparisons with kinetic features and coenzyme selectivity in other members of the ferredoxin reductase family of enzymes. Collectively, these studies advance our knowledge of the properties of two distinct types of P450-redox partner fusion enzymes, a growing class of enzymes with potential for biotechnological applications.
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Wang, Xiaodong. "Studies of CyP40 and β-tubulin in the Arnt-dependent signaling pathways". Scholarly Commons, 2006. https://scholarlycommons.pacific.edu/uop_etds/2634.
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Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner Ah receptor nuclear translocator (Arnt). The AhR/Arnt heterodimer binds to the enhancer element DRE to regulate target gene expression. It is known that the formation of the ligand-dependent AhR/Arnt/DRE complex requires protein factors in vitro. The first aim is to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR/Arnt/DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CYP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex. The second aim is to determine the role of β-tubulin in Amt-dependent signaling pathways. From the insect Sf9 cytosol, β-tubulin enriched fraction (F5) was isolated which suppresses the AhR/Arnt/DRE complex formation in a gel shift assay. Tubulin enriched from pig brain had a similar inhibition of the AhR gel shift complex, suggesting that β-tubulin in F5 is likely responsible for the action. Using the TALON resin, β-tubulin was co-precipitated with the baculovirus 6His-Arnt, showing that β-tubulin interacts with Arnt. β-tubulin was examined to decide its role in the hypoxia inducible factor-1α (HIF-1α) signaling which is also Arnt-dependent. Gel shift data using HIF-1α and Arnt showed that F5 suppressed the formation of the HIF-1α/Arnt/HRE complex. Subsequently the Sf9 β-tubulin was cloned and about 95% of its full-length sequence was identified. The amino acid sequence of Sf9 β-tubulin shares high sequence identity with human β-tubulin. Upon transient transfection of a plasmid containing a human β-tubulin cDNA into MGF7 or Hep3B cells, the HRE-driven luciferase activity was clearly suppressed. In conclusion, we have evidence supporting that β-tubulin inhibits the Arnt-dependent signaling and the mechanism may involve the interaction between Arnt and β-tubulin.
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Simpson, AnneMarie Elizabeth Claire Merryman. "The ontogeny of cytochrome P450 4A (CYP4A) gene expression in the rat." Thesis, University of Leicester, 1994. http://hdl.handle.net/2381/34231.
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Cytochrome P450s (P450s) play a major role in the metabolism of endogenous and exogenous chemicals. P450-metabolism generally converts compounds into more hydrophilic derivatives, which can then be excreted. In contrast, it can lead to the formation of reactive intermediates, which can attack cellular macromolecules leading to mutagenesis. The cytochrome P450 4A (CYP4A) subfamily encodes proteins involved in lipid metabolism. The CYP4A1, CYP4A2 and CYP4A3 genes are expressed constitutively in the adult rat, elevated expression being seen after administration of a hypolipidemic drug, clofibrate. The interest in the CYP4A subfamily is partly related to the apparent close association between CYP4A1 induction and the phenomenon of sustained peroxisome proliferation. It is also believed that the CYP4A proteins may be involved in the production of physiologically important renal metabolites. The aim of this thesis was a systematic study of the ontogeny and inducibility of CYP4A1 mRNA and protein levels in embryonic, fetal and post-natal rats. Constitutive and induced hepatic expression of the CYP4A1, CYP4A2 and CYP4A3 mRNAs was demonstrated in the 24 day, 10.5 day and day 1 neonates, in addition to the 18.5 day fetal expression. Renal expression was not detected prenatally, and was only apparent in the day 1 neonates following induction. The mRNAs were also present in induced adult brown fat; induced and control adult and 24 day neonatal small intestine; induced 18.5 day fetal placenta and, induced and control 11.5 day embryonic decidua. The demonstration of inducible CYP4A1 gene expression is potentially important, as in conjunction with peroxisome proliferation, it may result in an increased susceptibility of the tissue to carcinogenesis. The P450 inductive response may also play an important role in elevating the rate of metabolism of foreign compounds to detoxified forms, or in some cases to harmful reactive intermediates. If this were to occur during pregnancy it could potentially have important consequences for the developing embryo/fetus.
