Dissertations / Theses: 'In vitro biosynthesis'

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Smith, Kristina J. "In vitro biosynthesis of pectic polysaccharides." Thesis, University of Stirling, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322091.

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Gillespie, Charles Stewart. "Myelin membrane protein biosynthesis : an in vitro study." Thesis, University of Stirling, 1988. http://hdl.handle.net/1893/22868.

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The sites of biosynthesis and incorporation of the abundant CNS myelin proteins 2' , 3' -cyclic nucleotide-3'-phosphodiesterase (CNPase) and P2 protein into the growing myelin membrane were investigated. Cell-free translation systems programmed with mRNA from rat brain, rabbit spinal cord, free and bound polysomes and purified myelin demonstrated conclusively that both CNPase and P2 are synthesized on free polysomes like the myelin basic proteins (MBPs) but unlike the proteolipid protein (PLP), the major intrinsic membrane protein of CNS myelin, which is known to be synthesized at the oligodendrocyte endoplasmic reticulum on bound polysomes (Colman et al., 1982) . These observations were supported by labelling studies on rats in vivo during the period of maximal myelin deposition. Newly synthesized CNPase associated with the myelin membrane very rapidly after labelling (~2 minutes) and this is consistent with the view that there is only a brief delay between synthesis and incorporation into their target membrane for extrinsic-type plasma membrane proteins. An RNA fraction isolated from purified CNS myelin was not enriched in mRNAs coding for CNPase and P2 but a considerable enrichment of mRNAs coding for MBPs was observed. This phenomenon has important implications for the cell biology of myelination since it suggests that although MBPs, CNPase and P2 are all basic extrinsic membrane proteins, and synthesized on free polysomes, different mechanisms for their transport to the myelin membrane exist. The addition of dog pancreatic microsomes (DPM) during translation showed no membrane association for CNPase however, at least 50% of MBPs were observed to non-specifically associate with these membranes. When newly synthesized MBP and P2 were incubated post-translationally with DPM or rabbit spinal cord myelin P2 only associated with myelin whereas MBP showed an equal affinity for both types of membranes. The segregation of MBP free polysomes at the myelin membrane during synthesis ensures that the nascent MBP polypeptides associate with the correct membrane. Recent evidence has shown that the free polysome-mRNA complex is bound to the cytoskeleton during protein synthesis. After extensive characterization of the purified rat brain oligodendrocyte and myelin-associated cytoskeletons it was shown that the synthesis of MBPs and CNPase only occurs from mRNA that is associated with the cytoskeleton and not when it is part of the cytoplasmic mRNA pool. Lipid analysis of the purified rat brain myelin-associated cytoskeleton revealed the presence of tightly bound lipid with a considerable enrichment of cerebroside and sphingomyelin (the latter at the expense of phosphatidylethanolamine). These studies on the cytoskeletal involvement in myelinogenesis suggest that extrinsic CNS myelin proteins are synthesized on the cytoskeleton and that post-translational cytoskeletal transport of these proteins to the growing myelin membrane may take place.

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Smith, Susan Janette. "In vitro biosynthesis of steroids in equine testicular tissue." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328222.

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Nordman, Tomas. "In vitro studies on the biosynthesis and reduction of ubiquinone /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-475-5/.

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Lucas, Richard James. "In vitro peptidoglycan biosynthesis on silicon-supported tethered lipid bilayers." Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429714.

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Coxon, M. M. "Studies of pantothenate biosynthesis in Arabidopsis in vivo and in vitro." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598113.

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With a view to developing inhibitors, two enzymes of the Arabidopsis pantothenate pathway (KPHMT2 and PS) were expressed in E. coli, as MBP fusion proteins purified and used to perform kinetic studies to identify their characteristics. KPHMT2 showed an apparent Km, Vmax and kcat of 0.534 ± 0.089 mM, 0.802 ± 0.032 nmoles/min and 2.79 min/1 respectively for α-KIVA whilst the enzyme appeared to be inhibited by its co-factor, 5,10-methylene tetrahydrofolate, at levels above approximately 25μM. In addition to its potential as a target for herbicides and anti-microbials the pathway is also of commercial interest for the production of pantothenate for use as a supplement in animal feed and for addition to cosmetics. By constitutive expression of the genes of the E. coli pathway, Arabidopsis lines with higher levels of pantothenate than WT were obtained. Expression of E. coli panC resulted in increased levels of the vitamin in some lines, as did the expression of panD, however, expression of panB showed no increase. The β-alanine levels of the transgenic lines ere also assayed and showed hugely elevated levels in the lines expressing panD. To determine if expression of the E. coli genes or the accumulation of pantothenate effected the expression of the endogenous genes, RT-PCR was performed and showed no significant difference in the expression of endogenous panB2 or panC in lines expressing E. coli panB, panC or panD. Expression of the genes of the pathway was also studied using promoter:GUS fusions, RT-PCR, western blot analysis and enzyme assays. The results showed expression of the genes throughout the plant suggesting de novo pantothenate biosynthesis with panB1 and panB2 promoter activity showing up-regulation in flowers and siliques, tissues known to require large amounts of pantothenate.

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Jacobson, Annica. "Regulation of hyaluronan biosynthesis : Expression in vitro and importance for tumor progression." Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2004.

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Hyaluronan, a component of the extracellular matrix, is synthesized by either of three hyaluronan-synthesizing enzymes termed Has1, Has2 and Has3. The expression level of each Has gene varies between cell types of mesenchymal origin and is differentially regulated in response to external stimuli. For example, stimulation of mesothelial cells with PDGF-BB induced an up-regulation of the Has2 gene, whereas the Has1 and Has3 genes remained unaffected. The induction of Has2 gene expression correlated well with increased Has2 protein levels and accumulation of hyaluronan. Moreover, treatment of mesothelial cells with hydrocortisone suppressed hyaluronan synthesis in cell culture primarily through down-regulation of the Has2 gene. Thus, among the Has isoforms, Has2 seems to be most markedly regulated in response to external stimuli.

In an attempt to investigate the importance of hyaluronan in tumor progression, the hyaluronan synthesizing enzyme Has2 and the hyaluronan degrading enzyme Hyal1 were over-expressed in a rat colon adenocarcinoma cell line, PROb. We found that Has2 gene over-expression in colon carcinoma cells promoted cell growth in vitro and progression of transplantable tumors. In contrast, over-expression of Hyal1 lead to a considerable reduction of growth rates both in vivo and in vitro. A linear correlation between tumor growth rate and hyaluronan amount in tumor tissue was observed. In another tumor model, experimental anaplastic thyroid carcinoma, the effects of TGF-β inhibition on hyaluronan and collagen contents in tumor xenografts were investigated. We found that inhibition of TGF-β, a stimulator of hyaluronan and collagen synthesis, lead to reduced collagen deposition whereas the hyaluronan levels in stromal tissue only marginally differed. Our results indicate that a high ratio of collagen to hyaluronan may be characteristic of a pathogenic mechanism that leads to elevated interstitual tumor pressure.

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Champattanachai, Voraratt. "Effects of hexosamine biosynthesis on an in vitro model of cardiac ischemia." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/champattanachai.pdf.

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Chiang, Cheng Ching Kurt. "Natural Rubber Biosynthesis: Perspectives from Polymer Chemistry." University of Akron / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=akron1386367354.

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Cross, Stuart. "In vitro studies of the enzymes involved in fluorometabolite biosynthesis in Streptomyces cattleya /." St Andrews, 2009. http://hdl.handle.net/10023/720.

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Cross, Stuart M. "In vitro studies of the enzymes involved in fluorometabolite biosynthesis in Streptomyces cattleya." Thesis, University of St Andrews, 2009. http://hdl.handle.net/10023/720.

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Enzymatic fluorination of natural products is extremely rare. Of the 4000 halogenated natural products identified, only 13 possess a fluorine atom. The C-F bond forming enzyme from the soil bacterium, Streptomyces cattleya, remains the only native enzyme to be identified that is capable of such biochemistry. It generates 5’-fluoro-5-deoxyadenosine (5‘-FDA) from S-adenosyl-L-methionine (SAM) and F-. The “fluorinase” is the first committed step toward the biosynthesis of the two fluorometabolites, 4-fluorothreonine and fluoroacetate, via the common intermediate, fluoroacetaldehyde (FAld). The enzymatic steps responsible for the conversion of 5’-FDA to the fluorometabolites remained to be fully characterised when this project began. Previously, a purine nucleoside phosphorylase was identified that was capable of generating 5-fluorodeoxyribose-1-phosphate (5-FDRP) from 5’-FDA. 5-FDRP is subsequently isomerised to 5-fluorodeoxyribulose-1-phosphate (5-FDRulP) by an aldose-ketose isomerase enzyme. Chapter 2 describes the identification of the isomerase gene from the genomic DNA of S. cattleya and the corresponding protein product was capable of generating 5-FDRulP from 5-FDRP. The next intermediate, FAld, is generated from 5-FDRulP by a fuculose aldolase. Attempts to identify the aldolase gene from S. cattleya were unsuccessful, however a putative fuculose aldolase from Streptomyces coelicolor was isolated that could generate FAld from 5-FDRulP, which is described in Chapter 3. Following the identification and over expression of a PLP-dependant transaldolase, which generates 4-fluorothreonine (4-FT) from FAld and L-threonine in S. cattleya, Chapter 4 details the successful in vitro reconstitution of fluorometabolite biosynthesis using five over- expressed enzymes. In Chapter 5, attempts to develop a novel assay for fluorinase activity was explored. The colorimetric detection of L-methionine produced by the fluorinase in a coupled L-amino acid oxidase and horseradish peroxidase assay, leading to the oxidation of a dye substance. This was carried out with interest in developing a high-throughput assay for fluorinase mutants, generated by random mutagenesis, in order to identify those with increased activity. In the event, it proved unsuccessful.

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Chen, Haiyong. "Erxian decoction for menopause systematic review and mechanistic study in estradiol bio-synthesis in vitro /." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41290653.

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Abdel-Massih, Roula M. "In vitro biosynthesis of 1,4-#BETA#-galactan attached to a pectin-xyloglucan complex in peas." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366191.

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BARONE, FULVIO. "Role of nandrolone decanoate administration on testosterone biosynthesis: an in vitro and in vivo study." Doctoral thesis, Università di Foggia, 2017. http://hdl.handle.net/11369/363283.

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Gli steroidi androgeni anabolizzanti (SAA) sono una categoria di sostanze, analoghe degli ormoni sessuali maschili. Gli SAA vengono utilizzati molto comunemente da atleti per migliorare le performance, in modo fraudolento, e sempre più spesso anche da non professionisti a scopo puramente estetico. Tra gli SAA, il nandrolone decanoato (ND) è il più conosciuto e diffusamente utilizzato. Lo scopo di questa tesi di dottorato è quello di studiare gli effetti di un sovradosaggio di ND su due modelli sperimentali. La parte in vitro, svolta su una linea di cellule di Leydig tumorali, di Rattus norvegicus: le R2C; è stata volta a investigare gli effetti sulla produzione di testosterone, da parte di queste cellule e come il ND alterasse il pathway di questo ormone. La seconda parte è stata svolta su un modello murino (Mus musculus CD1) allenato secondo un programma di esercizio che mimasse l’allenamento di resistenza. Nella parte in vivo sono stati osservati, parallelamente alla prima parte, produzione di testosterone e pathway biosintetico, ma è stato anche svolto uno studio morfologico e funzionale per valutare i danni strutturali ai testicoli e in particolare alla barriera ematotesticolare.
Anabolic androgenic steroids (AAS) are a class of sbustances analogus to the male sexual hormones. AAS are very commonly used by professional athletes to improve performance, fraudulently, and increasingly also by non-professionals for purely aesthetic purposes. Among the AAS, nandrolone decanoate (ND) is the best known and widely used. The aim of this PhD thesis is to study the effects of supraphysiological administration of ND on two experimental models. The part in vitro, carried out on a line of tumor Leydig cells of Rattus norvegicus the R2C; It was designed to investigate the effects on testosterone production by these cells and how the ND alter the pathway of this hormone. The second part was conducted on a mouse model (Mus musculus CD1) trained according to an exercise program that mimic endurance training. In the in vivo were observed, parallel to the first part, the production of testosterone and biosynthetic pathway, but it was also carried out a morphological and functional study to assess the structural damage to the testicles and in particular the blood-testis barrier.

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Imler, Stacy Marie. "In Vitro Modulation of Meniscus Biosynthesis: a Basis for Understanding Cellular Response to Physiologically Relevant Stimuli." Diss., Available online, Georgia Institute of Technology, 2006, 2005. http://etd.gatech.edu/theses/available/etd-07152005-130841/.

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Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2006.
Dr. Marc E. Levenston, Committee Chair ; Dr. Lawrence J. Bonassar, Committee Member ; Dr. Robert E. Guldberg, Committee Member ; Dr. William J. Koros, Committee Member ; Dr. Christopher S. Lynch, Committee Member.

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Bacala, Ray. "In vitro studies of sex pheromone biosynthesis in the yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0010/MQ53083.pdf.

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Mir, Mohseni Mahsa [Verfasser]. "Analysis of the natural product biosynthesis in gliding bacteria using in vitro assays / Mahsa Mir Mohseni." Bonn : Universitäts- und Landesbibliothek Bonn, 2017. http://d-nb.info/115077780X/34.

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Cheng, Qian. "In Vitro Reconstitution of the Entire Enterocin Biosynthetic Pathway: New Insights into Type II PKS Enzymology." Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/195469.

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Type II polyketide synthases (PKSs) are responsible for the generation of structurally diverse and clinically important aromatic polyketides. The bacteriostatic agent enterocin (enc) isolated from the marine microbe "Streptomyces maritimus" is derived from a rare benzoate primer unit and contains a unique nonaromatic caged core structure resulting from a Favorskii-like carbon skeleton rearrangement. The apparent diversion between enterocin biosynthesis and all other type II PKS pathways offered the opportunity to discover novel enzymatic strategies that may be exploited to diversify the chemical structures of polyketides. A comprehensive biochemical analysis was performed in order to characterize the key steps in enterocin biosynthesis and finally to reconstitute the whole pathway in vitro using purified recombinant enzymes.A nonribosomal peptide synthetase (NRPS)-like priming mechanism was discovered for the selective activation of a benzoic acid starter unit and its subsequent attachment to the enc PKS to initiate polyketide biosynthesis. This is the first example of a type II PKS that employs an NPRS-like priming mechanism to utilize alternative non- acetate starter units. Secondly, the minimal enc PKS was reconstituted in vitro to give three novel acetate-primed metabolites that had never been identified by heterologous in vivo expression of recombinant enc PKS gene sets. The minimal enc PKS was then merged with the NRPS-like chain initiation module and the resulting multienzyme complex catalyzed the formation of benzoate-primed natural products wailupemycin F and wailupemycin G. Favorskii-like rearrangement of the nascent polyketide chain was replicated in vitro and the flavin-dependent oxygenase EncM was confirmed to be solely responsible for catalyzing this unprecedented rearrangement. Other biosynthetic steps in the late stage of the enc pathway were also replicated in vitro, including the methylation of desmethyl-5-deoxyenterocin to 5-deoxyenterocin and the hydroxylation of 5-deoxyenterocin to enterocin.Finally, the entire enc type II PKS pathway was successfully assembled in vitro using ten recombinant proteins and three commercial enzymes. Five enc-based natural products were generated from benzoic acid and malonyl-coenzyme A. This biochemical investigation on enterocin biosynthesis represents the first complete in vitro reconstitution of a type II PKS system and also provides an alternative strategy to create complex natural products by multienzyme synthesis.

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Chen, Haiyong, and 陳海勇. "Erxian decoction for menopause: systematic review and mechanistic study in estradiol bio-synthesis in vitro." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41290653.

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Conceicao, Ana Paula Santos da. "Estabelecimento de culturas in vitro de Pilocarpus pennatifolius Lemmaire e estudos iniciais sobre a biossíntese do alcalóide pilocarpina." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/46/46135/tde-07102014-115557/.