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Ning, Jia. "Allosteric effects of TPR domain-mediated protein-protein interactions." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31145.
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The tetratricopeptide repeat (TPR) motif contains 34 amino acids forming a helix-turn-helix structure. Different numbers of tandem TPR motifs assemble to form a TPR domain, thereby generating a polypeptide-binding interaction surface. The TPR domain provides a scaffold for mediating protein-protein interactions. Proteins that contain TPR domains exist in a broad range of organisms. These proteins have various functions. Cyclophilin 40 (Cyp40) and C-terminal Hsc70 interaction protein (CHIP) are two typical members of the family of TPR-containing proteins. Both proteins have the ability to bind the molecular chaperones Hsp70 and Hsp90. In most cases, TPR domains act as a scaffold to link chaperone and substrate or multi-protein complexes. Recent evidence suggests that Hsp90 binding to TPR domains can change the overall protein conformation but the allosteric mechanism triggered by ligand binding to the TPR domain remained unknown. This study focuses on using biophysical methods on the two TPR domain containing proteins Cyp40 and CHIP. In particular, this study reveals how the binding of the molecular chaperones Hsp70/90 to the TPR domains of Cyp40 and CHIP influences protein conformation and function. Here we show how conformational changes of the TPR domains affect structure and activity of Cyp40 and CHIP. By using biophysical methods, including thermal denaturation assay (TDA), differential scanning calorimetry (DSC), hydrogen deuterium exchange with mass spectrometry (HDX-MS) and small angle X-ray scattering (SAXS), together with enzymatic assays, we showed that (1) heat shock proteins allosterically affect the enzyme activity of both Cyp40 and CHIP, (2) heat shock proteins bind to the TPR domains of both Cyp40 and CHIP; (3) the binding increases the thermostability of both proteins. Further, by mutating an essential lysine in the TPR1 domain of both proteins (K30 for CHIP, and K227 for Cyp40) to alanine, the thermostability was significantly affected. The SAXS data showed in addition of the SRMEEVD peptide reduced the flexibility of CHIP. HDX-MS experiments suggest that the dynamic alteration due to binding with the Hsp90 peptide or the mutations further reduce the flexibility of the catalytic domains of both proteins. The results imply that the allosteric effects on the enzymatic activity are consequences of dynamic changes of the TPR domains. Hsp70 was also found to bind less tightly to CHIP-K30A than to wild-type CHIP, and thus showed less inhibition of enzymatic activity. These results further confirmed the discovery, that the dynamics of TPR domains allosterically affect enzymatic activity.
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Mohammed, Balarabe Rabiu. "Regulatory mechanisms involved in the control of CYP6M2 gene in insecticide resistant Anopheles gambiae (Diptera: culicidae)." Thesis, Abertay University, 2014. https://rke.abertay.ac.uk/en/studentTheses/75a1f78b-2d00-49d7-a564-d67ef369eb83.
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Cytochrome P450s, including CYP6M2 gene, are involved in the detoxification of permethrin. Some of these genes are regulated by CnCC / dKeap 1 and Spineless / Tango in Drosophila melanogaster. This mechanism is yet to be identified in Anopheles gambiae. Thus, we examine whether there is differential regulation of CYP6M2 gene between permethrin resistant Tiassalé and susceptible Kisumu strains of An. gambiae. Bioinformatics analysis was used to search for cis-acting elements within CYP6M2 (896 bp) region hypothesised to contain the promoter. Isolated and cloned CYP6M2 promoter reporter constructs were transfected into Anopheles gambiae Sua 5.1* cells to measure luciferase activity as a surrogate promoter activity. The WHO adult bioassay was used to expose adult females of the permethrin resistant Tiassalé and susceptible Kisumu strains of An. gambiae to discriminating doses of 0.75% permethrin. Uncharacterised strains from Auyo (Auyo-Nigeria) selected to 4% DDT and 0.1% Bendiocarb as recommended by WHO were also studied. Total RNA was isolated from the respective selected and unselected strains of An. gambiae and cDNA synthesised. Semi and Real time quantitative PCR (qPCR) using SYBR® Green were used to determine the gene expression and regulation levels. Results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) cis-acting elements within Anopheles gambiae CYP6M2 promoters in silico. Luciferase reporter gene assays revealed no promoter activity as confirmed by using CYP9M10 promoter from Culex quinquefasciatus with a known promoter activity as control. There is higher expression of Nf2e1 than AGAP010259 and a variable expression of CYP6M2 in all the insecticide selected individuals, which may potentially be associated with insecticide resistance. This study provides useful information on our understanding of the regulatory mechanisms involved in insecticide resistance. These results have implications for the control of mosquito populations and the global spread of human, livestock and poultry diseases.