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Pilocarpus pennatifolius Lemmaire (Rutaceae) é uma espécie nativa que ocorre nas regiões Sul, Sudeste, Centro-Oeste e Nordeste do Brasil, e é popularmente conhecida por jaborandi. Essa e outras espécies de Pilocarpus encontram-se em risco de extinção devido ao extrativismo (uso como planta medicinal) e ao desmatamento. O cultivo in vitro de P. pennatifolius foi realizado como uma alternativa para a produção de pilocarpina (princípio ativo do jaborandi). A cultura de células foi obtida em meio MS semi-sólido, suplementado com sacarose, como fonte de carbono, e tendo como reguladores de crescimento BAP, IAA e NAA. O tempo de duplicação da cultura de calos foi de 22 dias. A concentração do alcalóide, determinada por técnicas cromatográficas, foi de aproximadamente 0,1 µg/ mg de biomassa. Os ensaios enzimáticos referentes à biossíntese da pilocarpina tiveram como objetivo elucidar a etapa inicial de formação do anel imidazólico, além da localização desta reação na planta. Os experimentos realizados indicaram a presença da enzima histidina amino-transferase (HAT, EC 2.6.1.38) cuja atividade catalítica foi de 46,09 nKat/ mg de proteína, apenas no extrato protéico das raízes de P. pennatifolius. A determinação da composição do óleo essencial por CG e CG/EM indicou como constituintes majoritários os hidrocarbonetos tridecano (56,8 %) e pentadecano (25,5 %). Os sesquiterpenos não oxigenados constituíram cerca de 15 % do óleo obtido.
Pilocarpus pennatifolius Lemmaire (Rutaceae) is a native Brazilian species which is commonly known as jaborandi. This species is endangered due to its exploitation as medicinal plant and the increasingly deforestation. A callus culture of P. pennatifolius was established as an alternative source for pilocarpine production (the active compound of jaborandi). The cell culture was obtained in a semi-solid media, supplemented with sucrose as carbon source and as growth regulators, BAP, IAA e NAA. The doubling time for the callus culture was determined as 22 days. The concentration of the alkaloid, obtained by chromatographic techniques was ca. 0.1 µg/ mg dry weight. The enzymatic assays related to pilocarpine biosynthesis were carried out with the aim to elucidate the imidazole ring formation beyond identify the site were this reaction takes place. The results indicated the presence of the enzyme histidine ammonia transferase (HAT, EC 2.6.1.38) only in the protein extract obtained from the roots of P. pennatifolius. The catalytic activity for this enzyme was 46.09 nKat/ mg protein. Volatile constituents from the leaves were analyzed by GC and GC/MS and the major compounds were determined as the hydrocarbons tridecane (56.8 %) and pentadecane (25.5 %). Almost 15 % of the total composition was constituted of non-oxygenated sesquiterpenes.

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Tocci, Noemi [Verfasser], and Ludger [Akademischer Betreuer] Beerhues. "Hypericum perforatum subsp. angustifolium: study of xanthone biosynthesis in planta and in in vitro systems / Noemi Tocci ; Betreuer: Ludger Beerhues." Braunschweig : Technische Universität Braunschweig, 2013. http://d-nb.info/1175822159/34.

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Дідик, О. К. "Вплив опіоїдних пептидів на ендогенний біосинтез простацикліну і тромбоксану у нирках in vitro." Thesis, Сумський державний університет, 2017. http://essuir.sumdu.edu.ua/handle/123456789/54313.

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Вступ. Точний механізм або механізми фізіологічної дії опіоїдних пептидів до кінця не вивчені. Ланкою їх дії можуть бути метаболіти арахідонової кислоти - простациклін (PGI2) і тромбоксан (TxA2). Мета роботи. Дослідити вплив опіоїдних пептидів на ендогенний біосинтез PGI2 і TxA2 у нирках щурів.

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Lafleur, Christine. "The significance of enzyme 3-ß-hydroxysterol - delta24 reductase in cholesterol biosynthesis and steroidogenesis: an «in vitro» model to study Desmosterolosis." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=67031.

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Desmosterolosis is an autosomal recessive condition in which affected individuals lack expression of the final enzyme in the cholesterol biosynthetic pathway, 3β-hydroxyΔ²4cholesterol reductase (DHCR24). The enzyme is responsible for converting desmosterol to cholesterol. It is characterized by several congenital abnormalities, as well as high circulating levels of desmosterol accompanied by hypocholesterolemia. This study was intended to examine the molecular disruptions involved in desmosterolosis and determine the potential impact on steroidogenesis using an in vitro approach. Due to the importance of cholesterol in cell-signaling and cell membrane integrity, it was found that desmosterol was a poor substitute for cholesterol in cell membranes. Cells deprived of cholesterol for 45 minutes rapidly underwent morphological changes and presumably became apoptotic. This effect was potentiated in cells with DHCR24 knock-down. Additionally, cells responded by increasing levels of cholesterol biosynthetic factors such as, SREBP1, SREBP2, SCAP, S1P, as well as the enzyme HMG Co A reductase all of which were measured using real-time PCR. The increase in these transcripts indicates that desmosterol cannot be sensed by the sterol sensing domain (SSD). Following cholesterol repletion, cells were able to restore a certain level normalcy in terms of morphology and the production of the transcripts listed above. Cholesterol is required for steroidogenesis in the adrenal cortex. We examined whether or not desmosterol could be used as a steroidogenic substrate. We measured levels of steroidogenic acute regulatory protein (StAR) as well as measured cortisol production with DHCR24 knock-down following cholesterol depletion and repletion with and without trophic stimulation. We found that in cells stimulated with vasoactive intestinal polypeptide, devoid of cholesterol and with endogenous synthesis impaired at the leve
La desmostérolose est une maladie autosomale récessive dans laquelle les individus atteints ont un déficit de l'expression de l'enzyme terminale du processus de biosynthèse du cholestérol, connue sous le nom de 3β-hydroxyΔ ² 4cholesterol réductase (DHCR24). Cette enzyme est chargée de convertir le desmostérol en cholestérol. Cette maladie est caractérisée par plusieurs anomalies congénitales, ainsi que par des taux élevés de desmostérol dans le sang accompagnés d'hypocholestérolemie.L'objectif de cette étude a été d'examiner les anomalies provoquées par la desmostérolose au niveau moléculaire ainsi que son impact sur la stéroïdogenèse en utilisant une approche in vitro. Nous avons, pour cela, utilisé la technique d'interférence par des ARN pour diminuer l'expression de l'enzyme DHCR24 reproduisant ainsi les conditions de la desmostérolose dans notre modèle cellulaire.Le cholestérol participe à de nombreux processus de signalisation cellulaire et joue un rôle important dans la stabilité des membranes constituant les cellules. Au cours de notre étude nous avons constaté que le desmostérol du fait de sa structure n'est pas capable de se substituer au cholestérol pour remplir ces fonctions. En effet, les cellules dépourvues de cholestérol pendant 45 minutes ont systématiquement subi un changement de morphologie et ont par la suite présenté les caractéristiques de cellules en apoptose. Ce phénomène était encore plus prononcé dans les cellules où l'expression de DHCR24 avait été diminuée expérimentalement. Nous avons également examiné si le desmostérol pouvait être reconnu par la protéine dite 'domaine de reconnaissance des stérols' (SSD) qui in vivo stimule la synthèse endogène de cholestérol quand ses niveaux dans la circulation sanguine sont trop bas. Pour cela, nous avons mesuré les taux d'expression des protéines suivantes : 'sterol regula

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Mehtali, Abdel-Majid. "Les genes de maintenance : etude du gene hmgcoa reductase in vitro et dans les souris transgeniques." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13162.

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Les genes de maintenance sont exprimes de maniere ubiquitaire par un mecanisme encore incompris. Pour aborder ce probleme, les auteurs ont choisi comme modele d'etude un gene de maintenance typique, le gene de l'hydroxymethylglutaryl coa reductase (hmgcr), enzyme cle de la biosynthese du cholesterol. Le gene hmgcr de souris a ete isole et sa region promotrice caracterisee; l'expression et le profil de methylation de plusieurs genes chimeriques, dans lesquels le gene marqueur cat est sous le controle de sequences promotrices hmgcr de taille decroissante, ont ete etudies in vitro (par transformation de cellules en culture) et in vivo (dans les souris transgeniques)

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Wood, Derek William 1965. "Characterization of an N-acyl-L-homoserine lactone-mediated regulatory system controlling phenazine biosynthesis in Pseudomonas aureofaciens 30-84: In vitro and in situ analysis." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/282391.

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Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which are responsible for both suppression of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici and enhanced survival of 30-84 within the wheat rhizosphere in competition with other organisms. A gene (phzR) was identified just prior to the start of this work that is required for phenazine production by 30-84. PhzR was identified as a positive regulator of the phenazine biosynthetic operon. During the course of this dissertation it was discovered that PhzR belongs to the LuxR family of N-acyl- scL-homoserine lactone-responsive transcriptional regulators and that phenazine production in P. aureofaciens 30-84 is mediated by a diffusible signal molecule. The gene responsible for production of this signal (phzI) was identified. Both phzI and phzR are required for the production of phenazines in vitro. Together these two proteins (PhzR/PhzI) comprise a N-acyl- scL-homoserine lactone (AHL) response system that controls phenazine antibiotic production in P. aureofaciens 30-84. Classic AHL-mediated regulatory systems consist of two proteins, a LuxR homolog (PhzR) which transcriptionally activates target gene expression in response to AHL produced by the second protein, the LuxI homolog (PhzI). Using HPLC coupled with high resolution mass spectroscopy, the specific AHL produced via PhzI has been identified as N-hexanoyl- scL-homoserine lactone (HHL). It has been determined that PhzR activates phenazine production in conjunction with HHL produced by PhzI via transcriptional activation of the phenazine biosynthetic gene phzB. A variety of synthetic AHLs restore transcription of phzB and phenazine production in phzI mutants suggesting that phzI mutants can be used to detect the presence of exogenous AHLs. This ability was exploited to show that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere. The work presented in this dissertation is the first to show that AHL-mediated regulation, previously only examined in vitro, can operate within the natural habitat of a bacterium.

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Smeds, Emanuel. "In Vitro Studies of the Substrate Specificities of Heparan Sulfate 2-O- and 6-O-sulfotransferases." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ-.bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4738.

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Gautriaud, Emilie. "Review of natural rubber biosynthesis and synthesis of model intermediates for the preparation of a macroinitiator for the in vitro synthesis of polyisobutylene-polyisoprene diblock copolymer." Akron, OH : University of Akron, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=akron1154963470.

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Thesis (M.S.)--University of Akron, Dept. of Polymer Science, 2006.
"December, 2006." Title from electronic thesis title page (viewed 12/31/2008) Advisor, Judit E. Puskas; Faculty Reader, Coleen Pugh; Department Chair, Mark D. Foster; Dean of the College, Frank N. Kelley; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

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Schmidt, Renate Luise. "Der Einfluss von Clozapin, N-Desmethylclozapin und Chlorpromazin auf die in-vitro-Produktion von Thromboxan." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-149801.

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Die Hypothese, dass das AP Clozapin, ebenso wie dessen Metabolit NDMC die Produktion von TxA2 beeinfluss könnten, stellten wir nach ausführlicher Literaturrecherche auf. Letztere zeigte, dass bereits beim ersten AP CPZ eine reduzierende Wirkung auf die TxA2-Produktion nachgewiesen werden konnte. TxA2 und die Aktivierung seines Rezeptors modulieren Vasokonstriktion und Thrombozytenaggregation. Weiterhin nehmen sie Einfluss auf dopaminerge und serotonerge Signalwege. In der Pathophysiologie der Schizophrenie spielen eben diese eine bedeutende Rolle und stellen somit Zielstrukturen für APs dar. Um die Konzentration von TxB2, dem Metaboliten des instabilen Moleküls TxA2 in stimulierten und unstimulierten Blutproben 10 gesunder Probandinnen zu messen, verwendeten wir ein Vollblutverfahren. Um signifikante Ergebnisse zu erhalten, stimulierten wir die Proben mit TSST-1 oder dem monoklonalen Antikörper OKT3 (Muromonab-CD3), der gegen das Oberflächenantigen CD3 gerichtet ist, kombiniert mit dem monoklonalen Antikörper 5C3, der mit dem Protein CD40 interagiert und es stimuliert. Weiterhin versetzten wir das Blut mit den APs CPZ, Clozapin oder NDMC in einer von vier verschiedenen Konzentrationen. Außerdem wurden die Thromboxanspiegel im Blut ohne Zusatz von APs unter verschiedenen Stimulationskonditionen gemessen. Durch den Zusatz von Clozapin in den verschiedenen Konzentrationen kam es zu einer signifikanten (p<0.05) Verringerung der TxB2-Produktion in den mit TSST-1 und ebenso in den mit OKT3/5C3 versetzen Proben, was wir im Rahmen unserer Studie feststellen konnten. Weiterhin konnten wir zeigen, dass CPZ in sehr niedriger Konzentration die TxB2-Spiegel im unstimulierten und im mit TSST-1 stimulierten Blut reduziert. Daraus lässt sich schlussfolgern, dass Clozapin, NDMC und CPZ auch über eine Modulation der TxA2- und TxB2-Produktion das Neurotransmittersystem beeinflussen könnten. Auch typische Nebenwirkungen der AP, wie zum Beispiel die orthostatische Hypotension, könnten aus den Veränderungen der TxA2- und TxB2-Konzentrationen resultieren.

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Wang, Xiao Qiang. "Function and regulation of 17B-hydroxysteroid dehydrogenase type7 (17B-HSD7) in sex hormone biosynthesis and breast cancer : in vitro, in vivo, proteomic and three dimensional co-culture studies." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/27483.

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La 17β-hydroxystéroïde déshydrogénase humaine de type 7 (17β-HSD7) a une double fonction dans la cholestérologénèse et la stéroïdogénèse, et qui est impliquée à la fois dans la formation d’estradiol (E2) à partir de l’estrone (E1), et dans la dégradation de la dihydrotestostérone (DHT) en un œstrogène faible (3β-diol). Cependant, sa fonction dans le cancer du sein dépendant des œstrogènes (positif aux récepteurs oestrogéniques (RE+) n’a pas toujours été claire. L’E2 stimule la croissance des cellules cancéreuses du sein (CCS; cellules MCF-7) via les RE tandis que la DHT a un effet antiprolifératif via le récepteur des androgènes. Mes études in vitro, in vivo et de protéomique, ont apporté les résultats suivants : (1) L’inhibition de la 17β-HSD7 par un inhibiteur spécifique (INH7) dans les CCS a entrainé une baisse de l’E2, une augmentation de la DHT, une interruption du cycle cellulaire et une régulation négative de cette enzyme. De plus, l’INH7 a permis de réduire des tumeurs xénogreffes qui a été accompagnées d’une diminution de l’E2 et une augmentation de la DHT sériques. (2) L’INH7 a modulé des protéines impliquant différents processus biologiques. L’INH7 a supprimé l’expression de la protéine 78 régulée par le glucose (Grp78) et de fait a augmenté l’apoptose des CCS envers le Letrozole, un inhibiteur de l’aromatase. (3) Les interactions entre les CCS et les fibroblastes tumoraux montrent que la 17β-HSD7 était l’enzyme la plus régulée dans les CCS tandis que l’aromatase était l’enzyme les plus régulées dans les fibroblastes. De telles régulations ont mené à une augmentation de la conversion de l’E2 à partir de ses précurseurs, et a ainsi encouragé la prolifération cellulaire des CCS. Si l’augmentation de la prolifération cellulaire est bloquée par le Letrozole des résultats plus significatifs ont été observés par l’INH7 qui bloque la dégradation de la DHT. (4) L’analyse des données intégratives basée sur The Cancer Genome Atlas (TCGA) confirme l’amplification significative du gène HSD17B7 dans les divers cancers du sein comparé à des tissus mammaires sains. Ainsi, nous pensons que la 17β-HSD7 devrait être une nouvelle cible thérapeutique des cancers RE+ du sein.
Human 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7) displays a dual function in cholesterogenesis and steroidogenesis. In steroidogenesis, it is both involved in the formation of the estradiol (E2) from estrone (E1) and in the degradation of dihydroterstosterone (DHT) into weak estrogen 5α-androstane-3β, 17β-diol (3β-diol). However, its function in estrogen dependent breast cancer (estrogen receptor positive, ER+) has been unclear for many years. E2 stimulates breast cancer cells (BCCs, MCF-7 cells) growth via estrogen receptor (ER) whereas DHT displays anti-proliferative effects via androgen receptor (AR). In the present thesis, the function of 17β-HSD7 in ER+ breast cancer was studied with in vitro, in vivo, proteomics and three dimensional (3D) co-culture model and results were described: (1) Inhibition of 17β-HSD7 by its selective inhibitor (INH7) in BCCs induced significant lower E2, higher DHT, cell cycle arresting and negative regulating of the same enzyme. Such inhibition induced significant shrinkage of xenograft tumors accompanied by decreased E2 and elevated DHT in plasma. (2) Inhibition of 17β-HSD7modulated 104 proteins involved in different biological processes. INH7 especially suppresses the expression of glucose regulated protein 78 (GRP78) and consequently enhanced apoptosis of MCF-7 towards aromatase inhibitor. (3) The interactions between BCCs and tumor fibroblast modulate steroidogenic enzymes. 17β-HSD7 was the most modulated enzyme in MCF-7 cells whereas aromatase was the most regulated enzyme in fibroblast (Hs578Bst). Such regulations led to an increasing of E2 conversion from precursors and promoted MCF-7 cells’ proliferation. The increased cell proliferation was blocked by aromatase inhibitor in 3D co-culture system, but more significant results were observed with INH7 which blocked DHT degradation. (4) Integrative data analysis with The Cancer Genome Atlas (TCGA) confirmed the significant amplification of 17β-HSD7 in various breast cancers compared to normal breast tissue. Thus, in the present thesis, 17β-HSD7 was characterized as a novel therapeutic target for estrogen dependent breast cancer in postmenopausal women.