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Sommer, Karen Marie. "Identification and characterization of elements regulating the expression of the phenobarbital-inducible CYP2B1 and CYP2B2 genes /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/8477.
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Pirkl, Franziska. "Funktionelle Analyse der grossen Peptidyl-Prolyl-cis/trans-Isomerasen FKBP51, FKBP52 und Cyp40." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=96279127X.
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Luu, Tony C. "Investigation of the role of CyP40 in the aryl hydrocarbon receptor signaling pathway." Scholarly Commons, 2008. https://scholarlycommons.pacific.edu/uop_etds/2383.
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Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex containing baculovirus aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE). CyP40 was found to play a role in the AhR signaling since when the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylcholanthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidylprolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer. Coprecipitation data suggests CyP40 binds weakly to AhR, but not Arnt. We report on the progress of applying bioluminescence resonance energy transfer and chromatin immunoprecipitation techniques to further elucidate the role of CyP40 in the aryl hydrocarbon receptor signaling pathway.
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Kuo, Chien-Wen Sharon. "The genomic structure of the CYP4 gene family." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310930.
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Gillett, Lorna. "Function of cytochrome P450s in the CYP4 family." Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408051.
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Chan, C. Y. "Assessment of degradation rate constants for quantitative predictions of drug-drug interactions arising from CYP450 drug metabolising enzymes." Thesis, University of Liverpool, 2018. http://livrepository.liverpool.ac.uk/3020976/.
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The first-order degradation rate constant (kdeg) of drug metabolising enzymes (DMEs) is a known source of uncertainty in the prediction of time-dependent drug-drug interactions (DDIs) in physiologically-based pharmacokinetic (PBPK) modelling. There is a large disparity or paucity of published kdeg and related half-life (t1/2) values for DMEs. Physiologically-relevant kdeg values should ideally be derived in vivo to facilitate accurate DDI predictions. However, direct measurement of DME degradation in humans is not routinely possible and indirect measurements utilising changes in levels of specific endogenous substrates have only been described for a few DMEs. This thesis aims to develop an in vitro method of measuring DME protein degradation rates to improve the prediction accuracy of time-dependent DDIs. One in vitro approach of measuring protein degradation rates involves inhibiting de novo protein synthesis, followed by tracking residual protein or activity decline over time. Pharmacological protein synthesis inhibitor agents are commonly used for this purpose but may cause cytotoxicity. Four commonly used inhibitor agents were assessed for their capacity to inhibit protein synthesis without overt cytotoxicity. However, all selected compounds were too cytotoxic for subsequent use in kdeg studies. Small-interfering ribose nucleic acid (siRNA) can be added to in vitro systems to initiate gene-specific silencing by inhibiting messenger RNA (mRNA) translation. It was hypothesised that siRNA would inhibit de novo protein synthesis with less cytotoxicity owing to its specificity. CYP3A4 is the most widely studied cytochrome P450 (CYP) enzyme in terms of DDIs because of its well-recognised role in xenobiotic metabolism. Primary human hepatocytes were treated with CYP3A4-specific siRNA to suppress mRNA translation, followed by the tracking of enzyme activity and protein loss over time to derive kdeg. CYP3A4 kdeg was calculated at 0.019 (± 0.044) and 0.020 (± 0.0003) h-1 from protein and activity loss, respectively. These values were in good agreement with existing published values. The siRNA approach was subsequently applied to determine CYP2B6 kdeg. The CYP2B6 kdeg values derived from siRNA-treated hepatocytes were 0.081 (± 0.009) h-1 from protein loss and 0.058 (± 0.010) and 0.062 (± 0.006) h-1 from activity loss, which were assessed by different methods. The CYP2B6 kdeg values derived from untreated hepatocytes were similar to values in literature. This novel approach can now be used for other less well-characterised DMEs that are associated with time-dependent DDIs. Cellular protein abundance is a balance between synthesis and degradation. Dysregulation of the lysosomal or proteasomal protein degradation mechanisms affects steady-state protein levels and impacts on overall cellular functions. It was hypothesised that single nucleotide polymorphisms (SNPs) in the CYP3A4 protein degradation machinery could affect CYP3A4 protein abundance and downstream activity. Five SNPs were investigated for associations with plasma atazanair (ATV) concentrations, which was a surrogate measure for CYP3A4 activity. No associations were found and this was likely due to the lack of clear understanding of the specific mechanisms that commits CYP proteins for degradation. Further work in this field will identify targets that may be exploited in the future for more accurate measurements of DME kdeg. The data presented in this thesis enhances the understanding of methods used to study protein degradation and this can be applied to multiple fields of cellular research. Importantly, work herein has generated a novel approach to measuring kdeg of proteins that can be applied to other less-well characterised enzymes for better prediction of time-dependent DDIs.