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Star, Gregory. "The effects of bone morphogenic proteins and transforming growth factor [beta] on in-vitro endothelin-1 production by human pulmonary microvascular endothelial cells /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111942.

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Introduction: Idiopathic Pulmonary arteriole hypertension (IPAH) is a rare but severely debilitating disease that strikes women to men at a ratio of 3:1. Endothelial cell (EC) dysfunction is a hallmark of the disease. This includes rapid growth of the ECs until the occlusion of the vasculature as well as decreased blood levels of vasodilators. Markedly increased levels of endothelin-1, a potent vasoconstrictor and smooth muscle mitogen, have been noted in IPAH patients.
Recently mutations in the bone morphogenic protein receptor type II (BMPRII) have been linked to the disease. Interestingly mutations in activin-like kinase-1 (ALK-1) and endoglin have been linked to hereditary haemorrhagic telangiectasia (HHT), a disease that results in PAH clinically indistinguishable from IPAH. All of these proteins are either receptors or co-receptors to members of the TGFbeta superfamily. The connection of these mutations to the disease still remains largely a mystery to researchers and the effects of either bone morphogenic proteins 2, 4, 7 or TGFbeta levels on endothelin-1(ET-1) production in human microvascular endothelial cells cultured from normal lungs (HMVEC-LBI) are unknown.
Methods: HMVEC-LBI cells were cultured in the presence of various concentrations of BMP 2,4,7 and TGFbeta, in complete media or serum starved conditions. After allotted time points the media was collected and assayed by ELISA, meanwhile the cells were lysed and protein content assayed for normalization purposes. Small Mothers against Decapentaplegic (SMAD) 1/5 phosphorylation was also measured.
Results and Conclusions: Despite evidence that all BMPs used were biologically active, namely through SMAD phosphorylation studies, only BMP7 at very high dosages increased ET-1 production levels. TGFbeta had a more pronounced effect at earlier time points with lower concentrations. The results provide insights on the effects of an important group of proteins, the BMPs and TGFbeta, on lung microvascular ECs and which are likely the key cellular player In IPAH development. These findings may have clinical relevance in terms of control of the disease and understanding the normal response of these cells BMPs and TGFbeta.

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Briot, Pascal. "Etudes in vivo et in vitro de la biosynthèse des œstrogènes chez la hase (Lepus europaeus)." Paris 6, 1986. http://www.theses.fr/1986PA066159.

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Etude sur le sang des veines périphériques, ovariennes, utérines au cave de femelles gestantes ou non gestantes, stimulées par PMSG ou non stimulées. In vitro étude sur tissus incubés (follicules, corps jaune, surrénale, endomètre ou placenta).

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32

Couillaud, Julie. "The terpene mini-path : nouvel accès aux terpènes et exploration de l'espace chimique par une cascade enzymatique originale." Electronic Thesis or Diss., Aix-Marseille, 2021. http://www.theses.fr/2021AIXM0188.

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À ce jour, les terpénoïdes constituent la classe de produits naturels la plus abondante et diversifiée avec plus de 80000 composés décrits et dont les propriétés structurales, biologiques (antibiotique, anticancéreux, antipaludique…) et physico-chimiques (arôme, parfum, colorant…) retiennent l’attention de la communauté scientifique. Cependant, leur accès est limité par une faible disponibilité par extraction à partir de sources naturelles ; une synthèse chimique souvent coûteuse et laborieuse; et des voies de biosynthèses longues. En combinant des approches bioinformatiques, statistiques, biochimiques et de biologie moléculaire, nous avons développé la « mini-voie des terpènes », à seulement deux étapes enzymatiques, comme alternative synthétique et biosourçable pour l’accès aux DMAPP et IPP, précurseurs universels des terpènes. Cette nouvelle voie artificielle a permis la synthèse de différents terpénoïdes naturels et également non-naturels tels que des dérivés cyclobutyliques, en l’absence d’ingénierie métabolique et enzymatique. Ainsi, la mini-voie offre un accès facilité à l’ensemble des terpénoïdes et constitue un nouvel outil biosynthétique attractif pour l’exploration de la diversité de l’espace chimique des terpènes
To date, terpenoids form the most abundant and diversified class of natural products with more than 80,000 compounds whose structural, biological (antibiotic, anticancer, antimalarial, etc.) and physicochemical (flavor, fragrance, dye, etc.) properties hold the attention of the scientific community. However, their access is limited because of the low available quantity by extraction from natural sources; an often expensive and laborious chemical synthesis; and long biosynthetic pathways. By combining bioinformatic, statistical, biochemical and molecular biology approaches, we have developed the « Terpene mini-path », with only two enzymatic steps, as a synthetic and potentially bio-sourced alternative to access DMAPP and IPP, universal precursors of terpenes. This new artificial pathway allowed the synthesis of various natural and unnatural terpenoids, such as cyclobutylic derivatives, in the absence of any metabolic and enzymatic engineering. The mini-path provides thus an easy access to all terpenoids and represents an attractive new biosynthetic tool to explore the diversity of the terpene chemical space

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Dauchel, Hélène. "Le système du complément et la cellule endothéliale : biosynthèse in vitro des protéines de la voie alterne et activation pathologique au cours des vascularités leucocytoclasiques." Rouen, 1993. http://www.theses.fr/1993ROUES032.

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Ce travail est consacré à l'analyse des relations existant entre la cellule endothéliale et le complément au cours de l'inflammation. Une première approche a consisté en une étude in vitro de la synthèse constitutive et de sa régulation, sous l'influence de cytokines pro-inflammatoires et d'un agent pharmacologique anti-inflammatoire, par les cellules endothéliales, de protéines essentielles de la voie alterne: les protéines activatrices C3 et facteur B et les protéines inhibitrices facteur H et facteur I. Les résultats suggèrent que, in vivo, les cellules endothéliales seraient orientées, dans des conditions physiologiques normales, vers une fonction inhibitrice de l'activation spontanée du complément par la voie alterne, c'est-a-dire vers une fonction anti-inflammatoire. Cytokines et corticoïdes influenceraient les synthèses dans un sens, qui respectivement, favoriserait l'activation locale du complément ou au contraire renforcerait son inactivation constitutive. L'existence de ces synthèses locales hautement régulées des protéines de la voie alterne du complément constituerait de nouveaux éléments dans les processus d'activation de l'endothélium au cours de l'inflammation et d'inactivation de l'endothélium au cours de glucocorticothérapie. La deuxième approche est une étude clinique de l'implication du complément au cours de pathologies inflammatoires de l'endothélium à complexes immuns: les vascularités leucocytoclasiques cutanées. Elle a permis de démontrer: i) l'intérêt, dans l'exploration clinique du complément au cours de ces maladies, de nouveaux dosages plasmatiques des marqueurs d'activation initiale et finale, le fragment C3d,g et le complexe d'attaque membranaire (CAM); ii) l'implication de l'intégralité de la cascade, jusqu'à formation du CAM à la surface de l'endothélium, dans la genèse des lésions vasculaires; iii) la responsabilité potentielle prépondérante des immunoglobulines A dans l'activation pathologique du complément; iv) l'intervention d'un processus de contrôle de l'activation par les protéines régulatrices du complément. A la fois source et cible du complément, l'endothélium pourrait potentialiser le processus inflammatoire local, mais aussi subir les dommages consécutifs à son activation pathologique

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Gasque, Philippe. "Expression du complément par des cellules du système nerveux central : analyse in vitro de la biosynthèse des protéines du complément par un modèle de lignées d'astrocytes humains." Rouen, 1993. http://www.theses.fr/1993ROUES038.

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En 1987, Levi-Strauss et Mallat décrivirent, pour la première fois, la synthèse in vitro de deux composants du complément (C), le C3 et la facteur B (FB), par des astrocytes de rongeurs. Nous avons voulu poursuivre cette observation en réalisant une étude sur l'expression in vitro de 20 composants du C, de la voie alterne (C3, FB, FD, FP, FH, FI), de la voie classique (C1q, C1r, C1s, C1-Inh, C4, C4bp, C2, C3), et de la voie effectrice du complexe d'attaque membranaire (CAM) (C5, C6, C7, C8, C9, S-protein), par un modèle de lignées d'astrocytes humains. Ce mémoire de thèse décrit: 1) les différentes approches expérimentales qui ont été utilisées (Western blot, ELISA, immunoprécipitation, tests fonctionnels, Northern blot et RTPCR pour détecter les ARNm; 2) la régulation de ces biosynthèses par des cytokines (interféron-gamma, interleukine-1β, TNF-α). D'une manière générale, les cinq lignées d'astrocytes humains sont capables de synthétiser tous les composants nécessaires à l'activation des deux voies du C, mais également à la formation du CAM, responsable d'activité cytotoxique. Ces synthèses sont pour la plupart constitutives, avec cependant un rôle très important des cytokines (principalement l'interferon-gamma). Selon ces observations, nous pouvons considérer: 1) que, via l'astrocyte, le système nerveux central semble disposer d'une source locale en C; 2) qu'un tel potentiel pourrait être impliqué dans des activités neurodégénératives (maladie d'Alzheimer) et démyélinisantes (sclérose en plaques)

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Héron, Antoine. "Préparation et développement de nouveaux antisérums pour l'étude de la synthèse et de la maturation de l'inter-alpha-trypsine inhibiteur humain." Rouen, 1995. http://www.theses.fr/1995ROUES034.

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Les protéines humaines de la famille de l'inter-alpha-trypsine inhibiteur (ITI) sont structurées à partir de trois chaînes lourdes H1, H2 et H3 et d'une chaîne légère, la bikunine. Ces chaînes maturent (H→HC) et s'associent par l'intermédiaire d'une chaîne de chondroïtine sulfate pour donner naissance aux formes protéiques pluricaténaires actuellement décrites: l'ITI (HC1+HC2+bikunine), le pré-alpha-trypsine inhibiteur (HC3+bikunine) et le complexe HC2/bikunine. Il apparaît désormais qu'en plus d'une activité antiprotéasique portée par la bikunine, les chaînes lourdes et l'inhabituelle structure protéoglycannique participent aux fonctions physiologiques des protéines de la famille de l'ITI, en particulier dans le cadre de la stabilité de la matrice extracellulaire. Afin d'apporter des résultats précis concernant la localisation de synthèse et les modalités de la maturation de ces protéines dans la cellule, nous avons développé de nouveaux antisérums spécifiques de chacune des chaînes lourdes H1, H2 et H3. Pour cela, nous avons eu recours au développement d'un système d'expression bactérien permettant à partir de l'ADN, l'obtention de peptides immunogènes sous la forme de protéines hybrides, une alternative de choix pour la production d'anticorps spécifiques évitant de recourir à la purification des protéines natives. L'emploi de ces antisérums dans le cadre d'une étude de la maturation de l'ITI dans des modèles cellulaires d'hépatomes humains a apporté des résultats nouveaux. En particulier, les cellules HepG2 synthétisent une protéine dont les caractéristiques électrophorétiques sont semblables à celles de l'ITI plasmatique humain, mais dont les propriétés antigéniques démontrent que cette molécule ne comprend qu'un seul type de chaîne lourde associée à la bikunine (HC2#2+bikunine#2). L'assemblage de cette glycoprotéine ITI est spécifiquement localisé dans le réseau trans de l'appareil de Golgi après l'élongation et la sulfatation des structures O-glycanniques. La N-glycosylation n'intervient pas dans l'assemblage, la maturation et la sécrétion de cette protéine. Par ailleurs, nos antisérums permettent d'analyser spécifiquement les membres de la famille de l'ITI dans un mélange aussi complexe que le sérum. Cette analyse permet de penser qu'existent à côté des formes classiques d'ITI et de HC/bikunine, des espèces homologues comportant les autres chaînes lourdes. Ainsi, chaque chaîne H1, H2 et H3 semble pouvoir participer à l'assemblage de l'ITI comme de HC/bikunine ; les différences observées quant aux taux sériques respectifs de ces formes pourraient résulter de variations de stabilité de chaque structure