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Meyer, Anne Elisabeth [Verfasser], Helmut [Gutachter] Hanenberg, and Csaba [Gutachter] Mahotka. "Speziesübergreifende Charakterisierung des CYP4B1-Enzyms in Hinblick auf die Substratumsetzung / Anne Elisabeth Meyer ; Gutachter: Helmut Hanenberg, Csaba Mahotka." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2021. http://d-nb.info/1237495865/34.
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Jones, Paul S. "Expression and induction, by peroxisome proliferators, of the CYP4A and PPAR gene families in mouse." Thesis, University of Nottingham, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283640.
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GARCIA, Analia Nusya de Medeiros. "Polimorfismos dos genes CYP 46 e APOE e declínio cognitivo em idosos residentes no distrito de Fernando de Noronha-PE." Universidade Federal de Pernambuco, 2011. https://repositorio.ufpe.br/handle/123456789/8317.
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Previous issue date: 2011<br>O Declínio Cognitivo Leve (DCL) é um estado mental considerado a zona de transição entre o envelhecimento normal e a fase mais inicial de demência, sendo uma fase importante para a precocidade diagnóstica. Nos últimos anos, pesquisas estão sendo desenvolvidas na busca de marcadores genéticos para esta zona de pré-demência, como os polimorfismos dos genes da apolipoproteína E (APOE) representada por 3 alelos (E2, E3, E4) e do colesterol 24S-hidroxilase (CYP46) com alelos T e C. Indivíduos portadores do APOE E4 tem fator de risco quatro vezes maior de desenvolver a Demência de Alzheimer e dez vezes mais probabilidade se tiver associado os polimorfismos dos genes APOE e CYP46. O objetivo deste estudo foi investigar a possível associação entre o polimorfismo dos genes CYP46(T/C), APOE E4 e a presença de DCL na população idosa do Distrito de Fernando de Noronha, totalizando uma seleção de 52 indivíduos. A avaliação clínica foi realizada através de exame físico, funcional e mental. Foram aplicados testes neuropsiquiátricos (Mini Exame do Estado Mental, Teste de Fluência Verbal, Teste do Relógio) e a identificação do genótipo dos polimorfismos do APOE e CYP46 pelo método de PCR-RFLP. Como resultados observou-se que 87% da amostra apresentou declínio cognitivo leve. No Mini Exame do Estado Mental, Teste de Fluência Verbal e Teste do Relógio foi observado declínio cognitivo em 42,8%, 31,9% e 53,2% respectivamente. Foi observada uma frequência alélica de 10% para o alelo E4. Não foi observada associação entre APOE E4 e declínio cognitivo. Os alelos T (p = 0,628) e C (p = 0,2076) do gene Cyp46 não estão associadas ao DCL na população estudada. Não foi observada associação (p = 0,4286), quando analisado o sinergismo entre o polimorfismo dos genes Cyp46(T/C) e APOE E4 no desenvolvimento do DCL. Nesta população, os resultados sugerem que os polimorfismos dos genes Cyp46(T/C) e APOE E4 não estão associados ao DCL