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Maione-Silva, Lorena. "Encapsulação da vitamina c em lipossomas para o tratamento do envelhecimento cutâneo: desenvolvimento tecnológico, analítico e avaliação da performance biológica in vitro em modelos de permeação cutânea e em linhagens celulares de queratinócitos e fibroblastos." Universidade Federal de Goiás, 2016. http://repositorio.bc.ufg.br/tede/handle/tede/6415.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Skin aging involves events that lead to the reduction of its structural integrity and loss of biological functions. The reactive oxygen species (ROS) are potentially able to generate damage of tissues and are related with the cutaneous photoaging. Antioxidant molecules like vitamin C (VC) are capable of fighting these ROS. Besides, VC acts in the synthesis of collagen in the skin, the primary protein responsible for supporting its connective tissues. However, beneficial skin effects are only obtained when the VC is applied topically. In this work, liposomes containing VC for topical administration were developed and characterized. For quantification of VC in different matrixes, including pharmaceutical products, cosmetics, and porcine ear skin, a quantitative analytical method was developed and validated by high performance liquid chromatography with diode array detection (HPLC-DAD) using ion-pair reversed phase. The developed analytical method was capable of quantifying VC without the interference of the various components of the pharmaceutical formulations and the endogenous compounds of the biological matrix. The diluent chosen to extract and dilute VC was a mixture of water and methanol (4:1, v/v) acidified to pH 3.0 with phosphoric acid, with additional 0.02% sodium thiosulfate. This diluent was the most efficient to stabilize VC compared with other pH conditions and compositions, maintaining the amount of VC close to 100% after 10 days at 4°C. In this way, a method for quantification of VC that could be widely used by pharmaceutical companies and research laboratories was developed. It was precise and accurate in the evaluation of the content of VC in biological matrixes and different pharmaceutical formulations, making it advantageous towards other methods. Liposomes with VC were prepared by dehydration-rehydration vesicles method (DRV). Liposomes containing phosphatidylcholine (PC) or a mixture of PC and cholesterol and other electrically charged lipids were prepared, and liposomes with positive, negative and neutral charges were obtained. All formulations presented mean size inferior to 200 nm and low polydispersity index (<0.2). Encapsulation efficiency of VC was directly influenced by the amount of liposomes that were formed. In skin permeation studies, the association of VC in the liposomes only allowed greater retention in the dermis when negatively charged liposomes were used. After 6 hours, the application of this formulation promoted high skin retention of VC, with an accumulation of 37.9 ± 12.02 μg/cm2 and 73.95± 23.23 μg/cm2 in the epidermis and dermis, respectively. Liposomes were capable of increasing the flow of VC through the skin. The presence of cholesterol and negative charge in the liposomes promoted an increase in VC flow of 4 and 7 times, respectively, when compared to free drug (FD). The interaction of liposomes with live biological membranes was simulated in keratinocytes (HaCat) and fibroblasts (3T3) through the analysis of cell internalization of liposomes. For this assay, during the preparation of liposomes, fluorescent lipids were used to label the lipid membrane (coumarin and rhodamine). After treatment, the groups treated with negatively charged liposomal formulation presented superior fluorescence than the groups treated with other formulations and control, suggesting a higher interaction between the negatively charged liposomes and keratinocytes and fibroblasts. Thus, this negatively charged formulation was compared with free VC in the cell regeneration of keratinocytes after exposure to UVA radiation and in the production of collagen type I in fibroblasts. In both cases, the beneficial effect was only observed when VC was encapsulated in the liposomes. Therefore, the technological development of a liposomal formulation containing VC generated a formulation with a stability of at least 30 days and with characteristics that favored its retention and skin flow. Besides, the encapsulation of VC in negatively charged liposomes promoted an enhancement in the efficacy of regeneration of keratinocytes and the synthesis of collagen in fibroblasts.
O envelhecimento da pele envolve eventos que levam à redução da integridade estrutural e perda das suas funções biológicas. As espécies reativas de oxigênio (EROs) são potencialmente capazes de gerar danos teciduais e estão relacionadas com o fotoenvelhecimento cutâneo. Moléculas antioxidantes como a vitamina C (VC) são capazes de combater estes compostos. Além disso, a VC atua na síntese de colágeno na pele, proteína fundamental à sua sustentação. No entanto, efeitos benéficos cutâneos só são obtidos quando a VC é aplicada topicamente. Neste trabalho, foram desenvolvidos e caracterizados lipossomas contendo VC para a aplicação tópica. Para a quantificação da VC em diferentes matrizes, incluindo produtos farmacêuticos, cosméticos e pele de orelha de porco, foi desenvolvido e validado um método quantitativo por cromatografia líquida de alta eficiência acoplada à detector de arranjo de diodos (HPLC-DAD) por pareamento iônico em fase reversa. O método analítico desenvolvido foi capaz de quantificar a VC sem sofrer interferência dos diversos componentes das formulações farmacêuticas e dos compostos endógenos da matriz biológica. O diluente escolhido para extrair e diluir a VC foi a mistura contendo água e metanol (4:1, v/v) acidificada com ácido fosfórico para pH 3,0 com a adição de 0,02% de tiossulfato de sódio. Este diluente foi o mais eficaz na estabilização da VC, comparando-se com outras condições de pH e composição, com a manutenção da quantidade de VC próximo a 100% depois de 10 dias a 4ºC. Desta forma, foi desenvolvido um método de quantificação da VC que pode ser amplamente utilizado por indústrias farmacêuticas e laboratórios de pesquisa, uma vez que foi preciso e exato na avaliação do teor da VC em matriz biológica e em diferentes preparações farmacêuticas. Os lipossomas contendo VC foram produzidos pela técnica de dehydrationrehydration vesicles (DRV). Foram produzidos lipossomas contendo fosfatidilcolina (PC) ou mistura de PC com colesterol e outros lipídeos eletricamente carregados, obtendo-se assim lipossomas com carga elétrica neutra, positiva ou negativa. Todas as formulações apresentaram tamanho médio inferior a 200 nm e baixo índice de polidispersão (< 0,2). A eficiência de encapsulação da VC foi diretamente influenciada pela quantidade de lipossomas formados. Nos estudos de permeação cutânea, a associação da VC aos lipossomas só permitiu maior retenção na derme quando foram utilizados os lipossomas carregados negativamente. Após 6 horas, a aplicação desta formulação proporcionou alta retenção cutânea da VC, com acúmulo de 37,19 ± 12,02 μg/cm2 e 73,95 ± 23,23 μg/cm2 na epiderme e derme, respectivamente. Os lipossomas foram capazes de aumentar o fluxo de VC através da pele. A presença de colesterol e carga superficial negativa nos lipossomas provocaram aumento do fluxo de VC de 4 e 7 vezes, respectivamente, em relação ao fármaco livre (FL). A interação dos lipossomas com membranas biológicas vivas foi simulada em linhagens de queratinócitos (HaCat) e fibroblastos (3T3) através da análise da internalização celular dos lipossomas. Neste caso, durante o preparo dos lipossomas foram adicionados marcadores de membrana lipídica fluorescentes (rodamina e cumarina). Após tratamento, os grupos que receberam formulação lipossomal com carga superficial negativa apresentaram fluorescência superior aos grupos tratados com as outras formulações e o controle, sugerindo maior interação entre os lipossomas negativos e os queratinócitos e fibroblastos. Assim, a formulação com carga negativa foi comparada com a VC livre na regeneração celular de queratinócitos após exposição à radiação UVA e na produção de colágeno tipo I em fibroblastos. Nos dois casos, os efeitos benéficos só foram observados com a encapsulação da VC nos lipossomas. Desta forma, o desenvolvimento tecnológico de uma formulação lipossomal contendo VC, permitiu a obtenção de uma formulação com estabilidade de pelo menos 30 dias e com características que favoreceram sua retenção e fluxo cutâneo. Além disso, a encapsulação da VC em lipossomas negativos proporcionou aumento da sua eficácia na regeneração de queratinócitos e na síntese de colágeno em fibroblastos.

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37

Mesnard, François. "Etude par rmn du carbone 13 et de l'azote 15 du metabolisme de suspensions cellulaires de solanacees en relation avec la biosynthese des alcaloides (doctorat : genie enzymatique bioconversion et microbiologie)." Amiens, 1999. http://www.theses.fr/1999AMIEP066.

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38

Xiao, Zhe. "Biosynthetic studies of tetrodotoxin and its anticancer activities assessment in vitro." HKBU Institutional Repository, 2014. https://repository.hkbu.edu.hk/etd_oa/56.

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In this study, the synthesis of TTX by three species of TTX-producing bacteria (Vibrio alginolyticus, Microbacterium arabinogalactanolyticum and Serratia marcescens) was conducted in a 10-L fermentor under the same controlled fermentation conditions for each of a period of 60 hours. The bacterial growth curves were monitored and the TTX synthesized in the culture medium was determined by HPLC. The TTX biosynthesis was found limited at the microgram level per L of culture medium with toxicities 14.7 MU (mouse unit) and 13.0 MU per mL in the partially purified culture medium of V. alginolyticus and M. arabinogalactanolyticum respectively by mouse bioassay. In the studies on SW480 and SW620 colorectal carcinoma cell lines, the expression, distribution, invasion and proliferation of voltage-gated sodium channels (VGSCs) were investigated by MTT assay (24-48 hours) and wound healing assay (0-120 hours). The different subtypes of VGSCs were expressed by semiquantitative RT-PCR and the locations of Nav1.5 and Nav 1.7 were detected by immunofluorescence microscopy. In the MTT assay, 40μmol/L of TTX showed significant inhibitory effect on both cell lines, with maximum inhibition rate, 33% and 40%, in SW480 and SW620 respectively. In the wound-healing assay, the inhibitory rate of 80μmol/L of TTX on SW480 reached 22% after 120 hours, compared with 30% in the control group. Moreover, VGSCs were highly expressed in both SW480 and SW620, with the main subtypes of Nav1.5 and Nav1.7 located on the cell surface, which might increase the metastatic rate of the cell lines. Keywords: Tetrodotoxin (TTX), Bacterial synthesis, Anticancer, VGSCs

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39

Kandziora, Nadine [Verfasser]. "In vitro-Untersuchungen von Schlüsselschritten der Borrelidin- und Ambruticin-Biosynthesen / Nadine Kandziora." Hannover : Technische Informationsbibliothek (TIB), 2016. http://d-nb.info/1116960893/34.

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Friedrich, Steffen Christoph [Verfasser]. "Untersuchungen zur Biosynthese von Polyketiden : Studien zur in-vitro-Aktivität der Tailoring-Enzyme aus der Jerangolid-Biosynthese / Steffen Christoph Friedrich." Hannover : Technische Informationsbibliothek (TIB), 2016. http://d-nb.info/1107037034/34.

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41

Daniels, Kathy. "Characterisation of O-antigen biosynthesis genes in Vibro anguillarum and their association with IS1358 /." Title page, abstract and table of contents only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phd1861.pdf.

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42

De, Tapia Marc. "Proteines pr de haricot (var. Saxa) induites par traitement chimique ou infection virale : purification et proprietes." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13169.

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Burns, Kristi Lee. "An exploration of biochemistry including biotechnology, structural characterization, drug design, and chromatographic analyses." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/29593.

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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007.
Committee Chair: Sheldon W. May ; Committee Members: Donald F. Doyle, Leslie T. Gelbaum, Stanley H. Pollock, and James Powers. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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44

Veilleux, Nicole H. (Nicole Heather) 1979. "Proliferative, contractile, and biosynthetic activity of adult canine articular chondrocytes in Type I and II collagen-glycosaminoglycan matrices in vitro." Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/89919.

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45

Wunsch-Palasis, Julia [Verfasser], and Andreas [Akademischer Betreuer] Bechthold. "In silico und in vitro Untersuchungen zum Sekundärstoffwechsel in Aktinomyceten unter besonderer Berücksichtigung der Biosynthese der Rishirilide aus Streptomyces bottropensis." Freiburg : Universität, 2013. http://d-nb.info/1123477523/34.

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46

Vincent, Florence. "Biosynthese de certains composes du complement par les monocytes humains in vitro : relation avec la maturation/differenciation des monocytes normaux et pathologiques." Université Louis Pasteur (Strasbourg) (1971-2008), 1992. http://www.theses.fr/1992STR13203.

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La premiere partie du travail a ete consacree a l'etude de la differenciation des monocytes sanguins humains. Nous decrivons des conditions de culture n'utilisant pas de serum. Les cellules ont ete caracterisees tout au long de leur differenciation en milieu sans serum par differents tests (morphologie, cytochimie, analyse des antigenes membranaires, cytotoxicite. . . ). Cette methode de culture en milieu sans serum est simple et reproductible, elle permet la differenciation et la survie a long terme d'une grande quantite de monocytes humains en macrophages completement differencies et activables. Dans la deuxieme partie, l'etude de la biosynthese des composes c2, c4 et c9 du complement a ete entreprise chez les monocytes sanguins humains au cours de leur differenciation, chez des cellules pathologiques de leucemies monoblastiques, et chez des cellules de la lignee u937 (detection de la secretion des proteines c2 et c4 par des tests hemolytiques et detection de l'expression des genes par pcr. Par ces memes methodes, ni la proteine c9, ni son expression n'ont ete detectees. Les cellules de la lignee mono-macrophagique sont donc capables de produire certains composes du complement. Elles representent des composes cellulaires importants dans l'immunoregulation des phenomenes inflammatoires

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47

Saliba, Sahar. "Nouvelles approches biotechnologiques pour l’obtention d’alcaloïdes : culture in vitro de Leucojum aestivum L. et isolement d’endophytes bactériens d’Amaryllidaceae." Thesis, Université de Lorraine, 2015. http://www.theses.fr/2015LORR0107/document.

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Plus de 300 alcaloïdes d’Amaryllidaceae doués d’activités biologiques ont été isolés à partir des plantes appartenant à cette famille. De nos jours, seule la galanthamine, utilisée pour le traitement palliatif de la maladie d’Alzheimer, est commercialisée. L’accumulation de ces alcaloïdes dans les plantes est limitée. La culture in vitro est une méthode alternative intéressante pour l’obtention plus aisée de ces alcaloïdes à haute valeur ajoutée. Le premier objectif de ce travail vise à développer une méthode de purification efficace, simple et rapide des extraits de plantes préalablement à leur analyse en LCMS et GCMS. Le second objectif est d’étudier l’effet de plusieurs facteurs exogènes, ajoutés au milieu de culture de bulbilles de Leucojum aestivum et de sa variété Gravety Giant en bioréacteurs RITA®, sur les voies de biosynthèse de la galanthamine et de la lycorine. La variation des paramètres exogènes a permis une accumulation accrue en galanthamine et en lycorine (0,814 mg/g et 1,54 mg/g de matière sèche respectivement) dans les bulbilles. Le troisième objectif porte sur l’isolement et l’identification d’endophytes à partir de bulbes in vivo et in vitro de trois espèces d’Amaryllidaceae (L. aestivum, Narcissus pseudonarcissus et Galanthus elwesii). Des bactéries endophytes du genre Bacillus ont été identifiées. Un nouvel alcaloïde a été isolé à partir des cultures bactériennes
Over 300 Amaryllidaceae alkaloids possessing a wide range of biological activities have been isolated from plants belonging to this family. Galanthamine, used for the palliative treatment of Alzheimer’s disease, is the only one commercialized. The biodisponiblity of these alkaloids is low. In vitro culture offers an alternative yet interesting approach for the biotechnological production of these valuable alkaloids. The aim of this work was, first, to develop a fast, efficient and easy purification method of plant extracts prior to their phytochemical analysis both in LCMS and GCMS. Second, the combined effects of bioreactor RITA® culture and feeding with different exogenous factors on the biosynthetic pathway of both galanthamine and lycorine were studied. The experiments were conducted both with Leucojum aestivum and L. aestivum ‘Gravety Giant’ bulblets. The variation of several exogenous parameters resulted in a better accumulation of galanthamine and lycorine (0.814 mg/g and 1.54 mg/g dry weight respectively) in the bulblets. The third aim was to isolate and identify alkaloid producing endophytes from in vivo and in vitro bulbs of three Amaryllidaceae species (L. aestivum, Narcissus pseudonarcissus and Galanthus elwesii). Bacterial endophtes belonging to the Bacillus genus were identified. A new alkaloid was isolated from bacterial liquid cultures

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48

MAILLOT, VERNIER PASCALE. "Selection in vitro et caracterisation de mutants resistants a des inhibiteurs de la biosynthese des phytosterols, a partir de protoplastes de nicotiana tabacum dihaploide." Université Louis Pasteur (Strasbourg) (1971-2008), 1990. http://www.theses.fr/1990STR13058.

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Une strategie du mutagenese aux ultraviolets et de selection a l'aide d'un triazole-pesticide, appliquee a des cultures de protoplastes de tabac, a permis l'obtention de cals resistants. Ces cals, qui sont de trois types, presentent ou non des modifications de leur profil sterolique par rapport a un cal sauvage, sensible au triazole, indiquant que l'inhibition de la biosynthese des sterols est impliquee, peut-etre seulement en partie, dans la phytotoxicite du pesticide. La regeneration de plantes normales fertiles a pu etre realisee a partir de deux des trois types de cals selectionnes et l'heredite de ces deux types a ete demontree. Le premier est caracterise par une relative insensibilite de sa biosynthese des sterols a l'inhibiteur, montrant qu'il pourrait s'agir d'un mutant de detoxication ou d'un mutant presentant une cible modifiee. Le second genotype surproduit les sterols de fin de chaine et accumule de nombreux intermediaires biosynthetiques. Ce phenotype biochimique, qui se transmet comme un caractere mendelien monogenique codominant, pourrait etre code par une amplification genique. La resistance des cals de ces deux genotypes au triazole serait due a leur capacite a conserver une quantite suffisante de sterols habituels dans leurs membranes pour y assurer une fonction architecturale et peut-etre metabolique

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Soboh, Basem [Verfasser], Dietrich [Akademischer Betreuer] Nies, Rudolf [Akademischer Betreuer] Thauer, and Thomas [Akademischer Betreuer] Happe. "In vitro Biosynthese von komplexen Fe-S-Cluster-Cofaktoren der Fe-Mo-Nitrogenase und der [NiFe]-Hydrogenase / Basem Soboh ; Dietrich Nies, Rudolf Thauer, Thomas Happe." Halle, 2016. http://d-nb.info/1116950502/34.

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50

Schaller, Hubert. "Selection in vitro et caracterisation de mutants de nicotiana tabacum l. Resistant a des pesticides (de type n-alkyl-morpholine ou triazole) inhibiteurs de la biosynthese des sterols." Université Louis Pasteur (Strasbourg) (1971-2008), 1992. http://www.theses.fr/1992STR13121.

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Une strategie de mutagenese-selection de cultures de protoplastes de tabac a permis d'obtenir des cals resistant a des pesticides de type n-alkyl-morpholine ou triazole, inhibiteurs de la biosynthese des sterols. Les differentes classes de cals resistants, discrimines en fonction de leur composition sterolique, suggerent l'existence de plusieurs mecanismes de resistance a ces pesticides, mettant en jeu soit une modification de la voie des sterols, soit des modifications dans d'autres voies metaboliques. En particulier, un mutant surproducteur de sterols a ete caracterise. L'analyse genetique montre que cette surproduction segrege avec la resistance, ce qui suggere la coincidence des deux phenotypes. Le mutant parvient ainsi a contrer l'effet des inhibiteurs phytotoxiques en surproduisant les sterols puis en esterifiant avec des acides gras les intermediaires biosynthetiques accumules. Ce mecanisme de regulation permet de maintenir une fraction de sterols libres, destines a etre incorpores dans les membranes, approchant en quantite et en qualite celle du genotype sauvage cultive en l'absence d'inhibiteur

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Dissertations / Theses on the topic 'In vitro biosynthesis'

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